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Intra-articular sustained release carriers for osteoarthritis
management: formulation and bioactivity evaluation studies
DE LACERDA SALGADO, Carlota
Abstract
Les différentes études présentées dans cette thèse ont permis de faire face et ont tenté de
répondre à un certain nombre de défis rencontrés dans la recherche de l'IA DDS pour le
traitement de l'arthrose. Tout d'abord, le potentiel des molécules thérapeutiques pour
l'administration locale. Les produits naturels sont de riches sources de molécules
thérapeutiques ; les médicaments contre l'arthrose potentiellement modificateurs de la
maladie (DMOAD) et les voies moléculaires méritent d'être explorées plus avant en
appliquant des modèles précliniques précis de l'arthrose. Ensuite, le développement de
systèmes d'administration de médicaments par des vecteurs polymériques et enfin,
l'établissement de modèles in vitro précis d'arthrose pour l'évaluation des résultats de ce type
de formulations. Les microparticules polymères avec des nanoparticules de médicament
broyées par wet milling et des taux de chargement de médicament réglables semblent
répondre à plus d'une demande de traitement de l'arthrose par IA. Pour améliorer la
conception et le développement de ces DDS IA, il est nécessaire [...]
DE LACERDA SALGADO, Carlota. Intra-articular sustained release carriers for
osteoarthritis management: formulation and bioactivity evaluation studies. Thèse de
doctorat : Univ. Genève, 2021, no. Sc. 5546
DOI : 10.13097/archive-ouverte/unige:150608
URN : urn:nbn:ch:unige-1506080
Available at:
http://archive-ouverte.unige.ch/unige:150608
Disclaimer: layout of this document may differ from the published version.
1 / 1
UNIVERSITÉ DE GENÈVE
Section des Sciences Pharmaceutiques
Laboratoire de Technologie Pharmaceutique
FACULTÉ DES SCIENCES
Prof. Eric Allémann
Dr. Olivier Jordan
Intra-articular sustained release carriers for
osteoarthritis management: formulation and
bioactivity evaluation studies
THÈSE
présentée à la Faculté des Sciences de l’Université de Genève
pour obtenir le grade de Docteur ès Sciences, mention Sciences Pharmaceutiques
par
Carlota SALGADO
de
Lisboa (Portugal)
Thèse N°: 5546
Genève
Atelier de reproduction Repromail
2021
Ao meu querido avô João, para sempre o meu maior fã.
Saudações benfiquistas!
Table of contents
Acronyms
1
Introduction
5
Chapter I Osteoarthritis in vitro models: applications and implications in development of intra-articular drug delivery systems
11
Chapter II In vitro anti-inflammatory activity in arthritic synoviocytes of A. brachypoda root extracts and its unusual dimeric flavonoids
43
Chapter III Nano wet milled celecoxib extended release microparticles for local management of chronic inflammation
69
Chapter IV Sustained release carriers for osteoarthritis: in vitro evaluation of an anti-inflammatory drug and a chondrogenic drug on a 3D chondrocyte OA model
103
Conclusions and perspectives 135
French summary 141
Acknowledgements 143
1
Acronyms
ADAMTs Disintegrin and metalloproteinase with thrombospondin motifs
IL-6 Interleukin-6
COX-2 Cyclooxygenase 2 KGN Kartogenin CXB Celecoxib MMPs Matrix metalloproteinases
DAPI 4′,6-diamidino-2-phenylindole dihydrochloride
MPs Microparticles
DDS Drug delivery systems MPLC Medium pressure liquid chromatography
DLS Dynamic light scattering NMR Nuclear magnetic resonance
DMMB 1,9-dimethylmethylene blue
NSAIDs Nonsteroidal anti-inflammatory drugs
DMOADs Disease-modifying osteoarthritic drugs
OA Osteoarthritis
DMSO Dimethyl sulfoxide PDGF-BB Platelet-derived growth factor ECM Extracellular matrix PDLLA Poly D, L-lactic acid
ELISA Enzyme-linked immunosorbent assay
PGE2 Prostaglandin E2
FA Formic acid PLA Poly(lactic acid)
FGF-2 Basic fibroblast growth factor 2
PLGA Poly(lactic-co-glycolic acid)
GAG Glycosaminoglycans qPCR Quantitative polymerase chain
reaction
HA Hyaluronic acid
SEM Scanning electron microscopy
HBMSCs Human bone marrow mesenchymal stem cells
TFA Trifluoroacetic acid
HFLS Human fibroblast-like synoviocytes
TGFβ-1 Transforming growth factor beta 1
HPLCHigh performance liquid chromatography
TNFα Tumor necrosis factor alpha
HSCCC High-speed counter current chromatography
TPGS D-α-tocopherol polyethylene glycol 1000 succinate
IA Intra-articular UHPLC Ultra high performance liquid
chromatography IL-1β Interleukin-1 beta XRPD X-ray powder diffraction
Introduction
Introduction
5
BACKGROUND
Osteoarthritis (OA) is a chronic, degenerative disease. This most common form of
arthritis has a high and ever-increasing prevalence in the aging population
worldwide (> 65 years). As a disease of the whole joint, it is characterized by
inflammation and pain, ultimately leading to movement impairment. As one of the
leading disability causes in the elderly, the socio-economic burden caused by OA is
substantial [1,2]. Presently, pharmacological treatment approaches are based on
symptom management by oral analgesic and anti-inflammatory drugs [3]. However,
there is a current unmet need for a cure: by better, more efficacious
formulations/administration routes or disease-modifying drugs (DMOADs) that help
slow and potentially revert the disease. In recent years, significant efforts have been
made in the research and development of novel molecules and improved drug
delivery systems [4,5]. Natural products have become appealing in the field of OA
due to the potential to tackle both inflammatory and cartilage degradation pathways
[6]. Despite common drug development hurdles, like physicochemical issues such
as solubility and development of clinical trials, the complexity of OA as a disease
proves an obstacle for the successful development of DMOADs. A great number of
target tissues (synovium, cartilage, bone) plus a myriad of molecular targets of pain
and inflammation is involved in OA mechanisms, making drug discovery a
challenge. Lastly, the variety of OA disease phenotypes, onset and clinical
manifestations hampers the development of accurate and predictive pre-clinical and
clinical trials [4,7]. Additionally, the joint as a target, with poor irrigation and blood
supply, decreases the efficacy of systemic oral therapy. Intra-articular (IA) local
delivery of therapeutic molecules has been explored as an alternative that bypasses
issues of systemic oral therapy, such as low joint retention and adverse effects. A
controlled, sustained release of molecules directly into the joint capsule has been
sought after with the development of drug delivery systems. IA drug delivery
systems like nano- and micro-carriers, hydrogels or liposomes have been
investigated in recent years [5,8]. In both DMOADs and IA drug delivery systems
research, pre-clinical trials are an essential and crucial step. In this phase of
development, it is crucial to consider accurate and highly predictive in vitro and in
vivo OA models for increased translation into humans. Better design, prediction, and
translation into humans are key in furthering OA research [9,10].
Introduction
6
SCOPE AND AIMS
This thesis is based on three different axes: research of novel molecules with
potential as DMOADs; development of polymeric IA drug delivery systems for local
delivery of OA treatments and design and establishment of accurate in vitro OA
models. It follows previous projects led by Prof. Eric Allémann and Dr. Olivier
Jordan, thus continuing a 10-year research effort on novel OA treatments [11,12].
The main goal was the development of IA treatment options for OA by exploring
novel molecules and developing appropriate drug delivery systems. Throughout this
work, another aim was to accurately represent in vitro OA, with the establishment of
cellular models and adequate outcome evaluation.
GENERAL STRUCTURE
In an attempt to better design and formulate IA drug delivery systems for OA,
Chapter I reviews the literature on different in vitro models of OA. Models are
described and discussed regarding their specific application in the study and
development of IA drug delivery systems, such as polymeric micro and
nanoparticles. Chapter II explores the potential of natural plant extracts as local IA
treatment of OA. Root extracts of Arrabidaea brachypoda, a Brazilian shrub, are
known for their analgesic and anti-inflammatory properties in arthritics joints. In this
study, two different root extracts and three novel flavonoids isolated from one of the
extracts are evaluated in vitro, in human fibroblast-like synoviocytes, for their
potential as local OA therapeutic options. In Chapter III, approaches tackling the
inflammation component of OA are further explored. In this study, celecoxib, a non-
steroidal anti-inflammatory drug (NSAID) with established clinical use in OA, was
encapsulated into polymeric microparticles with the goal of developing a sustained,
controlled release IA drug delivery system. Using a spray-drying method, celecoxib
was incorporated, at three different drug loadings, as a drug solution or embedded
as nano-milled drug particles into PLA microparticles. Aiming at a long-lasting
therapeutic effect in OA, the main goal of this study was to assess the effects of the
incorporation of nano-milled drug, at high drug loadings, on the sustained prolonged
release of celecoxib. The same spray-drying method was applied to a different drug,
kartogenin, in Chapter IV. This chapter aimed to compare the performance of two
distinct molecules, incorporated in PLA microparticles as nano-milled drug at 10 %
drug loading: celecoxib and kartogenin. In the first part, both drug delivery systems
Introduction
7
were characterized. In the second part, human articular chondrocytes were
collected from three donors to form pellets of hyaline cartilage as a 3D in vitro model
of OA. The objective of this study was to determine the effects of a sustained
prolonged release of two molecules targeting two OA pathways (inflammation and
cartilage degradation) in a 3D in vitro model.
A final section of Conclusions and perspectives serves as a summary of all
described studies and explores progress in IA drug delivery systems research for
OA treatment.
REFERENCES
[1] D.J. Hunter, S. Bierma-Zeinstra, Osteoarthritis, Lancet. 393 (2019) 1745–1759. https://doi.org/10.1016/S0140-6736(19)30417-9.
[2] J. Puig-Junoy, A. Ruiz Zamora, Socio-economic costs of osteoarthritis: A systematic review of cost-of-illness studies, Semin. Arthritis Rheum. 44 (2015) 531–541. https://doi.org/10.1016/j.semarthrit.2014.10.012.
[3] S.L. Kolasinski, T. Neogi, M.C. Hochberg, C. Oatis, G. Guyatt, J. Block, L. Callahan, C. Copenhaver, C. Dodge, D. Felson, K. Gellar, W.F. Harvey, G. Hawker, E. Herzig, C.K. Kwoh, A.E. Nelson, J. Samuels, C. Scanzello, D. White, B. Wise, R.D. Altman, D. DiRenzo, J. Fontanarosa, G. Giradi, M. Ishimori, D. Misra, A.A. Shah, A.K. Shmagel, L.M. Thoma, M. Turgunbaev, A.S. Turner, J. Reston, 2019 American College of Rheumatology/Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand, Hip, and Knee, Arthritis Rheumatol. 72 (2020) 220–233. https://doi.org/10.1002/art.41142.
[4] A. Latourte, M. Kloppenburg, P. Richette, Emerging pharmaceutical therapies for osteoarthritis, Nat. Rev. Rheumatol. (2020). https://doi.org/10.1038/s41584-020-00518-6.
[5] P. Maudens, O. Jordan, E. Allémann, Recent advances in intra-articular drug delivery systems for osteoarthritis therapy, Drug Discov. Today. 23 (2018) 1761–1775. https://doi.org/10.1016/j.drudis.2018.05.023.
[6] G.-E. Deligiannidou, R.-E. Papadopoulos, C. Kontogiorgis, A. Detsi, E. Bezirtzoglou, T. Constantinides, Unraveling Natural Products’ Role in Osteoarthritis Management—An Overview, Antioxidants. 9 (2020) 348. https://doi.org/10.3390/antiox9040348.
[7] M.A. Tryfonidou, G. de Vries, W.E. Hennink, L.B. Creemers, “Old Drugs, New Tricks” – Local controlled drug release systems for treatment of degenerative joint disease, Adv. Drug Deliv. Rev. 160 (2020) 170–185. https://doi.org/10.1016/j.addr.2020.10.012.
[8] L.M. Mancipe Castro, A.J. García, R.E. Guldberg, Biomaterial strategies for improved intra‐articular drug delivery, J. Biomed. Mater. Res. Part A. (2020) 1–11. https://doi.org/10.1002/jbm.a.37074.
[9] T.L. Vincent, Of mice and men: converging on a common molecular understanding of osteoarthritis, Lancet Rheumatol. 2 (2020) e633–e645. https://doi.org/10.1016/S2665-9913(20)30279-4.
[10] Y. He, Z. Li, P.G. Alexander, B.D. Ocasio-Nieves, L. Yocum, H. Lin, R.S. Tuan, Pathogenesis of osteoarthritis: Risk factors, regulatory pathways in chondrocytes, and experimental models, Biology (Basel). 9 (2020) 1–32. https://doi.org/10.3390/biology9080194.
[11] J. Pradal, M.-F. Zuluaga, P. Maudens, J.-M. Waldburger, C.A. Seemayer, E. Doelker, C. Gabay, O. Jordan, E. Allémann, Intra-articular bioactivity of a p38 MAPK inhibitor and development of an extended-release system, 93 (2015) 110–117. https://doi.org/10.1016/j.ejpb.2015.03.017.
Introduction
8
[12] P. Maudens, C.A. Seemayer, C. Thauvin, C. Gabay, O. Jordan, E. Allémann, Nanocrystal-Polymer Particles: Extended Delivery Carriers for Osteoarthritis Treatment, Small. 14 (2018) 1703108. https://doi.org/10.1002/smll.201703108.
Chapter I
11
Chapter I
Osteoarthritis in vitro models: applications and implications
in development of intra-articular drug delivery systems Carlota Salgado a,b, Olivier Jordan a,b and Eric Allémann a,b
a School of Pharmaceutical Sciences, University of Geneva, 1 Rue Michel-Servet 1211 Geneva, Switzerland b Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, 1 Rue Michel-Servet 1211 Geneva, Switzerland
Published: Pharmaceutics, 2021, 13 (1), 60; https://doi.org/10.3390/pharmaceutics13010060
ABSTRACT
Osteoarthritis (OA) is a complex multi-target disease with an unmet medical need for the development of therapies that slow and potentially revert disease progression. Intra-articular (IA) delivery has seen a surge in osteoarthritis research in recent years. As local administration of molecules, this represents a way to circumvent systemic drug delivery struggles. When developing intra-articular formulations, the main goals are a sustained and controlled release of therapeutic drug doses, taking into account carrier choice, drug molecule, and articular joint tissue target. Therefore, the selection of models is critical when developing local administration formulation in terms of accurate outcome assessment, target and off-target effects and relevant translation to in vivo. The current review highlights the applications of OA in vitro models in the development of IA formulation by means of exploring their advantages and disadvantages. In vitro models are essential in studies of OA molecular pathways, understanding drug and target interactions, assessing cytotoxicity of carriers and drug molecules, and predicting in vivo behaviors. However, further understanding of molecular and tissue-specific intricacies of cellular models for 2D and 3D needs improvement to accurately portray in vivo conditions.
Keywords: osteoarthritis; intra-articular drug delivery systems; synovium; cartilage; in vitro cellular models; synoviocytes; chondrocytes
Chapter I
12
1. INTRODUCTION
Osteoarthritis (OA) is a chronic disease with worldwide incidence in the population
aged 65 years and higher, representing a significant economic burden in terms of
global health [1,2]. As the most common form of arthritis and one of the leading
causes of disability in the elderly population, OA is characterized by chronic
inflammation, articular cartilage degeneration and structural changes of whole joints.
There is currently an unmet need for disease-modifying drugs (DMOADs) that slow
or even revert disease progression [3–5]. Pharmacological treatment options focus
on symptom management. Oral analgesic and nonsteroidal anti-inflammatory drugs
(NSAIDs) are first-line treatments for pain and inflammation. However, since OA
mainly affects the joint as a whole closed structure, systemic drugs result in less
than optimal efficacy rates [6,7]. A known alternative that circumvents most of the
drawbacks associated with systemic drug administration is the delivery of drugs
locally, by intra-articular (IA) injection. IA allows for higher drug doses and prolonged
delivery of drug molecules directly into affected joints. By this approach, more
effective relief of symptoms may be attained, while systemic adverse effects are
generally avoided. Different drug delivery systems (DDSs) have grown in the field
to improve the delivery of small molecules locally to joints. These include different
formulations such as polymeric nano and microparticles, hydrogels, liposomes and
micelles, which have been extensively reviewed [8–11]. Due to its local
administration, maintaining the selectivity of drug molecules and the carrier system
towards biological tissue targets in the joint while avoiding off-target effects is critical
when developing IA formulations. In this regard, the design of predictive in vitro OA
models is crucial in characterizing and understanding the studied drug delivery
systems for OA treatment. Different cellular models represent different tissues of the
joint: synoviocytes, the synovium, and chondrocytes are used to model articular
cartilage [12]. The different types of in vitro cellular models (i.e., monolayer, three-
dimensional or explant) have various applications according to the final goals of IA
formulation. Thus, a deep understanding of their intricacies is very important in this
field. The purpose of the present review is to discuss the relevance of the different
in vitro OA models in the development of IA formulations for OA treatment. At first,
an overview of the latest (5 years) intra-articular DDSs is presented, highlighting the
choice of in vitro model for each formulation. In this review, viscosupplementation
OA in vitro models
13
formulations and delivery of cells (mesenchymal stem cells and platelet rich plasma)
have been excluded. The review focuses on nano and micro carriers, hydrogels and
liposomes containing drug molecules. Next, advantages and disadvantages, as well
as possible readable markers and targets of different in vitro OA models, are
discussed, based on their relevance for the development of intra-articular
formulations.
2. OSTEOARTHRITIS
Osteoarthritis is a chronic degenerative disease of the whole joint. It is characterized
by chronic inflammation, articular cartilage degeneration and structural changes in
several joint tissues. Age >65 years old, obesity, gender (double prevalence in
females), previous joint injuries and genetic predisposition to joint complications are
all considered risk factors in the development of mild to severe OA [2,13]. Other than
the economic burden it represents, OA is one of the primary causes of disability in
the elderly population. Considered the most common form of arthritis, its worldwide
incidence has repercussions on more than 100 million people [1,14]. The etiology of
OA is unknown (primary OA) in the majority of cases, with secondary OA (one that
follows joint injury) as an example of how trauma to the joint influences further
disease progression. Several biomechanical and molecular processes are known to
kick-start the pathology cycle. Tissue alterations of articular cartilage from increased
cell proliferation and microarchitectural changes to the structure of subchondral
bone are considered key events [15]. In early stages, degradation products of
proteoglycan and collagen are released into the joint cavity from hyaline cartilage.
This phenomenon stimulates immune cells from the synovial membrane to release
pro-inflammatory cytokines - mainly IL-1β, IL-6 and TNFα. This inflammatory state
induces catabolic mechanisms by the chondrocytes that produce matrix
metalloproteinase (MMPs) 1, 3 and 13 and aggrecanases 1 and 2 (disintegrin and
metalloproteinase with thrombospondin motifs - ADAMTS). Cartilage is further
degraded, and the inflammatory state is perpetuated. Due to its poor vascularization
and low cellular density, cartilage has a limited regeneration turnover. As disease
advances, catabolic mechanisms outweigh those of repair by the extracellular matrix
(ECM) [16–18]. As a result, there is a narrowing of the joint space due to cartilage
degradation, subchondral bone erosion with the formation of osteophytes and small
cysts, inflammation of the synovium (synovitis), and overall joint function loss (Figure
Chapter I
14
1). Clinical manifestations of the disease with well-established symptoms, mainly
joint pain, stiffness and, consequently, a decrease in daily movement, appear
relatively late. When detected and adequately diagnosed by physical assessment
and bioimaging (X-ray, MRI), OA has often progressed to a stage where preventive
and possibly reverting measures are no longer efficacious, leaving symptom
management as the only option [19]. Currently, there is a substantial unmet need
for disease-modifying OA drugs (DMOADs) that actively slow disease progression
as no molecule of the sort has been approved or introduced in the market.
Throughout the management of OA, different non-pharmacological treatments are
adopted, like physical therapy, weight management and the use of different dietary
supplements. Pharmacological treatment regimens depend on disease stage (I to
IV, minimal to severe). Analgesics like paracetamol and nonsteroidal anti-
inflammatory drugs (NSAIDs) such as diclofenac are first-line treatments. However,
the drawbacks and adverse effects associated with the use of these systemic drugs
are limiting [20–22]. In further stages of the disease, local administration (intra-
articular) is an alternative that circumvents these issues. This local administration of
hyaluronic acid derivatives, known as viscosupplementation, combines pain relief
and improvement of joint motion from the greater cushioning effect provided by the
hydrogels. Other biological compounds, like injections of autologous platelet-rich
plasma have also been explored as local treatment of OA [23,24]. When symptom
management is no longer viable in later stages, full joint replacement surgery of hip,
knee or heel is an option [5,6].
Figure 1. Schematic representation of the structural composition of healthy and osteoarthritic knee.
OA in vitro models
15
3. INTRA-ARTICULAR DRUG DELIVERY SYSTEMS AND
INTERACTIONS WITH OA JOINTS
In a clinical setting, despite progress in OA research and development of disease-
modifying drugs, joint anatomy and physiology still pose a challenge for effective
drug delivery. Systemic drug delivery is challenging due to the poor irrigation and
limited permeability of the synovial membrane and articular capsule of affected
joints. Local, intra-articular administration of small molecules and larger protein
products directly as solutions into joints is hindered by low retention times due to
fast clearance [25]. Therefore, IA administration is not used to deliver common
analgesic and anti-inflammatory drugs to the joint. As approved and in use, IA is
only applied to deliver glucocorticoids (GC) and hyaluronic acid (HA) for
viscosupplementation [26–28]. The lack of broader use of IA administration as a
drug delivery route for OA treatment might be due to some drawbacks such as
formulation issues and the invasiveness of the procedure, which limit the number of
yearly injections. Thus, attaining drug loadings high enough to release sufficient
therapeutic drug doses over extended periods represents a critical challenge [29].
Drug delivery systems with extended-release properties help circumvent these
issues and others, like potential low aqueous solubility of many molecules.
Comprehensive reviews on formulation aspects of IA DDSs have been published in
recent years [26,28,30,31]. Table 1 shows the drug delivery systems investigated in
the past five years to treat OA by IA administration. Different types of formulations—
micro- and nanoparticles, hydrogels, liposomes—allow for controlled and extended
release of drug and increased retention times in joints while avoiding systemic side
effects [10,32,33]. Various classes of molecules have been investigated as
DMOADs or for symptom management: analgesic/anti-inflammatory,
chondroprotective/regenerative and bone resorption inhibitors [28]. Each category
is linked to different target tissues in the joint. For example, anti-inflammatory drugs
like celecoxib target the synovium, and chondroprotective drugs like kartogenin
target articular cartilage tissue [34,35]. It is essential to consider tissue specificity
when formulating IA drug delivery systems as off-target effects may occur and
negatively impact OA progression. Understanding the structure of the different
tissues of joints is thus key for the design of DDSs. Human joints are complex
structures that connect bones, allowing the body’s movement. The main structures
Chapter I
16
of synovial joints (diarthrosis, joints with movements) (Figure 1) are joint capsule,
synovial membrane, joint cavity with synovial fluid, cartilage, ligaments, muscles,
bursae, tendons, subchondral bone, nerves and vessels [36,37]. Synovial joints are
the most affected by OA, with two of the main features that make them unique being
key explored targets of therapeutic treatments: the synovial membrane and the
articular cartilage. Synovium or synovial membrane is the connective tissue that
lines the joint cavity. This heterogeneous tissue mainly comprises two types of
synoviocytes: type A macrophage-like synoviocytes, lesser in number and
increased in inflammatory conditions, have an important role in phagocytosis and
production of pro-inflammatory cytokines; and type B fibroblast-like synoviocytes,
the structural cells of the synovium (75% of cellular total), producing synovial fluid
and ECM components. Collagen fibers, fenestrated blood capillaries and lymph
vessels are other structures found in the inner layers of the membrane. The synovial
fluid, produced by ultrafiltration of plasma, nourishes the non-irrigated articular
cartilage, lubricates and absorbs shock [36,38]. Drug delivery systems with drugs
targeting the synovium, like TSG-6 (TNFα gene precursor) or VX-745 (p38 MAPK
inhibitor) are active on type B synoviocytes and macrophages, mostly through
inflammatory and pain pathways [39,40]. On the other hand, articular cartilage
(hyaline cartilage) is a connective tissue layer that lines the ends of the bones of the
joint, serving as a barrier to friction and shock between them. Contrary to the
synovium, this is an avascular, alymphatic and aneural tissue. It is composed of
chondrocytes (differentiated mature cells) and ECM, mainly collagen and elastin
fibers, aggrecan and proteoglycans. Its form and elasticity are determined by the
organization of the collagen fibers, proteoglycans and diffusion of water molecules
during movement. The lubricants and hyaluronic acid secreted both by the synovial
fluid and chondrocytes are shock-absorbing and provide a cushioning effect. This
cross-talk between tissues and synovial fluid is driven by mechanical load caused
by body movement. Cartilage is part of the osteochondral unit as it covers the sub-
chondral bone plate [41,42]. Examples of drugs targeting cartilage and bone
delivered by IA administration of delivery systems include kartogenin (a
chondrogenesis inductor from the RUNX-1 pathway) and doxycycline (an antibiotic
with MMP inhibitor functions) [43,44].
The successful development of an IA drug delivery system formulation greatly
depends on its interaction with the target tissue in the joint. Accurate choice and
OA in vitro models
17
design of in vitro models of OA are crucial in understanding target interaction,
predicting in vivo outcomes, and developing effective IA formulations.
Chapter I
18
Table 1. Intra-articular small molecule drug delivery systems for OA treatment, developed in the past 5 years. (Acronyms defined at the bottom of table)
Formulation Drug Carrier Type of study Main target tissue In vitro model In vivo model Authors;
Year; References
Microparticles
Doxycycline PCL Pre-clinical studies Cartilage3D rabbit
chondrocyte agarose model
Rabbits Aydin et al. 2015 [44]
Celecoxib PEA Pre-clinical studies Synovium Differentiated HI-
60 cells and lysates
Human synovium and synovial fluid (ex vivo);
rat ACLT model
Janssen et al. 2016 [32]
Etoricoxib PCL Pre-clinical studies Synovium, cartilage Not reported Rats Arunkumar et al.
2016 [45]
Lornoxicam Chitosan/TPP Pre-clinical studies Synovium, cartilage Not reported Rat MIA model Abd-Allah et al.
2016 [46]
Fluvastatin PLGA Pre-clinical studies CartilageHuman primary chondrocytes Rabbit ACLT model
Goto et al. 2017 [47]
Rhein (cassic acid) PLGA Pre-clinical studies Synovium THP-1
macrophages Not reported
Gomez-Gaete et al. 2017 [8]
Kartogenin PLA Pre-clinical studies CartilageHuman
synoviocytes Mice DMM model Maudens et al.
2018 [43] PH-797804,
Dexamethasone PLA Pre-clinical studies Synovium
Human synoviocytes
Mice AIA model Maudens et al.
2018 [48]
Triamcinolone acetonide (Zilretta™)
PLGA Phase II/III clinical trials in
OA patients 1 Synovium, cartilage Not reported Rat knee model 2
Kumar et al. 2015 2;
Kraus et al. 2018 1[49,50]
TSG-6 (tumor necrosis factor-alpha stimulated
gene-6) Heparin Pre-clinical studies Cartilage Not reported Rat MMT model
Tellier et al. 2018 [39]
Fluticasone propionate PVA Pre-clinical studies Synovium Not reported Beagle dogs Getgood et al.
2019 [51]
Celecoxib PLA Pre-clinical studies Synovium Human synoviocytes
Not reported Salgado et al. 2020 [52]
Rapamycin PLGA Pre-clinical studies Cartilage Human immortal chondrocytes
Mice Dhanabalan et al. 2020 [53]
OA in vitro models
19
Nanoparticles
VX-745 (p38 MAPK inhibitor)
PLA and PLGA
Pre-clinical studies Synovium Human
synoviocytes Mice AIA model
Pradal et al. 2015 [40]
Dexamethasone Avidin/PEG Pre-clinical studies Synovium, cartilage Bovine knee
cartilage explants
Not reported Bajpayee et al.
2016 [54]
KAFAK (anti-inflammatory mitogen-
activated protein kinase-activated protein kinase 2
(MK2)-inhibiting cell-penetrating peptide)
pNiPAM-PEG Pre-clinical studies Synovium, cartilage Bovine knee
cartilage explants
Not reported Lin et al. 2016 [9]
Kartogenin; Diclofenac Chitosan/Plur
onic F127 Pre-clinical studies Synovium, cartilage
Human BMSCs (bone marrow mesenchymal
stem cells); Human primary chondrocytes
Rats Kang et al. 2016 [35]
Curcumin PLGA Pre-clinical studies Synovium, cartilage Not reported Rats Niazvand et al.
2017 [55]
Dexamethasone Avidin Pre-clinical studies Synovium Not reported Rabbit ACLT modelBajpayee et al.
2017 [56]
KAFAK pNiPAM-PEG Pre-clinical studies Synovium, cartilage
RAW 264.7 macrophages; Bovine knee
cartilage explants
Not reported McMasters et al.
2017 [57]
CAP (chondrocyte affinity peptide)
PEG-PAMAM Pre-clinical studies Cartilage Human primary chondrocytes
Rats Hu et al. 2018 [58]
Kartogenin Polyurethane Pre-clinical studies CartilageRat primary
chondrocytes Rat ACLT model
Fan et al. 2018 [59]
Adenosine PEG-b-PLA Pre-clinical studies Synovium, cartilage RAW 264.7
macrophages Rat ACLT model
Liu et al. 2019 [60]
Etoricoxib PLGA-PEG-
PLGA Pre-clinical studies Synovium, cartilage Human primary chondrocytes Rat ACLT model
Liu et al. 2019 [33]
Hyaluronic acid PLGA Pre-clinical studies CartilageRAW 264.7
macrophages Brine shrimp; Rats
Mota et al. 2019 [61]
Chapter I
20
Hyaluronic acid and near-infrared dye
PLGA Pre-clinical studies CartilageHuman primary chondrocytes
Mice DMM model Zerrillo et al.
2019 [62]
Celastrol Mesoporous silica
Pre-clinical studies Cartilage Rat primary chondrocytes
Rat MIA model Jin et al. 2020 [63]
Diacerein PLGA Pre-clinical studies Synovium, cartilage Rat synoviocytes Rat MIA model Jung et al. 2020 [64]
Etoricoxib PLA/Chitosan Pre-clinical studies Synovium
MC3T3-E1 cells (mouse
osteoblast precursor)
Not reported Salama et al.
2020 [65]
MK2i (anti-inflammatory MK2-inhibiting peptide)
Linked and non-linked
NIPAm Pre-clinical studies Synovium, cartilage
Bovine primary chondrocytes
Rats Deloney et al.
2020 [66]
Oxaceprol PLGA Pre-clinical studies Synovium
Human primary LCLs
(lymphoblastoid cell lines)
Not reported Alarçin et al.
2020 [67]
Triamcinolone acetonide Dextran sulfate
conjugated Pre-clinical studies Synovium
RAW 264.7 macrophages;
L929 cells (mouse
fibroblast)
Mice MIA model She et al. 2020 [68]
Hydrogels
Amphotericin B Hyaluronic
acid/glyceryl monooleate
Pre-clinical studies Synovium, cartilage Not reported Rabbits Shan-Bin et al.
2015 [69]
Celecoxib PCLA-PEG-
PCLA Pre-clinical studies Synovium Not reported Horse
Petit et al. 2015 [34]
Methotrexate/dexamethasone/near-infrared dye
Hyaluronic acid + PLGA microcapsule
s
Pre-clinical studies Synovium RAW 264.7
macrophages Rat RA model
Son et al. 2015 [70]
Sinomenine hydrochloride
Phytantriol Formulation studies Not reported Not reported Not reported Chen et al. 2015 [71]
Dexamethasone Hyaluronic acid
Pre-clinical studies Synovium, cartilage Human primary chondrocytes
Rat ACLT model Zhang et al. 2016 [72]
OA in vitro models
21
PEGylated Kartogenin Hyaluronic
acid Pre-clinical studies Cartilage
Human BMSCs; human primary chondrocytes
Rat ACLT model Kang et al. 2017 [73]
Celecoxib PCLA-PEG-
PCLA Pre-clinical studies Synovium Not reported Equine synovitis model
Cokeleare et al. 2018 [74]
Dexamethasone Hyaluronic acid/pNiPAM
Pre-clinical studies Synovium Human synoviocytes
Mice DMM model Maudens et al. 2018 [10]
Triamcinolone hexacetonide (Cingal®)
Hyaluronic acid
Phase II/III clinical trials in OA patients
Synovium, cartilage Not reported Not reported Hangody et al.
2018 [75]
Simvastatin Gelatin Pre-clinical studies CartilageMouse primary chondrocytes
Mice Tanaka et al.
2019 [76]
Dexamethasone Agarose gel +
PLGA microspheres
Pre-clinical studies Synovium, cartilage
3D canine articular
chondrocyte construct
Canine osteochondral autograft model
Stefani et al. 2020 [77]
Diclofenac
Hyalomer (HA and
poloxamer 407)
Pre-clinical studies Synovium, cartilage Not reported Rat MIA model Hanafy et al.
2020 [78]
Diclofenac
Linked PAPE (2-
Pyridylamino substituted 1-phenylethanol
)
Formulation studies Not reported Not reported Not reported Kawanami et al.
2020 [79]
Eicosapentanoic acid Gelatin Pre-clinical studies Synovium Human primary chondrocytes
Mouse DMM model Tsubosaka et al.
2020 [80]
Hyaluronic acid/diclofenac sodium
Silica colloidal crystal beads-
pNiPAM Pre-clinical studies Synovium, cartilage
Human primary chondrocytes
Rat DMM model Yang et al. 2020 [81]
Liposomes Quercetin
mPEG-PA (Methoxy-
poly(ethylene glycol)-l-
poly(alanine))
Pre-clinical studies Synovium, cartilage Human primary chondrocytes
Rat ACLT model Mok et al. 2020 [82]
Chapter I
22
Fish oil protein encapsulated in gold
nanoparticles DPPC Pre-clinical studies Synovium Not reported Rats
Sarkar et al. 2019 [83]
Glucosamine sulphate Distearoyl
phosphocholine
Pre-clinical studies CartilageMouse primary chondrocytes
Not reported Ji et al.
2019 [84]
Rapamycin
DSPC combined with low-intensity pulsed
ultrasound
Pre-clinical studies Cartilage Human primary chondrocytes
Guinea pigs Chen et al. 2020 [85]
Acronyms: PCL: polycaprolactone; PEA: polyetheramine; PLGA: poly(lactic-co-glycolic acid); PLA: polylactic acid; PVAL: poly(vinyl alcohol); PCLA: poly(ε-caprolactone-co-lactide); PEG: polyethylene glycol; pNiPAM: poly(N-isopropylacrylamide); PAMAM: poly(amidoamine); DPPC: dipalmitoylphosphatidylcholine; DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine; MIA: monoiodoacetate; AIA: antigen-induced arthritis; ACLT: anterior cruciate ligament transection; DMM: destabilization of medial meniscus.
OA in vitro models
23
4. IN VITRO MODELS OF OA
Grasping the complexity of OA pathophysiological mechanisms remains a challenge
in OA research and negatively reflects on the successful development of DMOADs.
Many OA in vitro and in vivo models have been developed and refined over the
years. However, there is still no confirmed gold standard in vitro model to apply when
developing OA drug molecules and/or drug delivery systems [12]. The
establishment of accurate in vitro models is crucial as these influence choice of in
vivo OA models. Although there are relevant in vivo OA animal models, major gaps
in translation from animal to human OA conditions still prevail. Smart design and
choice of in vitro models could potentially help bridge these gaps by enhancing
predictability of OA models. Current OA in vivo models have additional limitations.
These models often actively portray either post-traumatic and/or late-stage (III/IV)
OA, leaving a large gap in understanding the spontaneous occurring disease and
its early stages, where slowing disease progression would be an attractive treatment
strategy. Sustainability and 3R initiatives (refinement, reduction and replacement)
have to be considered to assess the usefulness of these in vivo animal models,
where in vitro OA models can become the best alternative [86]. In this case, result
translation and predictability from in vitro to in vivo models still lack refinement and
accuracy. The processes of naturally occurring OA in certain animal species have
been proven similar to those of humans. Therefore, tissue collection from affected
animals is essential in the development of in vitro and ex vivo early-stage OA
models. Tissue collection in humans (articular cartilage or synovium) is complex due
to several ethical and regulatory issues, and retrieval at early stages of the pathology
is nearly impossible. Recovery of samples is restricted to patients undergoing total
joint replacement surgeries where OA is far evolved [87,88]. In this context, various
in vitro OA cellular models have been designed and explored: monolayer (2D), 3D
with or without scaffolds and tissue explants. Each model is adapted to a unique
target tissue of the joint and yields quantification of different markers (e.g.,
inflammatory cytokines, collagen type II, aggrecan or MMPs). In addition, each
model has its intricacies with relevant advantages and disadvantages, discussed in
Table 2. In the field of IA DDSs, these are important, especially in characterizing
release mechanisms and cytotoxicity of carrier systems. In Table 2, the different
applications of each model in IA DDS pre-clinical development are further described.
Chapter I
24
Table 2. Overview of advantages and disadvantages of in vitro OA models and their application in IA DDS development
In Vitro OA Model Advantages Disadvantages
Models Applied in IA DDS
Development (as per Table 1)
Outcome Evaluation (as per Table 1)
2D cellular culture
Monolayer
High throughput, low cost. Homogenous cell exposition to nutrients. Allows for differences in cellular phenotype studies [12]
Furthest from natural in vivo tissue conditions. High variability (different passages). Better suited for synoviocytes than chondrocytes. 2D substrate induces de-differentiation and changes in morphology [12]
- Synoviocytes (human, mouse and rat)
- Chondrocytes (human, murine, rat and bovine)
- Macrophages (human and murine)
- BMSCs (human)
RAW 264.7 macrophages [33,57,60,68,70]: - Cytotoxicity assays - Quantification of NO. cAMP,
IL-6, IL-1β and TNF-α Synoviocytes [10,40,43,48,52,64]: - Cytotoxicity and proliferation - Quantification of IL-6, PGE2,
IL-1β, TNF-α, MMP-3, MMP-13, COX-2 and ADAMTS-5
Chondrocytes [47,53,59,62,63,66,72,73,81,84]: - Cytotoxicity, apoptosis and
proliferation assays - Quantification of IL-6, IL-1β,
TNF-α, GAG/DNA, Aggrecan, Collagen II, MMP-1, MMP-3, TAC-1, MMP-13, COX-2, PGE2, iNOS and ADAMTS-5
- Senescence assays after genotoxic and oxidative stress [53]
- Expression of inflammatory transcription factors: p-IKKα/β [80]
Co-culture
Important in studies of cell-to-cell interactions and studies of influence of different cellular phenotypes together [12]
Expensive and difficult to maintain. Lacks in three-dimensional characteristics of cartilage growth [87]
(examples not included in Table 1) - Synoviocytes-
chondrocytes - Chondrocytes-
osteoblasts [89,90]
3D cellular culture
Without Scaffold
High similarity with in vivo tissue conditions as it maintains structure from ECM growth. Cellular phenotype is preserved. Important in studies of intercellular and cell to ECM relationship and loading capacity assays [88,91]
Expensive and difficult to maintain. Restricted throughput (hard to propagate without compromising cell quality). Nature of scaffold plays role in cellular growth [92]
- Chondrocyte pellets
- Hanging drop BMSCs
- Quantification of: GAG/DNA, Collagen II, Aggrecan [35]
With Scaffold
- Hydrogels: biomaterial and synthetic
- Polymeric scaffolds (osteochondral plugs)
- Micro- and nanocarriers
- Fiber/Mesh scaffolds [88]
- GAG/DNA, MMP-13 and hydroxyproline quantification; proliferation in agarose assay by DNA quantification [44]
- GAG/DNA, Collagen II and Young’s/dynamic modulus (Eγ and G) [77]
- Proliferation in alginate beads - Quantification of IL-6, MMP-
13, Collagen II and Aggrecan [85]
OA in vitro models
25
Explants
Easy to obtain and inexpensive. Allows for studies of intercellular and cell to ECM relationship because it maintains tissue as a whole [93]
High variability and limited amounts of replicates from source. Cell death at edge of extracted tissues [12]
- Articular cartilage and synovial membrane (human and bovine)
- Osteochondral plugs (human)
- Femoral chondyles (human, murine and equine)
- Cytotoxicity and cartilage penetration assays
- Quantification of IL-6 [9,57]
4.1. 2D cellular models
Two-dimensional cellular models can be described as monolayer culture (4.1.1.),
when a single cell line is cultured or co-culture (4.1.2.) when two or more cell lines
are cultured together, in a monolayer.
4.1.1. Monolayer culture
Culturing cells in monolayer is a well-established, cost-effective method to obtain
relatively fast, reliable, and high-throughput results. As OA in vitro models, these
can be immortal lines or harvested primary cells cultured adherent to plastic flat
surfaces. Their source can vary from murine, bovine to human. Table 2 lists the use
of the most common cell lines: RAW 264.7 macrophages, human primary
synoviocytes and human articular chondrocytes. To evaluate IA delivery systems,
2D models with cells like synoviocytes or chondrocytes that respond to cytokine
stimulation (typically IL-1β, mimicking the inflammatory catabolic environment of
OA) are thus ideal for screening of either anti-inflammatory or chondroprotective
molecules from DDSs by quantification of several inflammatory and cartilage
degradation markers: IL-6, TNF-α, PGE2, COX-2, NO, iNOS, MMP-1, MMP-3,
MMP-13, and ADAMTS-5. These monolayer models (especially human cell lines)
are also useful and largely explored for cytotoxicity and proliferation testing in local
administration cases since they correspond to the direct cellular target
[40,53,62,64,84]. Additionally, different signaling pathways can be explored from
these models, such as the inflammatory NF-κB pathway in human chondrocytes
[94]. Human bone marrow mesenchymal stem cells (HBMSCs) have the ability to
de-differentiate to mature articular chondrocytes; thus, these are also used to
quantify chondrogenesis through sulfated glycosaminoglycans (GAG), abundant in
Chapter I
26
articular cartilage ECM content, gene expression of collagen II and aggrecan, in
addition to the cytotoxicity and screening of molecules [35,73]. However, problems
arise from culturing primary articular chondrocytes, as the actual cartilage tissue
would require a three-dimensional cell growth, interacting with the ECM, in contrast
to a flat surface (Table 2). Therefore, after a small number of passages, which limits
the number of experiments and length of studies, de-differentiation tends to occur
as cells change in phenotype and morphology from an orthogonal shape to an
elongated shape, resembling fibroblast-like chondrocytes. This phenotype is known
to produce collagen type I fibers instead of the collagen II fibers consistent with
articular cartilage, an issue when using this type of monolayer model to assess
cartilage growth from collagen II and aggrecan quantification. This lack of tissue-
mimicking properties prevents 2D in vitro models from accurately mimicking
intercellular and cell-to-ECM relationships. Not only this, but weight-bearing and
mechanical-loading experiments, crucial in the understanding of OA as a pathology,
are not easily explored using these models [12].
4.1.2. Co-culture
Monolayer culture of different joint cell lines is an alternative to improve intercellular
relationship studies. Differently from monolayers of a single cell line, in co-culture
where chondrocytes are incubated together with synoviocytes and stimulated by
pro-inflammatory cytokines, cross-talk between cells happens through intercellular
calcium and paracrine signaling, maintaining homeostasis of articular chondrocytes.
Evaluation of effects of anti-inflammatory or chondroprotective molecules in articular
cartilage is then higher in accuracy by the co-culturing of both cell types due to the
preservation of these intercellular signaling pathways [89]. Chondrocytes incubated
with osteoblasts help maintain cellular physiology and phenotype through paracrine
signaling. This is an useful model in investigating the effects of chondroprotection
(slowing of cartilage degradation) in bone remodeling [95]. Mesenchymal stem cells
are interesting in co-culture as pluripotency leads to specific de-differentiation,
allowing for different cellular pathways to be analyzed together with articular
chondrocytes from cellular secreted markers [96]. However, despite advantages
gained by culturing different types of cells together in terms of tissue-like
maintenance of homeostasis and phenotypes, this in vitro model is subject to some
of the same drawbacks as monolayer culture, notably, culturing in a flat surface and
OA in vitro models
27
lack of growth structure. In addition, maintaining different cellular environments at
the same time is expensive.
4.2. 3D cellular models
Three dimensional cellular models can be classified into models without scaffold
(4.2.1.), where cells are grown in pellets and models with scaffold (4.2.2.) where
cellular growth happens in an external platform (biologic or synthetic polymer).
4.2.1. 3D cellular models without scaffold
Three-dimensional cellular pellets circumvent some of the disadvantages of
monolayer cultures, especially as they allow a structure, maintaining cellular growth
in all dimensions and synthesis of articular cartilage ECM. In this approach,
chondrocytes can be centrifuged together in conical bottom wells or tubes or
cultured under stirring using bioreactors. Inducing cell clustering forms cartilage
tissue-like pellets, after a specific incubation time, with sizes up to 5 mm [97,98].
These pellets can mimic articular tissue as a whole, providing insights into cell-to-
cell and cell-to-ECM relationships. Like in a monolayer culture, HBMSCs pellets can
replace 3D chondrocyte pellets. As an in vitro model for IA DDSs development, 3D
pellets have been applied in the evaluation of chondrogenesis and
chondroprotective effects after IL-1β stimulation by GAG content quantification and
gene expression of collagen II, aggrecan, and MMPs [35]. A primary reason as to
why pellets are not a standard in vitro OA model is linked to difficulties in maintaining
3D cellular cultures in terms of cost and quantity. 3D articular dedifferentiated
chondrocytes are not associated with high proliferation rates and derive from low
monolayer passages restricting cellular amounts. Culture media is supplemented
with a high amount of growth factors and chondrogenic stabilizers, representing
higher costs compared to monolayer culture [99]. Additionally, pellets have short
viability spans, where nutrients have difficulties in penetrating the pellet, inducing
cell death at its core. As a model for IA DDSs, interaction of formulations with the
tissue as a whole is essential in characterizing target specificity. The inability to fully
penetrate the pellets poses a limitation to the use of this model in the IA setting [92].
Bypassing these shortcomings is, however, made possible by establishing this type
of 3D cellular growth in external structures - scaffolds.
Chapter I
28
4.2.2. 3D cellular models with scaffold
Cells can be cultured directly into external scaffolds, gaining three-dimensional
features. As an in vitro model for IA DDSs development, this alternative has great
potential for targeted delivery. Not only does it provide structural support for 3D
cellular growth by mimicking features of joint structure, making it a good model of
loading and weight-bearing in OA, as the nature of the scaffold (biologic or synthetic)
can play a role in cellular growth and maintenance. The most commonly used
scaffolds are hydrogels due to their high water content and the extensive ability to
tailor their mechanical and physicochemical properties. Biopolymers like agarose,
chitosan, alginate and hyaluronic acid have been applied to grow chondrocytes,
mimicking articular cartilage, and osteoblasts, aiming to model the osteochondral
plate. Combining the growth of both these types of cells has also been explored,
forming bilayer scaffolds, in an attempt to represent the whole articular joint [100].
As such, and after cytokine stimulation and exposure to therapeutic molecules,
different cartilage markers can be assessed by different assays: GAG content
(alcian blue assay), collagen II, aggrecan, MMPs (gene expression analysis) and
even pro-inflammatory cytokines (enzyme-linked immunosorbent assay ELISA)
[44,85]. Rheological measurements (elastic Young’s and G moduli) help investigate
the mechanical properties of chondrocytes in hydrogels (agarose) [77]. Synthetic
hydrogels and polymers can be applied as scaffolds, with advantages like
mechanical features and support. 3D printing has been applied in this field with
promising results in cartilage regeneration [101,102]. Compared to 2D models,
scaffold-based 3D culture is expensive, difficult to maintain and hard to standardize,
given the many options for scaffolds. Depending on their nature, problems may arise
with how these influence results. For example, biopolymer-based hydrogels may
themselves have a chondroprotective effect on cultured chondrocytes, skewing
effects of tested drugs. The nature of the scaffold may also translate into differences
between in vitro and in vivo models. For instance, hydrogels are rich in water, unlike
subchondral bones of joints; thus, the growth of osteoblasts in such scaffolds is not
an accurate representation of in vivo conditions [88].
4.3. Explants
Explants could be considered the most accurate in vitro model of OA as the whole
tissue is maintained in its form and function. Just like tissue where cells are
OA in vitro models
29
harvested from, their source can be both animal and human. Explants of both
cartilage and synovial membrane are useful to investigate anti-
inflammatory/chondroprotective effects of DDSs or molecules. Bovine cartilage is
also commonly harvested to test the permeation and distribution of a drug or DDSs
into the cartilage and/or subchondral bone, using fluorescent-dye-labeled-
nanoparticles drug molecules. Femoral heads are attractive in loading and weight-
bearing studies, whereas osteochondral plugs are used to investigate the balance
between cartilage and bone regeneration when exposed to chondroprotective
drugs. By measuring DNA and cellular turnover, cell viability and proliferation can
also be assessed using explants after exposure to DDSs and/or free drug molecules
[9,57]. Despite clear advantages (Table 2) from using explants where intercellular
and cell-to-ECM relationships are preserved; extraction induces cell death on the
outer layers of the tissue, compromising the model. Accurate induction of OA may
pose another limitation, as often harvested tissues are healthy specimens and not
pathological as the ones collected in other cellular models (monolayer, for example).
Additionally, the maintenance of tissues in culture can be expensive and difficult to
control, with explants lasting up to 10 days. Conditions such as temperature, pH,
humidity, culture medium and supplements like insulin plus light exposure are crucial
in maintaining the viability of explants. Another substantial limitation of this model is
that viable replicates are very difficult to achieve, as tissue sources are finite and
not abundant [87].
4.4. Considerations on OA in vitro models for development of IA DDSs
Understanding the advantages and disadvantages of the different types of OA in
vitro models is crucial when developing intra-articular drug delivery systems. The
choice of in vitro model is influenced by how effects of the delivered drug can be
assessed, be it anti-inflammatory by quantification of released cytokines or
chondroprotection by evaluating GAG content and collagen II mRNA expression.
Different cell lines such as macrophages, synoviocytes or chondrocytes secrete
different factors and/or respond differently to cytokine stimulation. When evaluating
the anti-inflammatory effects of therapeutic molecules in DDSs, macrophages and
synoviocytes represent the most accurate cellular model. For chondroprotective
effects and/or subchondral bone protection, it is important to test these in accurate
representations of articular cartilage and subchondral bone. For this, 3D
Chapter I
30
chondrocyte models or bilayer scaffolds for osteochondral defects are adequate
models. Furthermore, articular cartilage or subchondral bone cells/tissue do not
participate in inflammatory cascades directly, making these cellular models specific
for measuring cartilage degradation markers. When developing, for example, an IA
DDSs eluting a drug that has the synovium as a specific target, it is important to
measure not only off-target activity in the other joint tissues but also the response of
cartilage, for example, to the effects of the drug in the synovium. Monolayer models,
though abundantly investigated and easy to establish, fail in the evaluation of cross-
talk and molecular relationships within the different joint structures, especially
important when developing a local delivery system. However, 2D models are
relatively easy and accurate in assessing cytotoxicity and influence in cell
proliferation of both drug and carriers. In contrast, 3D models allow for a more
accurate and translational representation of joint tissues as phenotype and cellular
growth are preserved. 3D models are essential in evaluating cytokine stimulation,
cell-to-cell and cell-to-ECM relationships and, in the case of scaffold-based 3D
models, loading studies, as these have mechanical properties not found in
monolayer cultures. Nonetheless, their establishment requires highly specific
expertise and can be costly. In addition, the accuracy and reproducibility of the
outcomes, from cytotoxicity assays to gene expression analysis, can be high when
applying 3D models. Explants from specific tissues are good representations of in
vivo joints, as their intact features allow for loading and penetration studies of both
IA carriers and free drug molecules. However, representative experimental
replicates are not easily accessible, and molecular alterations may arise from the
extraction of the tissues. As mentioned previously, time and duration play an
important role in the development and application of in vitro models. OA is a slowly
progressing, chronic disease where molecular changes often only result in actual
physical symptoms very late. As such, tackling the effect over time on tissues is
crucial to understand disease mechanisms and potential therapeutic options.
However, experimentally, it is challenging to maintain cells and tissues viable for
long periods of time. Bioreactors or tissue-mimicking polymers could help
circumvent viability issues, maintaining OA conditions for slightly extended periods
[103].
Formulation aspects also influence the choice of in vitro OA model. The formulation
of DDSs (Table 1) implies the use of a carrier for a certain drug molecule. Carriers
OA in vitro models
31
have an impact in terms of size and nature. In terms of size, the local administration
of nano-range carriers (nanoparticles) can induce phagocytosis and inflammatory
cascade from synoviocytes in the joint capsule [104]. Therefore, interactions at the
cellular level when testing these DDSs are important to consider if
macrophages/synoviocytes are the chosen in vitro cellular models. As previously
discussed, most cellular OA models are cultured on plastic surfaces, in well plates,
dishes or tubes. Micro-range carriers (microparticles or larger liposomes) are prone
to sedimentation in these cell culture set-tings, especially polymeric carriers, which
display high density when in a culture medium suspension. This sedimentation may
negatively affect experimental result, by uneven drug molecule distribution,
heterogeneous presentation to test cells and lower contact surfaces between the
carrier–drug complex and cells [105]. This issue can be bypassed by performing
experiments in orbital shakers. However, as described for 3D cellular models,
altering centrifuge force and balance induces changes in cellular growth and
phenotype [100]. When evaluating hydrogels (Table 1), either in monolayer or 3D
cellular models, even when using explants, it is important to consider the nature of
the polymer (synthetic or bio) and the viscosity of the gel. Like for nano-
/microcarriers, choice of polymer will have an impact on cellular response. Thus,
biocompatibility and innocuousness of polymers are important characteristics,
particularly when testing inflammation and anti-inflammatory effects, as further
induction of inflammatory cascades is undesired. The majority of hydrogels being
explored for OA treatment are HA-based, a natural component of articular cartilage
[10,69,70,72,73,75,78]. As such, it is important to assess their impact as stand-alone
carrier vs. carrier with drug, as it is expected that this type of gel will have an
influence on chondrocyte growth by inducing chondroprotection through CD44
receptor interaction. Lastly, rheological properties of hydrogels need to be
considered when applying in vitro cellular models. High viscosity may induce
occlusion effects in either cultured cells or tissues, generating hypoxia phenomena
and thus lowering viability scores [106]. Consideration of all different formulation
aspects does not exclude testing of drug-alone controls in these cellular OA models,
as these dictate why and how DDSs are better alternatives in IA administration.
Chapter I
32
5. CONCLUSIONS AND FUTURE PERSPECTIVES
Improved design and development of efficacious IA DDSs relies on the use of
accurate, predictive in vitro and in vivo models. However, to date, there is no OA
gold standard in vitro model and few guidelines or models adapted specifically to IA
DDSs formulations. Presently, monolayer models, despite being easy to establish
and ideal for rapid screening of molecules, fail in representing ac-curate OA
conditions, such as cross-talk between different tissues. This could be bypassed by
co-culture of two types of cell lines, like synoviocytes and chondrocytes, but aspects
like cell de-differentiation and ECM growth are not negligible. Three-dimensional
models are considered better representations of in vivo OA, as in these models,
three-dimensional structures of tissues and cellular phenotype and growth are
preserved. However, with or without scaffold, 3D models are difficult to establish
and maintain, and outcomes vary greatly according to the source and nature of
scaffold. For studies in articular tissues, explants are considered best in correlation
to in vivo OA conditions. However, viable replicates and maintenance of tissues in
in vitro environments are important limitations. Recently, a bioengineering approach
combining 3D cell culture and microfluidics—organ-on-chip (OoC)—has been in the
field of OA. Cartilage-on-chip and osteochondral-tissue-on-chip have been
developed to perfectly mimic joint microenvironments, allowing for better
reproductions of in vivo conditions. Promising results have been described testing
the drug alone, making this a promising approach for the better development of IA
DDSs in the future [107–109]. In this context, considerations (Table 2) have to be
taken into account when designing and developing IA DDSs, especially when
deciding outcome readouts. To this extent, the type of formulation and mode of
action of drug molecules (Table 1) play a critical role. Monolayer models are better
suited for testing anti-inflammatory activity, whereas 3D chondrocyte models are
preferred to evaluate chondroprotection activities. When testing hydrogels, it is
important to assess the nature of the scaffold in 3D models and even occlusion in
explants. In the future, research advancements should focus on improving the
design and development of OA in vitro models for better prediction of in vivo and,
eventually, clinical results. This should be done while always considering the
tailoring of in vitro models to specific IA DDSs formulations, like maintaining cellular
viability conditions for testing of sustained prolonged drug release delivery systems.
OA in vitro models
33
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Chapter II
In vitro anti-inflammatory activity in arthritic synoviocytes of
A. brachypoda root extracts and its unusual dimeric
flavonoids
Carlota Salgado a,b, Hugo Morin a,b, Nayara Coriolano de Aquino c, Laurence Neff a,b, Cláudia Quintino da Rocha d, Wagner Vilegas e, Laurence Marcourt a,b, Jean-Luc Wolfender a,b, Olivier Jordan a,b, Emerson Ferreira Queiroz a,b and Eric Allémann a,b a School of Pharmaceutical Sciences, University of Geneva, 1 Rue Michel-Servet 1211 Geneva, Switzerland b Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, 1 Rue Michel-Servet 1211 Geneva, Switzerland c Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60450-765 Fortaleza-CE, Brazil d Laboratório de Produtos Naturais, Centro de Ciência Exatas e Tecnologia, Departamento de Química, 65080-805 São Luís-MA, Brazil e Experimental Campus of the Paulista Coast, UNESP-São Paulo State University, 11330-900 São Vicente-SP, Brazil
Published: Molecules 2020, 25 (21), 5219; https://doi.org/10.3390/molecules25215219
ABSTRACT
Arrabidaea brachypoda is a plant commonly used for the treatment of kidney stones, arthritis and pain in traditional Brazilian medicine. Different in vitro and in vivo activities, ranging from antinociceptive to anti-Trypanosoma cruzi, have been reported for the dichloromethane root extract of Arrabidaea brachypoda (DCMAB) and isolated compounds. This work aimed to assess the in vitro anti-inflammatory activity in arthritic synoviocytes of the DCMAB, the hydroethanolic extract (HEAB) and three dimeric flavonoids isolated from the DCMAB. These compounds, brachydin A (1), B (2) and C (3), were isolated both by medium pressure liquid and high-speed counter current chromatography. Their quantification was performed by mass spectrometry on both DCMAB and HEAB. IL-1β activated human fibroblast-like synoviocytes were incubated with both extracts and isolated compounds to determine the levels of pro-inflammatory cytokine IL-6 by enzyme-linked immunosorbent assay (ELISA). DCMAB inhibited 30% of IL-6 release at 25 µg/mL, when compared with controls while HEAB was inactive. IC50 values determined for 2 and 3 were 3-fold higher than 1. The DCMAB activity seems to be linked to higher proportions of compounds 2 and 3 in this extract. These observations could thus explain the traditional use of A. brachypoda roots in the treatment of osteoarthritis. Keywords: Arrabidaea brachypoda; flavonoids; anti-inflammatory activity; osteoarthritis; high-speed counter current chromatography; mass-spectrometric quantification
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1. INTRODUCTION
Arrabidaea brachypoda (D.C.) is a shrub native to the Brazilian region of Cerrado
(neotropical savanna). It belongs to the Bignoniaceae family, which includes 120
genera and nearly 800 species of different plants scattered in tropical and
subtropical regions worldwide [1]. Plants of Arrabidaea genus are known sources of
C-glucosylxanthones, phenylpropanoids, flavonoids, anthocyanidins, allantoins,
and triterpenes [2–4]. These molecules are linked to the astringent, anti-
inflammatory, antimicrobial, antitumoral and wound healing properties that plants of
this genus are known for in traditional medicine [5]. In Brazil, Arrabidaea brachypoda
is known as “cervejinha do campo”, a decoction of its roots used to treat kidney
stones and arthritic joints [6,7]. Different molecules have been isolated and identified
from the hydroethanolic extract (HEAB) and dichloromethane extract (DCMAB).
From this last extract, three isolated aglycones - brachydin A (1), brachydin B (2),
and brachydin C (3) - have been identified. In the HEAB, among the 14 described
molecules, these three dimeric flavonoids exist in their glucoronated form [8,9].
Antinociceptive, anti-inflammatory, anti-Trypanosoma cruzi, gastroprotective,
antileishmanial, and antimicrobial activities have been recently reported in different
in vitro and in vivo assays from both root extracts and isolated compounds [8–15].
In these previous studies, different oral and/or topical administration setups were
explored. Overall, studies reporting on anti-inflammatory and antinociceptive
activities of A. brachypoda have focused on general mechanisms of pain and
inflammation [10,12]. In this study, we aimed to test the in vitro anti-inflammatory
activity of A. brachypoda root extracts and three isolated dimeric flavonoids in the
context of osteoarthritis (OA). This chronic disease has worldwide incidence, in
particular in the aging population and is considered the most common form of
arthritis. Characterized by chronic pain and inflammation, articular cartilage
degeneration, and structural changes of whole joints, OA represents the main cause
of physical disability and a great health economic burden [16–19]. Presently,
treatment options are primarily based on non-pharmacological and symptom
management approaches. There is a need for long-acting, targeted local anti-
inflammatory drug products, in order to tackle the main OA complications: pain and
inflammation [20,21]. Human fibroblast-like synoviocytes activated for OA-like
inflammation were used as the in vitro cellular model. Interleukin-6 (IL-6) is a
Anti-inflammatory activity of A. brachypoda extracts
45
ubiquitous pro-inflammatory cytokine of acute inflammation, particularly involved in
the synovial hypertrophy, as well as an established therapeutic target for arthritis.
Therefore, IL-6 was selected as the molecular marker of inflammation in this study
[21]. Additionally, the isolated compounds were quantified in both HEAB and
DCMAB to establish a link between the chemical composition of the extracts and
anti-inflammatory properties of their individual constituents.
2. MATERIALS AND METHODS
2.1. Materials
2.1.1. General experimental procedures
All analytical HPLC-PDA analyses were performed using an Agilent Technologies
1260 Infinity system equipped with a photodiode array detector (Agilent
Technologies, Santa Clara, CA, USA). Preparative medium pressure liquid
chromatography (MPLC) was performed using a system equipped with a C-605
module pump, C-640 UV detector, and C-684 fraction collector all from Büchi (Flawil,
Switzerland). The system was controlled by the Sepacore Control software (Büchi
AG, Flawil, Switzerland). A coil connected to a resistance was used to control the
MPLC column temperature. The column (460 mm × 49 mm i.d.) was packed with
ZEOprep® C18 as the stationary phase (ZEOprep® C18, 15–25 µm; Zeochem,
Uetikon am See, Switzerland). Nuclear magnetic resonance (NMR) spectroscopic
data were recorded on a Bruker Avance III HD 600 MHz NMR spectrometer
equipped with a QCI 5 mm Cryoprobe and a SampleJet automated sample changer
(Bruker BioSpin, Rheinstetten, Germany). Chemical shifts were reported in parts per
million (δ) using the CD3OD residual signal (δH 3; δC 49) as internal standards for
1H and 13C NMR and coupling constants (J) were reported in hertz. High-speed
counter-current chromatography (HSCCC) coupled to UV was performed on a Tauto
TBE-300B instrument (Tauto Biotech, Shangai, China) equipped with two LC10AD
HPLC pumps (Shimadzu, Kyoto, Japan), a 20 mL injection loop, a Knauer K 2501
UV detector (Berlin, Germany) and a C-684 fraction collector from Büchi (Flawil,
Switzerland). Solvents used for extraction (methanol, ethyl acetate, hexane) were
all of analytical grade. Solvents used in the quantification of the isolated brachydins:
methanol, formic acid, acetonitrile and water were of LC-MS grade. All solvents were
Chapter II
46
purchased from Sigma-Aldrich (St. Louis, MO, USA). Milli-Q® water from Merck
(Burlington, MA, USA) was used throughout this study. All other chemical products
were obtained from Sigma-Aldrich (St. Louis, MO, USA). For the in vitro anti-
inflammatory assays, all media solutions and ELISA kits were purchased from
Invitrogen (Carlsbad, CA, USA). Fetal bovine serum was purchased from Eurobio
(Les Ulis, France). Interleukin 1 beta (IL-1β) was obtained from R&D Systems (Bio-
Techne, Abingdon, UK).
2.1.2. Plant material
Roots of Arrabidaea brachypoda were collected in April 2010 from the Sant’Ana da
Serra farm in João Pinheiro, Minas Gerais, Brazil. The plant was identified at the
ICEB of José Badine Herbarium of the Federal University of Ouro Preto by Prof.
Maria Cristina Teixeira Braga Messias. A voucher specimen (# 17935) was
deposited at the Herbarium of the Federal University of Ouro Preto, Brazil. The plant
was collected in accordance with Brazilian authorities (SISGEN # A451DE4).
2.2. Extraction and isolation of brachydins A (1), B (2) and C (3)
2.2.1. Plant extraction
Both HEAB and DCMAB of the roots of Arrabidaea brachypoda were obtained from
our previous study [9]. These extracts were stored and protected from light at -20
°C.
2.2.2. HPLC-PDA analysis
The DCMAB of A. brachypoda (Figure S1, Supplementary Material), the MPLC and
HSCCC fractions were analyzed by HPLC-PDA using a Waters X-Bridge C18
column (250 mm × 4.6 mm i.d., 5 μm; Waters, Milford, MA, USA) equipped with a
Waters C18 pre-column cartridge holder (10 mm × 2.1 mm i.d.). Water (A) and
methanol (B), both containing 0.1% of formic acid (FA) were used as the solvent
system. The column was equilibrated with 5% of B for 15 min. The separation was
performed in gradient mode, as follows: 5 to 100% of B in 60 min, and 100% of B
for 10 min. Flow rate 1 mL/min; injection volume 10 μL; sample concentration 10
mg/mL in methanol. The UV absorbance was measured at 254 nm and the UV-PDA
spectra were recorded between 190 and 600 nm (step 2 nm).
Anti-inflammatory activity of A. brachypoda extracts
47
2.2.3. Isolation of brachydins A (1), B (2), and C (3)
DCMAB was fractioned by MPLC, following a previously published protocol [22].
Fractionation conditions were optimized on an HPLC column (see Sections 2.1.1
and 2.2.2) using the same stationary phase and an acidic (0.1% FA) water (A) and
methanol (B) gradient. Solvent system gradient went as follows: isocratic 5% B in
56 min, 5 to 38% B in 5.5 h, isocratic 38% B in 4.7 h, 38 to 62% B in 3.2 h, 62 to
100% B in 9 min and isocratic 100% B during 1.5 h. A dry load injection of the sample
was performed by mixing 7 g of the dichloromethane extract with 35 g of Zeoprep®
C18 (40–63 µm). The dry-load cell (11.5 cm × 2.7 cm i.d.) was subsequently
connected between the pumps and the MPLC column. The flow rate was set to 20
mL/min and UV absorbance was monitored at 280 nm. MPLC separation yielded 90
fractions of 250 mL, which were analyzed by ultrahigh pressure liquid
chromatography coupled to an UV detector (UHPLC-UV). This approach yielded
gram amounts of compound 1. Fractions containing pure compounds were
combined, dried, analyzed by nuclear magnetic resonance (NMR) and properly
stored. Other MPLC fractions containing mixtures of compounds 2 and 3 were
combined and subjected to purification by HSCCC coupled to a UV detector. The
ARIZONA solvent system approach was applied to perform the separation of
compounds 2 and 3. This approach is based on the use of 23 solvent mixture
compositions of methanol/ethyl acetate/hexane/water. The mixtures are labeled with
letters from the alphabet from A to Z (except E, I, and O) [23]. To determine the best-
suited ARIZONA solvent mixture, the partition coefficient (Kp) of each compound
was determined. As so, few milligrams of the fraction containing 2 and 3 were
solubilized in a specific mix of the ARIZONA system. Upper and lower phases of
each mixture of solvents were separated and analyzed by HPLC-UV. The UV area
peak of each compound was used to determine its coefficient of partition (Kp)
according to the equation in Figure S2 (Supplementary Material). The best results
for complete separation of compounds 2 and 3 were obtained by solvent mixture P-
methanol/ethyl acetate/hexane/water (6:5:6:5; v/v/v/v). The coil was first filled with
the two phases (upper and lower, 1:1) and rotation was set to 1000 rpm. Lower
phase was then pumped into the column at a flow rate of 3 mL/min using the head-
to-tail mode (mobile phase = lower phase; stationary phase = upper phase) with
rotation set at 8000 rpm. After equilibrium between the two phases, 500 mg of
sample in 20 mL of upper and lower phase solution (1:1) were injected. Four
Chapter II
48
injections were performed. A total of 35 fractions (5 ml each) were obtained for each
injection. The fractions were combined according to the chemical composition
determined by HPLC-PDA analysis, dried, analyzed by NMR, and properly stored.
2.3. Quantification of compounds 1–3 from A. brachypoda root extracts by
uhplc-ms/ms
Sample preparation was carried out by dissolving the dried HEAB and DCMAB from
Arrabidaea brachypoda roots in 100% methanol. The final concentration of the
samples was 10 µg/mL, and three technical replicates were used for quantitative
analysis. The amounts of compounds 1, 2, and 3 in the extracts were quantified on
a QTRAP 4000 quadrupole linear ion-trap mass spectrometer (Sciex, Darmstadt,
Germany) with an ESI interface operating in positive ionization mode. UHPLC was
performed on an Acquity UPLC system (Waters, Milford, MA, USA) equipped with a
Waters UPLC BEH C18 column (50 mm × 2.1 mm ID, 1.7 µm). The solvent system
consisted of water: 0.1% FA for solvent A and acetonitrile: 0.1% FA for solvent B.
The elution was performed in gradient mode at 40 °C (400 µL/min flow rate) with the
following steps: 5 to 60% B in 4.2 min, 60 to 70% B in 1.3 min, 70 to 100% B in 1.5
min, maintaining B at 100% for 1 min and lastly, a reconditioning with 5% B for min.
The autosampler compartment was set at 10 °C throughout the analysis and the
injection volume was 5 µL. MS/MS quantitative analyses were performed in multiple
reaction monitoring (MRM) mode. Source and gas parameters were as follows:
curtain gas, 10 psi; collision gas, 10 psi; ionspray voltage, 3500 V; source
temperature, 400 °C; ion source gas 1, 40 psi; ion source gas 2, 50 psi. Nitrogen
was used as collision gas. MRM transitions and MRM parameters were all optimized
via flow injection analysis (FIA) technique with a mix containing 10 ng/mL of pure 1,
2, and 3 analytes (Table S1, Supplementary Material). Quantification was achieved
by constructing three separate external calibration curves from solutions of the
isolated pure compounds. The calibration curve concentrations for active
compounds were as follows: 500 ng/mL, 250 ng/mL, 125 ng/mL, 63 ng/mL, 31
ng/mL. As no analyte-free matrix was available, the displayed limits of quantification
(LOQ) were evaluated only on each pure standard. A signal-to-noise ratio of 10 was
considered as the approximate LOQ (Table S2, Supplementary Material).
Anti-inflammatory activity of A. brachypoda extracts
49
2.4. In vitro anti-inflammatory bioactivity
2.4.1. Human fibroblast-like synoviocytes (HFLS) isolation and culture
Hip synovial membrane was collected from three male adult patients with clinical
osteoarthritis (OA) at the time of hip replacement surgery. This protocol was
conducted under the approval of the local Ethics Committee (CCER, Geneva,
Switzerland) (Authorization # 2017-02234) and with informed and consenting
patients. Once collected, tissue samples were processed, according to previously
described protocols [25]. Briefly, samples were finely minced and digested for 3 h
(37 °C, 5% CO2 incubation) in a 3 mg/mL collagenase IX-RPMI 1640 solution. After
centrifugation (200 × g) and supernatant removal, the resuspended pellet was
cultured (37 °C, 5% CO2) in medium containing RPMI 1640, M199 (1:1), 1%
penicillin/streptomycin (100 IU/mL:100 g/mL), 2 mM L-glutamine and 20% fetal
bovine serum. After overnight culture, non-adherent cells were removed. At
confluence, cells were trypsinized and passaged to 75 cm2 culture flasks (Corning,
NY, USA) in complete medium containing 10% fetal bovine serum. A double control
was performed to confirm presence of HFLS after isolation: visual morphology
evaluation (Figure S3) and flow cytometry with synoviocyte specific surface markers
(CD14+ and VCAM-1). A representative sample of all cell populations was found to
have above 90% HFLS markers. All cellular assays were performed from passage
3 to 9. Experiments were conducted twice per donor (n = 6).
2.4.2. In vitro cytotoxicity assay and bioactivity assay with the determination of il-6
release in HFLS
Confluent HFLS cells (25,000 cells/well) were treated in a 96-well plate with
compounds 1, 2, and 3 (3 µM, 6 µM, 12 µM, 25 µM, 50 µM, and 100 µM), HEAB and
DCMAB (increasing concentrations from 3 to 100 µg/mL), or control vehicle for 1 h.
All tested compounds were dissolved in a dimethyl sulfoxide (DMSO) stock solution
and further diluted in culture medium. Final concentration of DMSO in vehicle of
tested concentrations was 0.01%. After 1 h incubation, cells were activated by
addition of IL-1β (1 µg/mL) (R&D Systems, Bio-Techne, Abingdon, UK) with
subsequent incubation for 23 h. All supernatants were kept at −80 °C, before further
testing. Remaining adherent cells were tested for viability using the cell proliferation
reagent WST-1 (Roche, Basel, Switzerland), according to the supplier instructions.
Pro-inflammatory cytokine IL-6 release in cell supernatant was assessed by ELISA.
Chapter II
50
For this, a human IL-6 ELISA kit from Invitrogen (ref # 88-7066-88; Thermo-Fisher
Scientific, Waltham, MA, USA) was used according to the manufacturer’s protocol.
All samples were diluted 90 times. Experiments were conducted twice per donor (n
= 6).
2.5. Half maximal inhibitory concentration (IC50) determination and
statistical analysis
In this study, data are presented as mean values ± standard deviation (s.d.). All
analyses were performed using GraphPad Prism 8.3 software. IC50 values were
determined using a nonlinear fitting of a log (inhibitor concentration) vs response
variable slope for each donor’s data set. Consequent values were then averaged.
After confirming the data normal distribution and homogeneity of variances (Brown-
Forsythe and Welch; Shapiro-Wilk, D’Agostino-Pearson and Anderson-Darling),
statistical analysis was performed using a two-way ANOVA with a Bonferroni
multiple comparisons test. Significance was determined at alpha level 0.05.
Significance values represented are * p < 0.05, ** p < 0.009 and ns for non-
significance.
3. RESULTS AND DISCUSSION
3.1. Isolation of compounds 1–3 from the DCMAB of A. brachypoda
The DCMAB of A. brachypoda was obtained according to the protocol described in
our previous study [8]. The high-performance liquid chromatography with
photodiode array detection (HPLC-PDA) analysis revealed three major compounds
(Figure 1) which were previously characterized as three unusual dimeric flavonoids
named brachydin A (1), brachydin B (2), and brachydin C (3) [8]. To study the anti-
inflammatory properties of these molecules, DCMAB was purified at large scale. As
a first step, separation conditions were optimized at HPLC-PDA analytical scale
(Figure S1, Supplementary Material) and transferred to medium pressure liquid
chromatography (MPLC) at a semi-preparative scale, using a gradient transfer
method. This yielded 4 g of brachydin A (compound 1; Figure 1A) in a single step.
Brachydin B (2) and brachydin C (3) were obtained in a mixture (4.9 g) (see Section
2.2.3), mostly due to a column overload, and were further purified by high-speed
counter current chromatography (HSCCC). The solvent system was determined
Anti-inflammatory activity of A. brachypoda extracts
51
using the ARIZONA solvent system approach. The separation was carried out
successfully as a result of the different Kp determined (compounds 2 (Kp = 1.5) and
3 (Kp = 2.2) (see Section 2.2.3). A full recovery of pure compound 2 (1.4 g) and 3
(3.2 g) (Figure 1C) resulted, as HSCCC is a support-free liquid-liquid partition
chromatography technique [24]. According to the literature, this is the first time this
technique has been described for the isolation of pure compounds from A.
brachypoda root extracts.
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52
Figure 1. (A) Structures of brachydin A (1), brachydin B (2), and brachydin C (3) isolated from the dichloromethane extract (DCMAB) of A. brachypoda roots (B) Determination of coefficient of partition Kp by high-performance liquid chromatography with photodiode array detection (HPLC-PDA) of the solvent mixture P: methanol/ethyl acetate/hexane/water (6:5:6:5; v/v/v/v) of the ARIZONA system [23]. (C) High-speed counter current chromatography (HSCCC)-UV chromatogram at 280 nm, with clear separation of compounds 2 and 3.
Anti-inflammatory activity of A. brachypoda extracts
53
3.2. Quantification of compounds 1–3 from A. brachypoda root extracts by
UHPLC-MS/MS
The amounts of compounds 1, 2, and 3 in the HEAB and DCMAB were determined
by ultrahigh pressure liquid chromatography coupled to mass spectrometer
(UHPLC-MS/MS) analysis, in the multiple reaction monitoring (MRM) mode. The
MRM parameters of each analyte were optimized to increase sensitivity for a specific
transition from mass spectrum2 (MS2) reported data (Table S1, Supplementary
Material) [8]. The range of the calibration curves was estimated for each compound
and was set to 31–500 ng/mL. This yielded five data point calibration curves with r2
> 0.99 (Table S2, Supplementary Material). The results obtained from the
quantitative analysis are displayed in Figure 2. The total content of compounds 1–3
was 3.35-fold higher in DCMAB than in HEAB. This was expected, since these
compounds are primarily occurring as glucoronated derivatives in HEAB [8], and
these are not detected by MRM. Compound 3 was the most abundant in both
DCMAB (123 mg/g (DW: dry weight) and HEAB (36 mg/g (DW)). Compounds 1 and
2 were present at 16 and 108 mg/g (DW) in DCMAB, respectively. Lower levels of
1 (5 mg/g) and 2 (33 mg/g (DW)) were found in HEAB. These results quantify, for
the first time, the amounts of the three isolated brachydins in both extracts.
Figure 2. Concentration (mg/g DW) of compounds 1–3 in hydroethanolic extract (HEAB) and dichloromethane extract (DCMAB), respectively. Bars correspond to mean values ± s.d.; n = 3.
Chapter II
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3.3. In vitro cytotoxicity and anti-inflammatory bioactivity of extracts and
isolated compounds
3.3.1. Arrabidaea bachypoda extracts (HEAB and DCMAB)
The cytotoxicity of both HEAB and DCMAB was assessed; results are shown in
Figure 3A (and corresponding scatter plot in Figure S4). Human fibroblast like
synoviocytes (HFLS) were viable after 24 h incubation, with tested concentrations
ranging from 3 to 100 µg/mL of HEAB. Conversely, at 50 μg/mL and 100 μg/mL of
DCMAB, HFLS presented viability values of 68% and 5%, respectively, compared
to controls (Figure 3A). Consequently, these two top concentrations were not
considered in the bioactivity assay, a sandwich enzyme-linked immunosorbent
assay (ELISA) (Figure 4A), due to toxicity. At the highest dose tested (100 μg/mL),
HEAB showed a 30% inhibition of IL-6 release. DCMAB yielded a similar inhibitory
effect at 25 μg/mL (31% inhibition; Table 1), the highest concentration inducing no
cytotoxicity. Both inhibition effects were non-significant (p value 0.27 and 0.19,
respectively) when compared to non-treated control: interleukin-1 beta (IL-1 β). In
line with these results, IC50 calculations (Table 1) show that an anti-inflammatory
effect occurs only above 100 µg/mL and 31 µg/mL for HEAB and DCMAB,
respectively.
3.3.2. Isolated brachydins from DCMAB (1–3)
The three isolated compounds 1, 2, and 3, were also tested for cytotoxicity (WST-
1) and anti-inflammatory activity (ELISA) and showed different activities (Figures
3B, S4 and 4B). Compound 1 was only cytotoxic at 100 µM, whereas compounds 2
and 3 induced a loss of viability at 50 µM, with no evident effect of dose increase,
unlike what was previously described for DCMAB in terms of cytotoxicity (Figure 3).
For this reason, the IL-6 ELISA assay was performed using supernatants recovered
after incubation with HFLS, up to 50 µM for 1 and only up to 25 µM for 2 and 3
(Figure 4B). At the highest concentration with remaining favorable viability (25 µM),
both 2 and 3 significantly decreased IL-6 release when compared to the activation
control IL-1β (80% and 94% inhibition, respectively (Table 1). Compound 1 showed
a non-significant inhibition of IL-6 release at 25 µM and 50 µM (10% and 24%,
respectively; Figure 4B and Figure S5, Supplementary Material). In Table 1, IC50
values (individual plots represented in Supplementary Material, Figure S6)
confirmed the increased anti-inflammatory effect of 2 (17 µM) and 3 (19 µM), in
Anti-inflammatory activity of A. brachypoda extracts
55
comparison to 1 (62 µM). These different outcomes between compounds could be
linked to the fact that both compounds 2 and 3 are of higher structural similarity
between each other than compound 1, that exhibits higher polarity influencing
effects. Regarding cell viability of HFLS, HEAB and compound 1 presented the least
cytotoxicity and DCMAB, compounds 2 and 3, showed higher cytotoxicity. This is
expected, since these are the major two components of this extract, as above
mentioned (see Section 3.2). In order to compare the bioactivity results between the
tested compounds, IC50 results in µM (isolated compounds) were transformed into
µg/mL to match those of the extracts, that represent mixtures of several compounds
(Table 1). In terms of the anti-inflammatory effect, results are in line with those of
cytotoxicity. HEAB (IC50 > 100 µg/mL) was inactive in decreasing release of pro-
inflammatory cytokine IL-6 in HFLS. According to what was previously reported for
HEAB [9–11], this extract is comprised by the dimeric flavonoid aglycones (1–3) and
their various glucoronated derivatives [9]. The glucoronated derivatives appear in
higher amount than the corresponding aglycones. In an HFLS in vitro setting, the
metabolic pathways that hydrolyze the glucoronated forms into simple aglycones
(as it would in the acidic pH of the stomach) do not occur and the compounds are
absorbed as is [26]. Based on such considerations, the lack of activity of HEAB can
be thus correlated to the low amounts of 1, 2, and 3 (Figure 2) and the glucoronated
derivatives themselves, inactive in this experimental setup. This is also the
reasoning as to why all current studies and evaluations of this extract in vivo occur
after per os administration and not local [8–10,12]. DCMAB on the other hand, only
contains the three isolated brachydins A, B, and C in their non-glucoronated forms.
Such extract is thus more interesting to explore in this experimental setup and when
seeking local administration of anti-inflammatory treatments for OA. Albeit through
unknown pathways, this study is the first analyzing the anti-inflammatory activity in
the specific context of OA. Additionally, it compares effects and quantifies the
presence of isolated compounds in both A. brachypoda extracts in order to assess
their potential as OA therapeutic alternatives. Compound 1 exhibited similar anti-
inflammatory activity (IC50) when compared to DCMAB (33 µg/mL and 31 µg/mL,
respectively), whereas compounds 2 (9 µg/mL) and 3 (10 µg/mL) showed a 3-fold
higher activity. This suggests that the extracts activity results mainly from the
presence of the two latter compounds. Taking into account the amounts of each
compound 1-3 in the DCMAB (Table 1), the cumulative IC50 of each individual
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56
isolated compound (27 µg/mL) does not greatly differ from DCMAB alone - 31
µg/mL. Based on these results, no investigation on synergistic effect was pursued.
Differences between the anti-inflammatory activities between the three compounds
potentially stem from the higher polarity of compound 1, in comparison to 2 and 3.
Figure 3. The cellular viability of human fibroblast like synoviocytes (HFLS) incubated with all tested compounds at increasing concentrations, after 24 h. Root extracts (A) and isolated compounds from dichloromethane extract (DCMAB) (B). Bars correspond to mean values ± s.d.; n = 6; V = vehicle, 0.01% dimethyl sulfoxide (DMSO). Additional scatter plot with individual data in Supplementary Material, Figure S4.
Anti-inflammatory activity of A. brachypoda extracts
57
Figure 4. Release of pro-inflammatory cytokine IL-6 from IL-1β activated HFLS incubated with root extracts (A) and isolated compounds 1–3 (B), after 24 h. Bars correspond to mean values ± s.d.; n = 6. *p < 0.05, **p < 0.009 and ns = no significance. Dotted lines represent cut-off of maximum IL-6 release by Il-1β stimulation.
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Table 1. IL-6 inhibitory activity and anti-inflammatory IC50 of all tested compounds.
Extracts IC50 (µg/mL) Inhibition of IL-6 release at highest non-toxic
concentration (%) [25 µg/ml] HEAB >100 2 ± 12
DCMAB 31 ± 1 31 ± 8
Compounds Molecular
weight (g/mol)
IC50
Inhibition of IL-6 release at
highest non-toxic
concentration (%)
[25 µM = 13 µg/ml *]
Compound in DCMAB
(% of DW **)
Compound concentration in IC50 of DCMAB
(µg/mL) (µg/mL) (µM)
1 524 33 ± 2 62 ± 2 10 ± 11 2 0.5 2 538 9 ± 3 17 ± 3 80 ± 9 10 3.4 3 508 10 ± 3 19 ± 3 94 ± 5 12 3.8
* Mean value for equivalence to 25 µM of Compound 1, 2 and 3. ** Dry Weight (DW).
4. CONCLUSIONS
Arrabidaea brachypoda is a plant commonly used in Brazilian traditional medicine
to treat pain and inflammation related to arthritic joints. The effects of HEAB,
DCMAB and three isolated dimeric flavonoids were assessed. In this study, specific
in vitro anti-inflammatory activity was tested using primary human arthritic
synoviocytes, in order to understand the mechanisms involved and potentially
identify molecules of interest for local drug delivery in OA. The present study is the
first comparing the anti-inflammatory effect and providing a quantification of the
three isolated compounds from the DCMAB. Despite high cellular viability scores at
tested concentrations, HEAB was not performant in terms of anti-inflammatory
effect. Studies suggest that the acidic hydrolysis in the stomach is responsible for
the releasing of aglycones deriving from glucoronated forms of not only brachydins
A, B, and C, but also other molecules of the extract [8,26]. In this study setup
however, hydrolysis does not take place, which explains the lack of activity of HEAB.
Conversely, DCMAB showed an effect against the release of pro-inflammatory
cytokine IL-6. Quantification of 1, 2, and 3 in this extract showed higher proportions
of 2 and 3 compared to 1. The bioactivity results are in line with these findings, where
1 alone is considerably less active than to 2 and 3 alone. Compound 3 presented
as both the most effective and cytotoxic compound, the fact that it is the main
Anti-inflammatory activity of A. brachypoda extracts
59
component of DCMAB could account for the extract’s enhanced effect over both 1
and 2 and, partly, HEAB. Differences in polarity between compound 1 versus 2 and
3 can affect bioavailability and bioactivity. Compounds 2 and 3 seem to have a major
role in the anti-inflammatory effects of DCMAB and, potentially, other aglycones
present in HEAB deserve further exploring in terms of anti-inflammatory bioactivity.
Additionally, further research of in vivo studies and potential drug delivery systems
would enrich the understanding of the mechanisms underlying A. brachypoda roots
and its treatment of arthritic joints.
5. REFERENCES
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[11] F.A. Resende, C.H. Nogueira, L.G. Espanha, P.K. Boldrin, A.P. Oliveira-Höhne, M. Santoro de Camargo, C. Quintino da Rocha, W. Vilegas, E.A. Varanda, In vitro toxicological assessment of Arrabidaea brachypoda (DC.) Bureau: Mutagenicity and estrogenicity studies, Regul. Toxicol. Pharmacol. 90 (2017) 29–35. https://doi.org/10.1016/j.yrtph.2017.08.010. [12] V. Rodrigues, C. Rocha, L. Périco, R. Santos, R. Ohara, C. Nishijima, E. Ferreira Queiroz, J.-L. Wolfender, L. Rocha, A. Santos, W. Vilegas, C. Hiruma-Lima, Involvement of Opioid System, TRPM8, and ASIC Receptors in Antinociceptive Effect of Arrabidaea brachypoda (DC) Bureau, Int. J. Mol. Sci. 18 (2017) 2304. https://doi.org/10.3390/ijms18112304. [13] V. Rocha, C. Quintino da Rocha, E. Ferreira Queiroz, L. Marcourt, W. Vilegas, G. Grimaldi, P. Furrer, É. Allémann, J.-L. Wolfender, M. Soares, Antileishmanial Activity of Dimeric Flavonoids Isolated from Arrabidaea brachypoda, Molecules. 24 (2018) 1. https://doi.org/10.3390/molecules24010001. [14] L.M. de Sousa Andrade, A.B.M. de Oliveira, A.L.A.B. Leal, F.A. de Alcântara Oliveira, A.L. Portela, J. de Sousa Lima Neto, J.P. de Siqueira-Júnior, G.W. Kaatz, C.Q. da Rocha, H.M. Barreto, Antimicrobial activity and inhibition of the NorA efflux pump of Staphylococcus aureus by extract and isolated compounds from Arrabidaea brachypoda, Microb. Pathog. 140 (2020) 103935. https://doi.org/10.1016/j.micpath.2019.103935. [15] C.Q. Da Rocha, F.C. Vilela, F. V Santa-Cecília, G.P. Cavalcante, W. Vilegas, A. Giusti-Paiva, M.H. Dos Santos, Oleanane-type triterpenoid: an anti-inflammatory compound of the roots Arrabidaea brachypoda, Rev. Bras. Farmacogn. 25 (2015) 228–232. https://doi.org/10.1016/j.bjp.2015.03.005. [16] M.B. Goldring, S.R. Goldring, Osteoarthritis, J. Cell. Physiol. 213 (2007) 626–634. https://doi.org/10.1002/jcp.21258. [17] A.E. Nelson, K.D. Allen, Y.M. Golightly, A.P. Goode, J.M. Jordan, A systematic review of recommendations and guidelines for the management of osteoarthritis: The Chronic Osteoarthritis Management Initiative of the U.S. Bone and Joint Initiative, Semin. Arthritis Rheum. 43 (2014) 701–712. https://doi.org/10.1016/j.semarthrit.2013.11.012. [18] A. Koszowska, R. Hawranek, J. Nowak, Osteoarthritis -a multifactorial issue, Reumatologia. 52 (2014) 319–325. https://doi.org/10.5114/reum.2014.46670. [19] D.J. Hunter, D. Schofield, E. Callander, The individual and socioeconomic impact of osteoarthritis, Nat. Rev. Rheumatol. 10 (2014) 437–441. https://doi.org/10.1038/nrrheum.2014.44. [20] S.L. Kolasinski, T. Neogi, M.C. Hochberg, C. Oatis, G. Guyatt, J. Block, L. Callahan, C. Copenhaver, C. Dodge, D. Felson, K. Gellar, W.F. Harvey, G. Hawker, E. Herzig, C.K. Kwoh, A.E. Nelson, J. Samuels, C. Scanzello, D. White, B. Wise, R.D. Altman, D. DiRenzo, J. Fontanarosa, G. Giradi, M. Ishimori, D. Misra, A.A. Shah, A.K. Shmagel, L.M. Thoma, M. Turgunbaev, A.S. Turner, J. Reston, 2019 American College of Rheumatology/Arthritis Foundation Guideline for the Management of Osteoarthritis of the Hand, Hip, and Knee, Arthritis Rheumatol. 72 (2020) 220–233. https://doi.org/10.1002/art.41142. [21] B. Abramoff, F.E. Caldera, Osteoarthritis: Pathology, Diagnosis, and Treatment Options, Med. Clin. North Am. 104 (2020) 293–211. https://doi.org/10.1016/j.mcna.2019.10.007. [22] S. Challal, E.F. Queiroz, B. Debrus, W. Kloeti, D. Guillarme, M.P. Gupta, J.L. Wolfender, Rational and Efficient Preparative Isolation of Natural Products by MPLC-UV-ELSD based on HPLC to MPLC Gradient Transfer, Planta Med. 81 (2015) 1636–1643. https://doi.org/10.1055/s-0035-1545912. [23] A.P. Foucault, Centrifugal partition chromatography - Chromatographic Science Series, Taylor & Francis, 1995. [24] K. Hostettmann, A. Marston, M. Hostettmann, Countercurrent Chromatography, in: Prep. Chromatogr. Tech., Springer Berlin Heidelberg, Berlin, Heidelberg, 1998: pp. 135–201. https://doi.org/10.1007/978-3-662-03631-0_7. [25] L. Neff, M. Zeisel, V. Druet, K. Takeda, J.P. Klein, J. Sibilia, D. Wachsmann, ERK 1/2- and JNKs-dependent synthesis of interleukins 6 and 8 by fibroblast-like synoviocytes stimulated with
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protein I/II, a modulin from oral streptococci, requires focal adhesion kinase, J. Biol. Chem. 278 (2003) 27721–27728. https://doi.org/10.1074/jbc.M212065200. [26] R.M. Hill, H.B. Lewis, The hydrolysis of sucrose in the human stomach, Am. J. Physiol. Content. 59 (1922) 413–420. https://doi.org/10.1152/ajplegacy.1922.59.1.413.
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Supplementary material
Figure S1. Reverse phase HPLC-UV analysis at 254 nm - optimized conditions for MPLC separation. Brachydin A (1), Brachydin B (2) and Brachydin C (3).
Figure S2. Coefficient of partition Kp equation (ARIZONA system).
Figure S3. Human fibroblast like synoviocytes (optical microscope). Scale bar: 200 µm.
Anti-inflammatory activity of A. brachypoda extracts
63
Figure S4. Scatter plot of Figure 3, showing cellular viability of human fibroblast like synoviocytes (HFLS) incubated with all tested compounds at increasing concentrations, after 24 h. Root extracts (A) and isolated compounds from dichloromethane extract (DCMAB) (B). Individual plotted values (n = 6) with mean values ± s.d.. V = vehicle, 0.01 % dimethyl sulfoxide (DMSO).
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Figure S5. Inhibition percentage of IL-6 release normalized to 100 % activation, as a function of each compound concentration. Bars correspond to mean values ± s.d.; n = 6.**** p <0.0001 and *** p = 0.0007 and ns = no significance.
Figure S6. IC50 calculations of hydroethanolic extract (HEAB) (A), dichloromethane extract (DCMAB) (B), 1 (C), 2 (D) and 3 (E). Points correspond to mean values ± s.d.; n = 6.
Anti-inflammatory activity of A. brachypoda extracts
65
SRM, Selected reaction monitoring; DP, Declustering potential; EP, Entrance potential; CE, Collision energy; CXP, Collision cell exit.
r2, Correlation coefficient; LOD, Limit of Detection; LOQ, limit of quantification
Table S1. Optimized MRM parameters for the quantification of active compounds by UHPLC-MS/MS.
Compound Precursor
Ion SRM
transition DP (V)
EP (V)
CE (eV)
CXP (V)
Dwell time (ms)
1 525 525 ➝ 271 30
10 10 7
125 2 539 539 ➝ 285 3 509 509 ➝ 255 80 50 12
Table S2. Calibration curves parameters for active compounds and determination of the LOD and LOQ in ng/mL.
Compound Linear function r2 LOD (ng/mL) LOQ (ng/mL)
1 𝑦 = 816𝓍 + 6,06.103
0.998
0.21 0.64
2 𝑦 = 422𝓍 + 3,51.103 0.47 1.41
3 𝑦 = 122𝓍 + 639 1.42 4.25
66
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69
Chapter III
Nano wet milled celecoxib extended release microparticles
for local management of chronic inflammation
Carlota Salgado a,b, Laure Guénée c, Radovan Černý c, Eric Allémann a,b and Olivier Jordan a,b a School of Pharmaceutical Sciences, University of Geneva, 1 Rue Michel-Servet 1211 Geneva, Switzerland b Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, 1 Rue Michel-Servet 1211 Geneva, Switzerland c Department of Quantum Matter Physics, Laboratory of Crystallography, University of Geneva, 24 Quai Ernest Ansermet 1211 Geneva, Switzerland
Published: International Journal of Pharmaceutics 2020, 589, 119783; https://doi.org/10.1016/j.ijpharm.2020.119783
ABSTRACT
Osteoarthritis (OA), the most common form of arthritis, is characterized by chronic inflammation, degeneration of articular cartilage and whole joints. Local delivery by intra-articular (IA) injection of small molecules is an established treatment to relieve pain and improve joint motion, requiring month-lasting release of therapeutic drug doses. We incorporated anti-inflammatory drug celecoxib in poly (D, L-lactic acid) microparticles using two spray-drying approaches - either as a solid solution or embedded as milled nano drug. Differential scanning calorimetry, X-ray powder diffraction, electron microscopy and in vitro drug release allowed comparison of the microparticles. Both types resulted in spherical particles ranging from 20 to 40 μm mean size, with high drug loadings (10 % to 50 % w/w) and entrapment efficiencies > 80 %. However, after 90 days, in vitro celecoxib release from nano drug embedded microparticles presented a significantly slower release in comparison to drug in solution microparticles, attributed to the presence of stabilized amorphous drug. No cytotoxicity was observed in human articular synoviocytes and PGE2 release was fully suppressed at low doses of both microparticulate systems. This study provides techniques to release high drug loads over months in a tunable manner, providing valuable options for the IA management of osteoarthritis. Keywords: celecoxib; crystalline behavior; spray-dried microparticles; intra-articular injection; drug delivery systems; osteoarthritis
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1. INTRODUCTION
Osteoarthritis (OA), the most common form of arthritis, is a chronic disease with
worldwide incidence in the population aged 65 years and higher [1,2]. It is
characterized by chronic inflammation, articular cartilage degeneration and
structural changes of whole joints. Symptoms range from mild to incapacitating,
mostly in terms of pain, inflammation and loss of movement. Risk factors include
age, gender (higher prevalence in females), overweight/obesity, previous joint
injuries and genetic predisposition to joint complications. Other than the economic
burden it represents, OA is one of the main causes of disability in the elderly
population [3–6]. Since this is a multifactorial, complex disease there is an unmet
need for disease modifying drug that target multiple tissues and mechanism, slowing
disease progression. Current treatment options are mainly based on non-
pharmacological and symptoms management approaches [7,8]. Joints are poorly
irrigated closed structures, making systemic drugs a liability. In this field, intra-
articular (IA) administration plays an important role. Local delivery of small
molecules not only circumvents systemic adverse effects but also allows for a more
effective relief of symptoms. However, drawbacks include fast joint space clearance
and limited number of yearly injections [9,10]. Polymeric microparticulate drug
delivery systems have grown in the field as a response to such disadvantages,
primarily by allowing higher drug loading and prolonged delivery of drug molecules
directly into the joint [11,12]. However, attaining drug loadings high enough for
release of effective therapeutic drug concentrations over extended periods remains
a challenge. In this study, to achieve high drug loadings required to deliver
therapeutic doses in a sustained manner, spray drying was selected as the
formulation technique. Spray drying is an established technique applied in the
formulation of drug encapsulating polymeric microparticles [13]. This continuous,
single step manufacturing process is an attractive technique due to its cost-
effectiveness, upscaling potential, versatility and high reproducibility [14,15]. The
process is based on the atomization of a liquid feed, forming droplets. Aided by a
drying gas and in a closed chamber of controlled environment, the droplets
transform to dry particles by solvent evaporation. The final product is separated and
collected according to size [16,17]. In this work, this technique was applied using
poly D,L-Lactic Acid (PDLLA) as the matrix polymer. A biodegradable material, this
Nano wet milled celecoxib microparticles
71
polymer was selected due to its high molecular weight and its known properties of
extended drug release [18,19]. The incorporated drug, celecoxib, is a crystalline
poorly soluble drug compound commonly prescribed in the symptomatic oral
treatment of OA. A strong anti-inflammatory nonsteroidal drug, the gastrointestinal
and cardiovascular side effects of this COX-2 specific inhibitor are well established
[20–23]. With the main goal of tailoring drug release to ensure therapeutic anti-
inflammatory activity over an extended period of time, in this study, celecoxib was
encapsulated into polymeric microparticles using two different approaches. Drug in
solution and nano wet milled drug particles were incorporated by spray drying into
microparticles sized 20 to 30 µm. Three different initial drug concentrations were
tested in order to evaluate the impact of drug loading in the delivery systems.
2. MATERIALS AND METHODS
2.1. Materials
Poly (D,L-Lactic Acid) (PDLLA - Resomer 205 S®) was purchased from Evonik
(Essen, Germany). Celecoxib was purchased from LC Laboratories® (Woburn, MA,
USA). Solvents dichloromethane, acetone, trifluoroacetic acid and acetonitrile were
all of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). Milli-
Q® water from Merck (Burlington, MA, USA) was used throughout this study. All
other chemical products were commercially obtained from Sigma-Aldrich (St. Louis,
MO, USA). For the in vitro anti-inflammatory assays, all media solutions were
purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum was purchased
from Eurobio (Les Ulis, France). Interleukin 1 beta (IL-1β) and prostaglandin PGE2
quantification assay were purchased from R&D Systems (Bio-Techne, Abingdon,
UK).
2.2. Nano wet milled drug
Commercial celecoxib was first recrystallized by a non-solvent precipitation method
and then wet-milled to nano size. Briefly, 5 mg of drug were dissolved in 300 µL of
acetone in a 2 mL tube. Water was added dropwise in a 1:2 solvent/water volume
ratio. The organic solvent was evaporated at 4 °C from open tubes, overnight. Next,
100 µL of a 2 % w/v aqueous solution D-α-tocopherol polyethylene glycol 1000
succinate (TPGS) were incorporated in the tube, as a stabilizer. Lastly, 580 mg of
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BeadBug™ 0.5 mm zirconium beads from the same supplier were added to each
tube. Size reduction was performed using a homogenizer (Precellys 24®) from Bertin
Instruments (Montigny-le-Bretonneux, France). Five cycles of 90 s at 10000 rpm
followed by a 5 min ice bath were completed twice. An extra 10 cycles of 60 s at
6600 rpm and ice bath succeeded. Once separated from the zirconium beads, all
nano drug suspensions were centrifuged and freeze-dried. Size determination was
done by dynamic light scattering (DLS) using a Nanosizer (Malvern Panalytical,
Malvern, England).
2.3. Spray dried polymeric microparticles
PDLLA microparticles were formulated using a 4M8-TriX spray dryer (ProCepT,
Zelzate, Belgium). After process optimization with unloaded particles, a set of
variable and fixed process parameters was selected. These parameters were:
polymer concentration 7 % w/v; feed rate 5 mL/min; bi-fluid nozzle 0.6 mm (internal
diameter); atomizing air force 6 L/min; inlet temperature 45 °C; outlet temperature
34 °C; cyclone size medium and inlet air flow 0.3 m3/min. Air was chosen as the
processing gas. Using dichloromethane as solvent, three different proportions of
celecoxib - 10, 25 and 50 % (weight percentage of total dry content) - were loaded
into two different types of microparticles (MPs): drug in solution microparticles and
nano drug particles embedded into microparticles. In the first case, both drug and
polymer were dissolved simultaneously in dichloromethane and the resulting
solution was sprayed. The second type of microparticles was obtained after spray
drying a suspension, using the same nominal drug loadings (10, 25 and 50 %).The
nano wet milled drug powder was extemporaneously dispersed into a
dichloromethane polymer solution, in an ice bath, just before spraying.
2.4. Drug and polymeric microparticles characterization
2.4.1. Size and size distribution
Particle size determination was conducted by dynamic light scattering (DLS) or laser
light diffraction using, a Nanosizer and a Mastersizer S from Malvern Instruments
Ltd (Malvern Pananalytical, Malvern, England), respectively. All measurements
were done in triplicate.
Nano wet milled celecoxib microparticles
73
2.4.2. Surface, morphology and drug incorporation
Morphology and surface analysis were conducted by optical microscopy using a
Carl Zeiss Axio microscope (Oberkochen, Germany) and scanning electron
microscopy (SEM) using a Jeol JSM-7001TA microscope (Tokyo, Japan). For SEM,
all samples were deposited onto carbon tape covered metal studs. Studs were
allowed to dry overnight under vacuum. Prior to analysis, studs were sputter coated
with gold. All measurements were conducted at a 5 kV voltage. To further
understand drug incorporation into the polymeric microparticles, drug release
mechanisms and water intake during in vitro release studies, a number of particles
were cross sectionned transversally. Briefly, a small amount of powder was
dispersed into a drop of OCT embedding matrix for frozen sections (CellPath,
Newtown, England) onto a metal stub. The mounted samples were frozen at -80 °C
for 24 h. Before cutting, using a CryoStar NX70 microtome (Thermo Fisher
Scientific; Waltham, MA, United States), all stubs were allowed to acclimate for 30
min in the cooling chamber of the apparatus, at -20 °C. All cuts were directly
mounted onto silicone wafers and analyzed by SEM using the same procedure
above described.
2.4.3. Drug loading and encapsulation efficiency
Drug content of all microparticulate systems formulated was quantified by ultra-high
performance liquid chromatography (UHPLC). All samples were analyzed using a
Waters Acquity™ Ultraperformance LC (Milford, MA, USA) equipped with an Acquity
UPLC® BEH C18 column (2.1 mm x 50 mm, 1.7 µm, Waters, USA) equilibrated at
40 °C. The mobile phase was composed of acetonitrile and water to which 0.1 %
trifluoroacetic acid (TFA) was added. A 2 min solvent gradient, at a constant flow
rate of 0.5 mL/min, was used. Detection was performed with a PDA detector set at
254 nm. The volume of injected sample was 2 µL. All samples were analyzed in
triplicate. Amount of encapsulated drug (drug loading, DL %) and the encapsulation
efficiency (EE %) were calculated by the following equations:
[1] DL %= Mass of CXB in MPs
Total mass of MPs ×100
[2] EE % = DL %
Theoretical DL %
Chapter III
74
2.4.4. Differential scanning calorimetry (DSC)
The different drug forms (commercial and nano wet milled) and all formulated
microparticles were analyzed using a Mettler Toledo DSC 3 Stare System differential
scanning calorimeter (Columbus, OH, USA). Sealed 40 µL aluminum crucibles were
used for all measurements. In a first instance, in order to delete humidity and thermal
history, all polymeric samples were heated from 25 to 100 °C. A cooling cycle from
100 to -10 °C followed. In a second cycle, samples were heated from -10 to 200 °C.
The same heating/cooling rate of 10 K/min was applied throughout and all
experiments were conducted under nitrogen flow (80 mL/min). Non-polymeric
samples, like commercial celecoxib, were only submitted to the cooling and second
heating cycles, under the same conditions. Glass transition temperature (Tg),
melting temperature (Tm), enthalpy (∆H) and crystallinity rates were calculated from
the last heating cycle’s resulting curve.
2.4.5. X-ray powder diffraction (XRPD)
Amorphous celecoxib was obtained from the commercial product by quench cooling.
Briefly, 25 mg of powder were melted in a tempered glass tube. Immediately after
reaching 192 °C, the tube was immersed into liquid nitrogen for 5 min. The resulting
glassy product was stored at -20 °C. X-ray powder diffraction data were collected
on an Empyrean diffractometer (Malvern Pananalytical, Malvern, England) equipped
with Cu K1,2 radiation and focusing mirror. Samples were enclosed in a glass
capillary (0.8 mm outer diameter). Throughout data collection temperature was
controlled between room temperature and 500 K using a 700+ Cryostream cooler
(OxfordCryosystems, Oxford, England). All collected data were analyzed using
TOPAS software [24].
2.5. In vitro drug release studies
In vitro drug release studies were carried over 3 months under sink conditions in
triplicates. Celecoxib solubility was determined to be 80 mg/L in phosphate buffer
saline (PBS) containing 0.5 % w/v of polysorbate 80 (Tween 80®) and 0.025 % w/v
of sodium azide. A weighed amount of microparticles was dispersed in 50 mL of
medium. Assay was performed in closed vials to avoid evaporation of water. At
alloted time points, 1 mL was withdrawn and replaced with fresh medium.
Quantification of released celecoxib was performed by UHPLC using the same
method described above.
Nano wet milled celecoxib microparticles
75
2.6. In vitro anti-inflammatory bioactivity assays
2.6.1. Human fibroblast-like synoviocytes (HFLS) isolation and culture
Hip synovial membrane was collected from three male adult patients with clinical
osteoarthritis (OA) at the time of hip replacement surgery. This protocol was
conducted under the approval of the local Ethics Committee (CCER, Geneva,
Switzerland, authorization # 2017-02234) with informed and consenting patients.
Once collected, tissue samples were finely minced and digested for 3 h (37 °C, 5 %
CO2 incubation) in a 3 mg/mL collagenase IX - RPMI 1640 solution. After
centrifugation (200 g), the resuspended pellet was cultured (37 °C, 5 % CO2) in
medium containing RPMI 1640, M199 (1:1), 1 % penicillin/streptomycin (100
IU/mL:100 g/mL), 2 mM L-glutamine and 20 % fetal bovine serum. After overnight
culture, non-adherent cells were removed. At confluence, cells were trypsinized and
passaged in 75 cm2 culture flasks (Corning, NY, USA), in complete medium
containing 10 % fetal bovine serum. All cellular assays were performed from
passage 3 until 9. Experiments were conducted twice per donor (n = 6).
2.6.2. In vitro cytotoxicity assay and determination of PGE2 levels in HFLS
supernatant by enzyme-linked immunosorbent assay (ELISA)
Confluent HFLS cells were treated in a 96-well plate for 1 h. Commercial celecoxib
in a dimethyl sulfoxide (DMSO) stock solution was further diluted in complete culture
medium and incubated at 1, 10 and 30 µM (DMSO final concentration at 0.1 %,
considered vehicle). Drug in solution and nano drug embedded microparticles at 10
% DL were resuspended in complete culture medium at drug equivalent
concentrations of1, 10 and 30 µM, based on microparticle drug loading. After 1 h
incubation, cells were activated by addition of IL-1β (1 µg/mL) (R&D Systems, Bio-
Techne, Abingdon, UK) with subsequent incubation for 23 h. All supernatants were
kept at -80 °C for further testing. Remaining adherent cells were tested for viability
using the cell proliferation reagent WST-1 (Roche, Basel, Switzerland), according to
the provider instructions. Prostaglandin E2 release in cell supernatant was assessed
by a sandwich enzyme-linked immunosorbent assay (ELISA). For this, a human
PGE2 parameter assay kit from R&D Systems (ref # KGE004B; Bio-Techne,
Abingdon, UK) was used according to the manufacturer’s protocol. All samples were
diluted 3 times. Experiments were conducted twice per donor (n = 6).
Chapter III
76
2.7. Statistical analysis
Data are shown in values of mean ± standard deviation (s.d.). Statistical analysis
was performed using a two-way variance analysis by a multiple groups ANOVA
(Bonferroni multiple comparisons; GraphPad Prism 8.3 software) after confirming
normal distribution and homogeneity of variances by Shapiro-Wilk, D’Agostino-
Pearson and Anderson-Darling tests. Significance was determined at alpha level
0.05. P values represented are ****p < 0.0001. A Student’s t-test - homoscedastic,
two tails – was performed in Microsoft Office Excel for the in vitro release studies
data (p < 0.05).
3. RESULTS AND DISCUSSION
3.1. Nano wet milled drug
Commercial celecoxib (Figure 1B) was recrystallized in water/acetone mixture prior
to wet milling in order to control the homogeneity and quality of the starting material.
Purity and crystallinity of the recrystallized compound were assessed by differential
scanning calorimetry and X-ray powder diffraction. As shown in Table 1 (Figure S1),
the differential scanning calorimetric analysis of both celecoxib forms presented
comparable values of Tm and enthalpy. Both values of 160 °C and 92 J/g have been
previously reported in the literature for pure celecoxib [25,26]. Displayed in Figure
1A are the XRPD patterns of commercial and recrystallized celecoxib. The same
diffraction pattern was observed for both samples with pronounced intensity at 2θ
values of 5.4°, 10.7°, 11.0°, 13.0°, 14.9°, 16.1°, 17.9°, 19.7°, 21.5°, 22.2°, 25.4° and
29.5°. Both crystalline compounds contained the triclinic (P1) polymorph III of
celecoxib, as verified by Rietveld refinement using the model from CSD database
[20,27]. The content of the amorphous fraction of the sample was lower in the
recrystallized celecoxib as observed by the lower background in this sample (Figure
S5 and S6). These results confirmed the quality and crystallinity of the drug
compound used for the wet milling procedure. The amorphous form of celecoxib
was analyzed in parallel for comparison. DSC analysis (Table 1, Figure S2) showed
an exothermic event (112 °C, ∆H 61.6 J/g) followed by an endothermic peak (161
°C, ∆H 72.5 J/g). This sequence corresponds to recrystallization of celecoxib above
its Tg, followed by melting, consistent with literature reports [25]. It is known that the
Nano wet milled celecoxib microparticles
77
phase which recrystallizes is the polymorph III [27]. Upon heating, the form III
transforms into the polymorph I, which also recrystallizes upon cooling of the melted
sample, as seen in XRPD patterns of the amorphous compound at different
temperatures (Figure S7). On the XRPD pattern (Figure 1A) the amorphous state of
the compound was confirmed with no distinct peaks observed. The resulting curve
had a wide broad band centered at around 2θ value of 20°, consistent with an
amorphous compound. After wet milling, the resulting product was also
characterized. As determined by dynamic light scattering, the newly produced drug
particles (Figure 1C) were of an average size of 593 nm (Z-average; PDI < 0.39). In
Table 1, the evaluation of the endothermic event of the DSC analysis showed a Tm
with an onset of 154 °C and 34.08 J/g enthalpy. The XRPD pattern of the wet milled
sample (Figure 1A) shows the presence of the polymorph III in the recrystallized
celecoxib (Figure S7). However, a decrease in intensity of all peaks was observed
at all 2θ values including a complete loss of the sharp intensity of values 10.7°, 11.0°
and 13.0°. This result is in line with the 63 % loss of the crystalline fraction in the
sample (∆H 93.8 J/g = 100 % crystalline CXB) observed in the DSC analysis results
(∆H 34.1 J/g = 36.3 %). Related to the wet milling process and contributing to these
results is the role of TPGS added to stabilize the particles. D-α-tocopherol
polyethylene glycol 1000 succinate is an amphiphilic, highly stable molecule. This
surfactant is commonly used in the drug delivery field as a solubilizer and
permeation enhancer of poorly soluble drugs [28–30]. In this case, due to
incorporation and coating of the compound in the nano wet milled drug particles, a
solubilization effect resulted in a loss of crystallinity. Another contribution to this is a
result of the high pressure, high entropy environment of the wet nano milling
technique. This induced the celecoxib size decrease and rearranged the crystal
formation to smaller, more fragile and brittle crystals (Figure 1C). These high impact
collision energies between zirconium beads during wet milling explain the zirconium
dioxide peaks (ZrO2) observed in the XRPD pattern of the nano wet milled drug
(Figure 1A) at 2θ values of 23.5°, 28.2° and 31.5° [31].
Chapter III
78
3.2. Polymeric microparticles: formulation and characterization
3.2.1. Size, morphology and drug encapsulation
After spray drying, all dry products were immediately characterized and stored at 4
°C. Residual solvent (DCM) was below 600 ppm in all the final dry products [data
not shown] [32]. Spray drying process yields were calculated based on the weight
of dry matter sprayed (Table 2). With overall values ranging from 7 to 35 %, drug in
Table 1. Differential scanning calorimetry endothermic events for all forms of celecoxib.
Endothermic thermal event
Celecoxib sample Onset (°C) ΔH (J/g)
Commercial 161.8 92.2
Recrystallized 160.2 93.8
Amorphous 161.0 72.5
Nano wet milled 154.3 34.1
Figure 1. X-ray powder diffraction patterns of all celecoxib forms and the nano wet milled drug (A); SEM micrographs of commercial (B; scale bar: 10 µm) and nano wet milled celecoxib (C; scale bar: 10 µm).
Nano wet milled celecoxib microparticles
79
solution microparticles batches exhibited higher production yields when compared
to nano drug embedded microparticles. High yields at increasing drug loadings and
different feed solutions, supported the selected spray drying process parameters,
choice of polymer, solvent and suggested the overall robustness of the method. The
lower yields can be explained by the small size of batches (few mL) prepared using
the ProCept spray drying equipment – pilot plant sized. Particle size and size
distribution were determined by laser light diffraction (Table 2). The three studied
loadings (10, 25 and 50 %) of drug in solution microparticles showed sizes (D50) of
21.3, 28.8 and 34.3 µm, respectively. In size distribution, span values were ≤ 3 for
this type of particles. In parallel, as shown in Table 2, nano drug embedded
microparticles presented sizes 31.3, 32.6 and 38.3 µm corresponding to the three
values of increasing drug loading. A higher polydispersity was observed where span
values ranged from 2.2 to 4.7. A trend of a slight increase in size of the embedded
particles was observed. This could relate to numerous phenomena such as feed
viscosity, solvent evaporation, shrinkage and surface tension. An increase in size
was also visible when increasing drug loading for drug in solution microparticles,
attributed to the increased viscosity of the consequent feed solution. Solubility of
celecoxib in the polymer was assessed [Data not shown] using a film method
described in the literature [33]. No visible precipitation of drug/ formation of particles
in the dried droplets, at any drug loading (10, 25 and 50 %) was observed under
optical microscopy. In comparison to dichloromethane alone (recrystallization after
drying), the polymer acted as a solubility enhancer for CXB (55 mg/mL = 7.5 % w/w,
as measured by UHPLC titration of a saturated solution). Thus, a full solubilization
in the polymer/dichloromethane solution as well as in the dried polymer was
confirmed. In addition, drug loadings of 10, 25 and 50 % were confirmed by UHPLC
(Table 2), values higher than those reported for celecoxib microparticles formulated
by spray drying [34] and those formulated by traditional methods [35]. High
entrapment efficiencies (> 83 %) were calculated for all studied microparticles. In
order to confirm these findings, SEM analysis (Figure 2) was performed in both dry
powder form and cross sectional transversal cuts of the particles at the time of
formulation (T=0). As shown in Figure 2 (a1 to a3), all drug in solution microparticles
were spherical. Despite no evidence of irregular features, different degrees of
surface smoothness, correlated to the increase in drug loading, were observed.
Polydispersity was also observed, where particles of approximately 10 µm and
Chapter III
80
smaller were found in all analyzed batches. In analyzing the inner core of drug in
solution microparticles (Figure 2a4, a5 and a6), no evident macrostructures (drug or
polymer) were found. Indeed, what was observed were homogeneous polymer
matrix cores. Porosity was identified at 10 % DL. At 50 % drug loading the inner core
of the particles presented as dense polymer-drug spheres. In the same analysis for
nano drug embedded microparticles (Figure 2B), the same spherical shape was
observed. Likewise, size polydispersity was confirmed (Figure 2b1). However,
observations of the outer layer of this type of microparticles revealed coarser and
rougher surfaces, unrelated to drug loading and observed in all batches (Figure
2b1b2b3). As shown in Figure 2b4 to b6, the inner cores of nano drug embedded
microparticles exhibited different features from those of drug in solution MPs. The
same degree of polymer matrix porosity was found throughout the three different
drug loadings. The outer layer (Figure 2b5) was also found to be more irregular than
those of drug in solution MPs (Figure 2a5) that presented as a continuous, even
line. The main difference, however, is the visible nano drug incorporation into these
particles. Increasing amounts of embedded nano celecoxib clusters were observed,
once again directly in relation to increasing drug loadings. As shown by the
micrographs in Figure 2B, at 10 % drug loading, sparse nano drug clusters were
found located near the inner surface of the particles. At 25 % DL (Figure 2b5), the
clusters appeared both near the surface and as well the center of the polymer
particle. At the highest loading (50 %; Figure 2b6) the observed clusters were found
to be embedded in all areas: surface, center and in the outer layer of the
microstructure.
Nano wet milled celecoxib microparticles
81
3.2.2. DSC and XRPD
Following size and morphology studies and as for the starting materials (Table 1)
the microparticles were evaluated by differential scanning calorimetry. In Table 3 (T
= 0), the evaluation of the resulting thermograms (Figure 3C and D) is shown. For
all six formulations, the first thermal event was the glass transition. All determined
values are in proximity with the Tg value reported for the chosen polymer – 52 °C.
This result illustrates no influence of the spray drying process in polymer integrity
since process temperature (45 °C) is lower than Tg. Glass transition temperature of
celecoxib is 53.8 °C, thus overlapping with the polymer Tg making it difficult to assert
the compounds presence by DSC.
3.2.2.1. Drug in solution microparticles
In the formulation of drug in solution microparticles, commercial celecoxib was
completely solubilized in the polymer solution prior to spray drying. The analysis of
the thermograms (Figure 3C) of drug in solution microparticles showed an evident
influence of the increase in drug loading (Table 3). At 10 % drug loading no other
thermal event than glass transition of the polymer was detected. Increasing the
amount of drug in solution had an impact on the solid-state of the polymeric matrix
incorporating drug molecules. For microparticles loaded 25 % and 50 %, at 110 °C,
an exothermic event was observed. This is consistent with a spontaneous
recrystallization of solubilized celecoxib induced by temperatures higher than the
polymer’s glass transition temperature and thus promoted by the consequent
Table 2. Size and loading characterization of all microparticulate systems formulated. Values
shown are mean values (n = 3).
Nominal
drug
loading
(%)
Microparticle sample Size D50
(µm) Span
Drug loading
(% w/w)
Entrapment
efficiency (%)
Process
yield
(%)
10 Drug in solution 21.3 2.9 10 100 35
Nano drug embedded 31.3 3.8 14 95 9
25 Drug in solution 28.8 3.0 22 87 13
Nano drug embedded 32.6 2.2 18 83 7
50 Drug in solution 34.3 3.0 50 100 15
Nano drug embedded 38.3 4.7 51 96 10
Chapter III
82
polymer relaxation, in agreement with values reported in literature [36]. A second
endothermic event consistent with melting of celecoxib was found at 118.5 °C for 25
% DL and 114.0 °C for 50 % DL. The respective enthalpies were 4.8 and 34.3 J/g.
In comparison to commercial celecoxib (Table 1), the observed crystallization
enthalpies were found to be consistent with the contents of drug inside these
microparticles. A normalization of the enthalpies to the corresponding drug amounts
resulted in values 21.7 J/g (25 % DL; 24 % crystallinity) and 68.5 J/g (50 % DL; 74
% crystallinity). The increase in crystallization enthalpy was observed in direct linear
correlation with increasing drug loading percentages (r2 = 0.99). In order to confirm
these results, the same drug in solution microparticles were analyzed by XRPD
(Figure 3A). No distinct diffraction pattern was found, in comparison to polymer
alone. In fact, a superimposition of the amorphous polymer’s background was
observed for all three tested drug loadings. The polymer curve showed an
amorphous broad band centered at 2θ value of 21° and the microparticles at 18°.
Amorphous state was also confirmed by comparison to commercial celecoxib
(Figure 1A), where a specific diffraction pattern would have been observed if the
drug presented in its crystalline form. Considering that this XRPD analysis was
performed at room temperature, these results are in line with those of DSC were
spontaneous drug recrystallization, and consequent crystalline behavior, occurred
only at temperatures above those of the polymer’s and celecoxib glass transition
temperatures (110 vs 52 and 54 °C, respectively). As well, the ability of the spray
drying process to preserve the physical state of drug was confirmed.
3.2.2.2. Nano drug embedded microparticles
The formulation of nano drug embedded microparticles was based on a different
process than that of drug in solution microparticles. In this case, celecoxib was first
wet milled. Then, the nano wet milled celecoxib was suspended in a polymer
solution, immediately prior to spraying. The DSC thermograms of this type of
microparticles were different from particles obtained with a solution of drug (Figure
3C and D). As shown in Table 3, no thermal event was detected at any drug loading.
However, the incorporated nano wet milled celecoxib, though not 100 % crystalline
exhibited a crystallinity of 37 % (Table 1). An effect of drug loading was expected to
be observable, in regards to the consequent increase of crystalline material
embedded inside the microparticles. Though polymer relaxation occurred, drug
Nano wet milled celecoxib microparticles
83
recrystallization and subsequent endothermic event were not visible in the
thermograms, in contrast to the drug in solution microparticles. In this case, the
presence of TPGS potentially acted as a recrystallization inhibitor. After size
reduction, newly formed nano drug particles were partly coated with, partly
entrapping the compound. An XRPD analysis was also performed in these
microparticles (Figure 3B). The resulting diffraction patterns resembled those of
drug in solution microparticles (Figure 3A). The same superimposition of the
amorphous polymer’s background was found for all drug loadings. In this case, the
resulting curves presented as broad bands, similar to those of polymer and other
type of microparticles. Incorporation of nano wet milled drug was confirmed by the
presence of the specific diffraction patterns of zirconium dioxide, also found in the
diffraction pattern of the starting material (Figure 1B). However, the diffraction
pattern of the starting nano wet milled celecoxib was not detected.
Figure 2. SEM micrographs, at t = 0, of formulated microparticles and cross sectional cuts demonstrating their inner core. Drug in solution microparticles (A) and nano drug embedded microparticles (B). Scale bars: 10 µm.
Chapter III
84
* No observable melting event from T0; solid-state solution (10 % loading). ** Nano drug embedded
microparticles: No observable event from T0 to T90. *** Remaining drug loading below 10 % - no observable
peak. Behavior relates to 10 % DL drug in solution microparticles.
Table 3. Differential scanning calorimetry thermograms evaluation for all formulated
microparticles, at different time points. Time points 45 and 90 days were samples taken from the in
vitro release study (Figure 4); these values are further discussed in section 3.3.
Time (days) 0 45 90
MPs sample
Glass
transition
Tg (°C)
Melting Glass
transition
Tg (°C)
Melting Glass
transition
Tg (°C)
Melting
Onset
(°C)
ΔH
(J/g)
Onset
(°C)
ΔH
(J/g)
Onset
(°C)
ΔH
(J/g)
10 % Drug in
solution
50.9 * 49.6 * 48.2 *
25 % 53.4 118.5 4.8 47.6 *** 46.1 ***
50 % 52.7 114.0 34.3 44.1 102.6 4.7 45.9 ***
10 % Nano drug
embedded
51.8
**
50.5
**
50.3
** 25 % 51.6 50.3 50.0
50 % 51.5 49.7 49.5
Figure 3. X-ray powder diffraction patterns of drug in solution microparticles (A) and nano drug embedded microparticles (B), compared to polymer PDLLA. Differential scanning calorimetry thermograms of drug in solution microparticles (solid lines, C) and nano drug embedded microparticles (dashed lines, D) at different drug loadings.
Nano wet milled celecoxib microparticles
85
3.3. In vitro drug release studies
Release of celecoxib from the different formulated microparticles was assessed in
sink conditions. Over 90 days, a quantification of the released drug in the PBS
medium was performed by UHPLC. Results are given in Figure 4 as percentage of
cumulative release normalized to the initial microparticle drug load. In parallel, at
four time points chosen to represent time zero, beginning, mid- and end of the
release study, an amount of particles was removed from the media; freeze-dried
and analyzed using SEM. As previously done in the characterization, the particles
were evaluated as both dry powder form and cross sectional transversal cuts (Figure
5).
3.3.1. Drug in solution microparticles
At lowest drug loading 10 %, a cumulative release of approximately 61 % was
attained after 3 months. Microparticles with higher drug loadings reached 64 and 74
%, respectively for 50 % and 25 % DL (Figure 4, solid lines). At this particular time
point (90 days), the value of cumulative release of these microparticles, 25 % DL, is
statistically different from the other two loadings by approximately 10 %. There was
no difference between the values of loadings 10 % and 50 % at the end of the study.
No apparent burst like effect was observed in the first days of the release study, at
any drug loading. Notwithstanding, at 50 % DL, a plateau in cumulative from 30 days
was detected, in comparison to the other two loadings. In order to further understand
their release mechanism, the resulting curve were fitted using different models: zero
and first order, Higuchi, Hixson and Korsmeyer-Peppas [37]. Until 30 days, before
plateauing, 50 % DL drug in solution microparticles demonstrated the strongest fit
with the Higuchi diffusion model (r2 = 0.98). Both 10 % and 25 % DL profiles, up to
90 days, also fitted with the Higuchi diffusion model (r2 = 0.97 and r2 = 0.98,
respectively). These outcomes indicate that celecoxib loading in the polymer matrix
may be a tunable parameter for controlling drug release. Analyzing the SEM
micrographs (Figure 5A) throughout the release study, it appeared evident the
impact of drug loading in the release of celecoxib from the microparticles. For all
drug loadings, morphological surface changes, tackiness and appearance of
crevices/pores were observed as early as the one-week time point (Figure S8). This
is consistent with water uptake and a plasticization of the polymer matrix. This
impact of water uptake and its effect on the polymer is well known and has been
Chapter III
86
extensively reported in the literature, for polymeric microparticles in similar release
media [38,39].The water uptake was supported by the decrease of Tg over time, with
greater impact at the higher drug loadings (Table 3). Induced changes in the
polymer’s conformation by possible plasticization may have translated into release
of drug by diffusion transport. The 10 % DL presented, at time zero, a Tg of 50.9 °C
that decreased to 48.2 °C after 90 days. Over the same period, the observed
decrease at 25 % DL went from 53.4 to 46.1 °C and from 52.7 to 45.9 °C at 50 %
DL. This suggests that the higher the drug content, the higher the polymer matrix
susceptibility to water uptake. Drug exited the particles facilitated by the erosion
provoked by the mentioned water intake and consequent polymer relaxation.
Indeed, as time progressed, visible inner pores and tears were observed, particularly
from 45 days and at 50 % DL (Figure 5A). At 10 % drug loading, this effect was not
as noticeable. In fact, these microparticles, at 90 days resembled those of 25 and
50 % DL at 45 days. This erosion and consequent drug diffusion may happen at
later stages.
3.3.2. Nano drug embedded microparticles
Nano drug embedded microparticles showed drastically different cumulative release
profiles compared to drug in solution microparticles (Figure 4, dashed lines). After
90 days, a considerably slower release was observed. Additionally, no burst like
effect was found. At 10 % DL a 90 days cumulated drug release of 22.8 % was
reached, 27.8 and 19.2 % for 25 % and 50 % DL, respectively. A small but significant
difference was observed between 25 % DL and the other two drug loadings.
Nonetheless, after 30 days, the release from all three drug loadings remained very
slow, with a possible tendency to a plateau until the end of the study. For this reason,
only the points up to day 30 were considered for curve fitting calculations. All drug
loadings had the strongest fit with the Higuchi model, with similar slopes and r2
(0.038, 0.94; 0.043, 0.92; 0.033, 0.93 for 10, 25 and 50 % respectively). In this case,
no clear impact of drug loading was found. The fact that drug was not solubilized in
the polymer matrix, but embedded, had an influence in the release, suggesting that
its mechanism was drug particle-dependent and not relying on the matrix itself.
Indeed, water uptake was less pronounced in these microparticles. The relatively
constant values of the Tg of polymer over time (Table 3) corroborates these findings.
A decrease of approximately 1 °C was found in all drug loadings. As shown in Figure
Nano wet milled celecoxib microparticles
87
5B and Figure S8, the impact of water uptake in the particles morphology was also
visible in this type of microparticles. Despite visible pores and tears at the later
stages of the release, drug particles remain visible embedded in the polymer matrix.
Celecoxib is a poorly soluble drug (water solubility: 4.3 mg/L [40]) and as previously
mentioned, showed high solubility to the polymer solution (DCM). This, translates in
a higher affinity of the drug to the polymer matrix than to aqueous medium, slowing
down its release from the microparticles. Both size (nm) and the incorporation of
TPGS play a role in the reduced release rate. A similar slowing down of the release
rate upon drug nano wet milling and TPGS stabilization was observed using a p38
MAPK inhibitor [41]. As before mentioned, TPGS acts as a drug stabilizer, facilitating
crystalline formation and preserving this physical conformation thus altering the
effect of aqueous media in the drug particles. To our knowledge, the combination of
TPGS with a biodegradable polymer matrix to modulate and extend the release
kinetics has not yet been reported. Noteworthy, a long-term (6 months) complete
release (100 %) was observed for drug in solution microparticles whereas for nano
drug embedded microparticles only 55 % were attained, confirming the afore
mentioned ability to sustain the release of the later nano drug embedded
microparticles.
The overall slow release observed in both type of microparticles, could be possibly
explained by the mid to high molecular weight - 77kDa - of the polymer, as well as
its ester termination. The high solubility of drug in polymer solution could also play
a role. Celecoxib was incorporated in two different ways into the same polymer
matrix. In the presence of solubilized drug, the polymer may undergo conformation
changes making it more vulnerable to water uptake thus erosion over time that
promoted drug diffusion from the microparticles. This effect was directly correlated
to the increase of drug amounts. When drug particles were simply embedded, the
same effect was not noticeable, illustrating the polymer’s ability to sustain the
release of drug. The extended drug release observed in our microparticles herein
outperforms that of typical CXB microparticles, that release their drug content from
hours [42] up to a few weeks [43].
Chapter III
88
3.4. In vitro anti-inflammatory bioactivity
Different in vitro cellular assays were performed, in order to assess both the safety
and efficacy of the formulated drug eluting microparticulate systems. Cytotoxicity of
commercial celecoxib and both drug in solution and nano drug embedded
microparticles at three drug-equivalent concentrations (1, 10 and 30 µM) was
assessed using a WST-1 assay (Figure 6A). The primary human fibroblast-like
synoviocytes isolated from 3 donors were incubated with a suspension of
microparticles or drug directly diluted in culture medium for 24h. No cytotoxicity was
Figure 4. Celecoxib cumulative in vitro release from all formulated microparticles over 3 months. Solid lines: drug in solution microparticles; dashed lines: embedded nano drug microparticles. Symbols correspond to mean values ± S.D.; n = 3.
Figure 5. SEM micrographs of cross sectional cuts of all formulated microparticles. During the in vitro release study, microparticles were freeze-dried and cut at different time points. Drug in solution microparticles (A) and nano drug embedded microparticles (B). Scale bars: 10 µm.
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observed when compared to dead (DMSO) and alive (culture medium) controls. All
cellular viability values were > 96 %. Since no cytotoxicity was found, the three
products were tested at the same concentrations for their anti-inflammatory
bioactivity (Figure 6B). Prostaglandin E2 release was measured in the resulting
supernatants by ELISA. This prostaglandin was selected as the protein of interest
due to it being a direct product of COX-2 stimulation. Celecoxib, a specific COX-2
inhibitor, will suppress the release of this prostaglandin by blocking COX-2
expression [20,44]. Commercial celecoxib was effective in suppressing PGE2
release at all tested concentrations. Inhibitions of PGE2 release of 88, 89 and 92 %
(ns; > 0.999) were detected at 1, 10 and 30 µM, respectively (Figure S9). This
complete inhibition at all concentrations is consistent with IC50 values reported for
other human cell line, below the µM range [45]. Both drug in solution and nano drug
embedded microparticles were not effective in suppressing prostaglandin release at
1 µM (Figure 6B, Figure S9). Conversely, at 10 and 30 µM a dose response effect
was found. A 66 % statistically significant inhibition at 10 µM was calculated for both
type of particles. At 30 µM, drug in solution microparticles significantly inhibited 86
% of the PGE2 release and nano drug embedded microparticles 83 %. At this drug-
equivalent concentration, both types of microparticulate systems performed equally
to commercial drug alone in fully suppressing PGE2 release. An analysis of the in
vitro drug release study from the tested microparticles (Figure 4) might explain the
observed dose response effect. At 24 h, in the release medium, drug in solution and
nano drug embedded microparticles released 10.9 ± 0.5 % and 7.3 ± 2 % of total
amount of celecoxib, respectively. Therefore, no effect at a drug-equivalent
concentration of 1 µM is expected. Correspondingly, at 10 µM, the full amount of
drug is attained thus the effect of drug eluted from the microparticles should mimic
that of commercial celecoxib at this concentration. Indeed the inhibition of PGE2
release increased by 62 %. Nevertheless, no true correlation between the two
results is possible due to the differences of the PBS + surfactant release medium
and the complete culture medium, which may explain that a significant matching of
the effects of drug alone, at 24 h, is only observed (Figure 6) at a 3-fold concentration
increase (30 µM). This significantly comparable performance between the two types
of microparticles validates their drug loading (10 %) despite the different formulation
processes. Additionally, it confirms the release of therapeutic concentrations of
celecoxib from both types of systems, at very low amounts of microparticles in
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suspension. Using these particles loaded at 10 %, in order to achieve 30 µM of drug-
equivalent concentration, a suspension of 110 µg/mL of microparticles was used. In
this case, the formulation would be performant at 0.011 % (w/v) of microparticles in
suspension. As per the literature [46,47], in a clinical setting, this is an acceptable –
even low - concentration of solid components of a formulation knee IA injection. At
higher drug loadings (25 and 50 %), this amount would further decrease, thus
augmenting the anti-inflammatory effect at low amounts of the proposed drug
delivery systems.
4. CONCLUSIONS
Local intra-articular administration of molecules is proving to be an answer to deliver
effective therapeutic doses to the joint space reducing/avoiding issues with systemic
adverse effects of said drug molecules. In this study, we have developed
microparticulate drug delivery systems encapsulating celecoxib into biocompatible
polymer by two different approaches – drug in solution and embedded nano-milled
Figure 6. In vitro cellular viability (WST- 1) of primary HFLS incubated with commercial celecoxib, drug in solution and nano drug embedded microparticles (10% DL) for 24 h. Vehicle – 0.1% DMSO (A). Release of PGE2 by IL- 1 β activated primary HFLS confirmed anti-inflammatory activity of commercial celecoxib and drug eluted from drug in solution and nano drug embedded microparticles (10% DL) (B). Bars correspond to mean values ± s.d.; n = 6; ****p < 0.0001.
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drug. An optimized spray drying process allowed for high drug loadings that did not
compromise the toxicity of both type of microparticles when in an in vitro culture with
primary human fibroblast-like synoviocytes. As so, and in the same cellular system,
effective anti-inflammatory bioactivity of eluted drug from 10 % DL microparticles
was proven by the suppression of PGE2 release. This efficacy at the lowest drug
loading tested, allows for minimal amounts of dry solid material in final injectable
formulations. Both microparticulate systems showed an extended controlled release
of celecoxib over three months. The embedding of nano wet milled drug
considerably slowed down the release of drug. As a potential OA therapeutic poorly
soluble drug delivery system, the described microparticles allow for a tailoring of
drug release profile by fine-tuning both drug loading percentages and type of
incorporation into the polymeric matrix.
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Supplementary material
Figure S1. Differential scanning calorimetry analysis of commercial celecoxib.
Figure S2. Differential scanning calorimetry analysis of amorphous celecoxib.
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Figure S3. Differential scanning calorimetry analysis of polymer Resomer 205 S.
Figure S4. Differential scanning calorimetry analysis of the physical mixture of celecoxib (50 % DL) and polymer Resomer 205 S.
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Figure S5. Rietveld plot of commercial celecoxib.
Figure S6. Rietveld plot of celecoxib recrystallized in acetone.
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Figure S7. XRPD patterns of amorphous celecoxib at different temperatures. Heating from room temperature until 200 °C followed by cooling back to room temperature.
Figure S8. SEM micrographs for surface morphology assessments. Dry powder of the sectioned (Figure 5) drug in solution microparticles (A) and nano drug embedded microparticles (B). Each time point corresponds to a sample taken from the in vitro release study (Figure 4); these results are further discussed in section 3.3.1. and 3.3.2.. Scale bar: 10 µm.
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Figure S9. Inhibition percentage of PGE2 release normalized to 100 % activation, as a function of celecoxib concentration. Bars correspond to mean values ± S.D.; n = 6.
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Chapter IV
Sustained release carriers for osteoarthritis: in vitro
evaluation of an anti-inflammatory drug and a chondrogenic
drug on a 3D chondrocyte OA model
Carlota Salgado a,b, Olivier Jordan a,b and Eric Allémann a,b a School of Pharmaceutical Sciences, University of Geneva, 1 Rue Michel-Servet 1211 Geneva, Switzerland b Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, 1 Rue Michel-Servet 1211 Geneva, Switzerland
ABSTRACT
Osteoarthritis (OA), the most common form of arthritis, causes the narrowing of the joint space and it is considered the number one cause of disability worldwide in the elder population. With no known cure, local administration of drug compounds by intra-articular (IA) injection represents an alternative to conventional oral OA treatment. In previous studies, the incorporation of nano drug particles in polymeric microparticles has proven to allow a sustained controlled release of therapeutic drug compounds for IA delivery. This chapter, divided in two sections, aimed at characterizing and comparing the incorporation as nano wet milled particles of two drug compounds – celecoxib and kartogenin. Secondly, it aimed at assessing the potential of an in vitro 3D chondrocyte OA model in the evaluation of polymeric microparticulate carriers incorporating these compounds. Both drug compounds were wet milled and incorporated with comparable results, despite different in vitro drug release profiles. Human chondrocyte pellets were successfully cultured and established as an accurate in vitro model. However, due to the sustained release nature of the evaluated carriers, clear optimization steps are imperative to fully characterize the bioactivity of the eluted drug compounds over long periods of time. Keywords: osteoarthritis; intra-articular drug delivery systems; cartilage; celecoxib; kartogenin; in vitro 3D model
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INTRODUCTION
Osteoarthritis (OA) is a chronic disease that affects whole joints. Considered the
most common form of arthritis, it is the number one cause of disability worldwide for
people above 65 years old. Often described as a “wear and tear” disease, OA
causes the narrowing of the joint space due to chronic inflammation of the synovium
and synovial fluid, leading to cartilage degradation of articular joints. In more
advanced stages, it leads to structural changes and damage like subchondral bone
remodeling and erosion with formation of osteophytes and overall loss of joint
function. Clinically, it is generally associated with pain, inflammation, and loss in
amplitude of movement [1–3]. Current treatment approaches are predominantly
based on symptom management - acetaminophen and nonsteroidal anti-
inflammatory drugs (NSAIDs) are common treatment options for pain and
inflammation – rely on several non-pharmacological measures (physical therapy)
and often lead to joint replacement surgeries. Yet, due to the complex and
multifactorial nature of OA, there is a great need for disease modifying therapies
that effectively slow disease progression [4,5]. However, the closed features of the
joint structure result in low distribution of systemic drugs thus a suboptimal efficacy
of treatment by this delivery route. Local administration, by intra-articular injection
(IA), represents an alternative to the shortcomings of oral drug administration. Not
only this is an interesting approach in terms of circumventing systemic adverse
effects commonly associated to pain and inflammation treatment options, but due to
the fact that it can be directly administered to diseased joints, IA can provide a more
sustained and prolonged delivery of drug compounds [6–8]. In this field, multiple
types of formulations like polymeric nano and microparticles, liposomes, micelles
and hydrogels, have been explored in order to answer to the different critical IA
predicaments [9]. Once in the joint, different structures and tissues can represent
the targets of these drug delivery systems. The two main components of the articular
joint are the synovium and the cartilage itself. The first is associated to the
inflammatory component of OA, and is formed by synoviocytes and different lymph
and blood vessels that produce the synovial fluid, which irrigates cartilage. This is a
connective tissue formed by extracellular matrix and chondrocytes, responsible for
absorbing friction and shock between bones during movement [10,11]. In order to
develop safer and more efficacious IA treatment options for OA, adequate in vitro,
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105
ex vivo and in vivo OA models are required. Established preclinical models
encompass mouse, rat, rabbit and larger animals such as dogs and horses [12].
Different in vitro established OA models mimic partial joint structures (ex.: bovine
cartilage explants and monolayer cultured synoviocytes) making it challenging to
assess effects of IA drug delivery systems in the joint, as a whole. Currently, main
options for in vitro testing of OA include 2D and 3D cellular cultures. 2D monolayer
cultures of synoviocytes are applied to evaluate cytokine release and synovium
characterization; co-culturing different cells of different articular tissues such as
synoviocytes, chondrocytes and osteoblast is helpful in understanding crosstalk
between different tissues in response to therapies. Explants (mostly bovine and
equine) of whole joints or articular cartilage are helpful in permeation and drug
delivery studies. Cellular 3D cultures maintain cellular phenotype and cell to cell
interactions, preserving tissue connectors. As so, these are considered more
accurate and actual cell models of articular cartilage in OA than monolayer cultures
[13–15]. In this chapter, a scaffold free 3D human chondrocyte model (pellets) was
developed and applied to evaluate the anti-inflammatory and/or chondroprotective
effects of polymeric (poly-lactic acid) microparticulate drug delivery systems. The
potential of this model in assessing combination of drugs and/or different aspects of
OA is also discussed. Two different drug compounds were embedded in the
microparticles. The first drug compound, celecoxib (CXB), is a NSAID COX-2
inhibitor that to our knowledge is firstly tested here using the 3D human chondrocyte
pellet model [16]. Second drug compound, is the small molecule kartogenin (KGN),
a drug linked to articular cartilage regeneration by activation of collagen type II,
aggrecan and TIMPs. The increased expression of these important proteins stems
from the CBFβ-RUNX1 complex – formed by the translocation of CBFβ to the
nucleus, once freed from filamin A by kartogenin [17,18]. Kartogenin has been
extensively tested with positive outcomes in 3D cellular cultures using human
mesenchymal stem cells pellets but no accounts have been found describing effects
in human articular chondrocyte pellets [19].
This chapter comprises two parts: 1 – that elaborates on the spray dried polymeric
microparticles with embedded nano wet milled celecoxib and kartogenin; and 2 –
where a 3D human chondrocyte model is described and the effects of the two types
of microparticles are discussed.
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1. NANO WET MILLING AND SPRAY DRIED POLYMERIC
MICROPARTICLES
This first part describes the development of spray dried polymeric microparticles
loaded with nano wet milled celecoxib and kartogenin. Characterization and in vitro
drug release are explaining and a comparison between the two types of formulated
microparticles is provided.
1.1. Materials and methods
1.1.1. Materials
The base polymer Poly (D,L-Lactic Acid) (Resomer 205 S®) was purchased from
Evonik (Essen, Germany). Celecoxib was acquired from LC Laboratories® (Woburn,
MA, USA) and Kartogenin from Merck (Buchs, Switzerland). Analytical grade
working solvents dichloromethane, acetone, tetrahydrofuran, trifluoroacetic acid and
acetonitrile were all purchased from Sigma-Aldrich (St. Louis, MO, USA), as well as
all other chemical products. In all experimental setups, Milli-Q® water was employed
(Merck, Burlington, MA, USA).
1.1.2. Nano wet milling
In a first step, both commercial drug compounds were recrystallized using a non-
solvent precipitation method described in detail in Chapter 3 [20]. Kartogenin was
processed in a similar method, based on previous works in this lab [21]. For that, 5
mg of drug were dissolved in 300 µL of organic solvent (CXB: acetone; KGN:
tetrahydrofuran) in a 2 mL tube. Using a 1:2 solvent/water volume ratio, water was
added dropwise (600 µL). Open tubes were left overnight at 4 °C, for the evaporation
of organic solvent. Immediately before wet milling, 100 µL of D-α-tocopherol
polyethylene glycol 1000 succinate (TPGS) aqueous solution (CXB: 2 % w/v; KGN:
4 % w/v) were added to the 2 mL tube. Then, 580 mg of BeadBug™ 0.5 mm
zirconium beads from Sigma-Aldrich (St. Louis, MO, USA) were added. Wet milling
was carried out using the Precellys 24® homogenizer from Bertin Instruments
(Montigny-le-Bretonneux, France). For CXB: two times five cycles of 90 s at 10000
rpm were completed, followed by a 5 min ice bath. At these time and speed settings,
only 5 cycle repetitions are allowed. An ice bath was added in order to control
temperature increase inside the tubes. This was succeeded by 10 cycles of 60 s at
In vitro evaluation of sustained release carriers for OA
107
6600 rpm and another 5 min ice bath. For KGN: two times five cycles of 90 s at
10000 rpm were performed, with 5 min ice baths in between cycles. Then, zirconium
beads were removed and nano drug suspensions were washed three times with
water, and separated by centrifugation (10000 x g; 15 min; 4 °C). Resulting
suspensions were frozen at -80 °C, then freeze-dried overnight and stored at 4 °C
for further analysis and incorporation in polymeric microparticles.
1.1.3. Spray drying
Polymeric microparticles were formulated using a 4M8-TriX spray dryer (ProCepT,
Zelzate, Belgium). Settings for spray drying were optimized in a previous study [20]
and included: 7 % w/v polymer concentration; 5 mL/min feed rate; 0.6 mm (internal
diameter) bi-fluid nozzle; 6 L/min atomizing air force; air as the processing gas; 45
°C inlet temperature; 34 °C outlet temperature; medium cyclone and 0.3 m3/min inlet
air flow. Dichloromethane was selected as solvent for polymer solution. Immediately
before spray drying, nano wet milled drug powder amounts corresponding to 10 %
nominal drug loading (weight percentage of total dry content) were dispersed into
the polymer solution, in an ice bath. Collected dry products were further
characterized and stored at 4 °C.
1.1.4. Characterization of drug products and polymeric microparticles
1.1.4.1. Size and distribution
Particle size determination of nano milled drug products was performed by dynamic
light scattering (DLS) using a Nanosizer. Laser light diffraction was used to
determine size and distribution of formulated microparticles in a Mastersizer S. Both
equipment from Malvern Instruments Ltd (Malvern Pananalytical, Malvern,
England). All measurements were taken in triplicate.
1.1.4.2. Surface, morphology and drug incorporation
Morphology and surface analysis were analyzed by optical microscopy with a Carl
Zeiss Axio microscope (Oberkochen, Germany) and scanning electron microscopy
(SEM) using a Jeol JSM-7001TA microscope (Tokyo, Japan). In this technique, dry
samples were deposited onto carbon tape covered metal studs. Studs were allowed
to dry overnight under vacuum and were sputter coated with gold, prior to analysis
at a 5 kV voltage. To observe drug incorporation into the microparticles, a small
amount of particle powder was transversally cross-sectioned using a CryoStar NX70
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microtome (Thermo Fisher Scientific; Waltham, MA, United States). For this,
samples were prepared by dispersing a small amount of powder into a drop of OCT
embedding matrix for frozen sections (CellPath, Newtown, England) onto a metal
stub. Mounted samples were frozen at - 80 °C for 24 h. Before cutting, all stubs were
allowed to acclimate for 30 min in the cooling chamber of the apparatus, at - 20 °C.
All cuts were directly mounted onto silicon wafers and analyzed by SEM using the
same procedure above described.
1.1.4.3. Drug loading and encapsulation efficiency
Ultra-high performance liquid chromatography (UHPLC) was applied to quantify
drug content of all formulated microparticles. Analysis were performed in a Waters
Acquity™ Ultraperformance LC (Milford, MA, USA) equipped with an Acquity UPLC®
BEH C18 column (2.1 mm x 50 mm, 1.7 µm, Waters, USA) equilibrated at 40 °C.
The selected mobile phase: acetonitrile and water with added 0.1 % formic acid
(FA), was run in a 2 min gradient at 0.5 mL/min constant flow rate. Detection by a
PDA detector was set at 254 nm for samples containing CXB and 280 nm for
samples containing KGN. The volume of injected sample was 2 µL. All samples
were analyzed in triplicate. Amount of encapsulated drug (drug loading, DL %) was
determined by a ratio between the mass of drug determined in the microparticles
and the total mass of particles, transformed into percentage. Encapsulation
efficiency (EE %) was calculated by dividing calculated DL % values by the
theoretical drug loading, in this case 10 %.
1.1.5. In vitro drug release
In vitro drug release studies were carried over 2 months under sink conditions, in
triplicates.
1.1.5.1. Celecoxib (CXB)
Prior to establishing the release assay, the solubility of celecoxib was determined to
be 80 mg/L in the release medium - phosphate buffer saline (PBS) containing 0.5 %
w/v of polysorbate 80 (Tween 80®) - to ensure solubility in sink conditions - and
0.025 % w/v of sodium azide – to preserve medium over extended periods of time -
at pH 7.4. A weighed amount of microparticles with equivalent CXB amounts based
on drug loading (10 %) was dispersed in 50 mL of medium. At allotted time points,
1 mL was withdrawn and replaced with fresh medium.
In vitro evaluation of sustained release carriers for OA
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1.1.5.2. Kartogenin (KGN)
Solubility of kartogenin (850 mg/L) was determined in the release medium -
phosphate buffer saline (PBS) at pH 7.4 containing 0.1 % w/v of sodium dodecyl
sulfate (SDS) by a saturated solution. SDS was found to be a more suitable
surfactant to ensure sink conditions of KGN throughout the study. As for celecoxib,
an amount of microparticles with equivalent KGN loading based on DL 10 %, was
dispersed in 2 mL of the buffer. At each time point, total volume was removed,
centrifuged prior to analysis and replaced by fresh conditioned medium.
In an orbital shaker (80 rpm), both assays were performed at 37 °C, in closed, amber
vials to avoid evaporation of water and to protect samples from light degradation.
Once collected at each given time point, the different aliquots were centrifuged
(10000 x g; 15 min; 4 °C) in order to ensure no polymeric particles were present.
Quantification of released drug from microparticulate systems was carried out by
UHPLC detection, using the methods above described.
1.2. Results and discussion
1.2.1. Nano wet milling
Prior to wet milling, both CXB and KGN were recrystallized by non-solvent
precipitation to ensure quality and homogeneity. Recrystallization of commercial
celecoxib in acetone resulted in large (approx. 30 µm) brittle, clear crystals, with an
acicular habit (Figure 1A). Once wet milled, these crystals were reduced to 593 nm
and gained a rod like appearance (Figure 1a2; Table 1). Commercial kartogenin
crystals had a polyhedric habit, as a thin white powder. Once wet milled, although
the shape resembled the celecoxib rod-shaped particles (Figure 1B), kartogenin had
lower mean size (350 nm) (Table 1). The wet milling processes were adapted to the
recrystallized drug products. Process optimization was performed based on crystal
appearance and general physical chemical properties of the drug compounds, such
as melting point. Precipitation solvent, TPGS concentration, temperature, time and
speed settings of tissue homogenizer were all adjusted accordingly. Due to the fact
that the recrystallized CXB presented as brittle large crystals, a 2 % (w/v) TPGS
solution was sufficient to stabilize crystal properties [22,23] thus size of the drug
particles considerably decreased by wet milling. However, with a low melting point
at 160 °C, it was important to control the temperature of the wet milling process,
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hence why ice bath dippings were introduced between cycles. Due to the lower
amount of TPGS (for KGN a 4 % (w/v) was used), which acts as a crystal stabilizer,
an extra round of 10 cycles at lower speed (6600 rpm) was added to the two main
rounds of long and fast cycles (10000 rpm). For KGN, that recrystallized in THF as
hard, white crystals, the higher melting temperature (272 °C) and the higher
concentration of TPGS solution (4 % w/v) facilitated the wet milling. In this case,
only two rounds of the high energy/high speed cycles were required to decrease
drug particles size to the nano range. Considering that individual drug properties
(solubility, melting point, and shape) are taken into account when optimizing the wet
milling process, using a tissue homogenizer for wet milling of powder products can
be widely applied to different drug compounds. As previously reported by our group
[20], despite size reduction, no changes were observed in other drug properties like
crystallinity and polymorphism. Additionally, it has convenient advantages such as
controlled speed (4000 - 10000 rpm), temperature (possibility to incorporate an ice
cooler or cool the rack externally) and duration (cycles from 5 to 90 s). The volume
of wet milled material can also be adjusted if different tubes and tube holders are
selected, our equipment allows for 24x 2 mL tubes yielding approximately 100 mg
of milled drug product in roughly 30 min of process. When compared to techniques
developed previously in our lab namely, vortexing [21,24], and other techniques
described in the literature (for CXB) like grinding mills, dispersing and high pressure
homogenizers [25,26], our method shows comparable results in less steps, with an
easier and considerably faster and cost-effective process. These advantages make
for high reproducibility between batches (Table 1) and thus, an ideal technique for
scale-up in drug product manufacturing using wet media mills, cooling settings and
zirconium beads [27].
In vitro evaluation of sustained release carriers for OA
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Table 1. Properties of nano wet milled drug products and spray dried microparticles.
Experiments were carried out in triplicates.
Characterization
Drug compound
CXB KGN
Nano wet milled drug Size Z-average [nm] 593 ± 120 350 ± 89
PDI < 0.39
Microparticles 10 % DL
Size D50 [µm] 31.3 ± 12 30.6 ± 14.2
Span 3.8 2.2
Drug loading [% w/w] 12 ± 10 8.3 ± 7.2
Entrapment efficiency [%] 95 ± 5 83 ± 6
Cumulative drug release at 2 months [%] 32.8 ± 0.95 67.2 ± 0.13
Figure 1. Scanning electron micrographs of commercial celecoxib (a1), kartogenin (b1) and the corresponding wet milled drug products (a2 and b2). Scale bars: 10 µm (a1) and 5 µm (a2, b1 and b2).
Chapter IV
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1.2.2. Polymeric microparticles: Celecoxib and Kartogenin
1.2.2.1. Characterization
Microparticles incorporating nano milled CXB have been extensively characterized
in Chapter 3 [20]. At 10 % drug loading, these microparticles presented as spherical
with a porous, rough outer surface when compared to unloaded microparticles (Fig.
2 a1,b1), that present a smooth surface. This difference can be linked to the
presence of drug particles in suspension, at the moment of spray drying that
changes the physical properties of the feed solution. Incorporation of drug was
confirmed visually by the presence of embedded nano drug clusters at the outer
areas of the polymer matrix (Figure 2b2). The polymer matrix of the unloaded
microparticles was porous and homogeneous, as can be observed in Fig. 2b1 in the
cross sectional cut micrograph. Drug loading was confirmed by UPLC with
entrapment efficiency of 95 % (Table 1). Polydispersity was as well observed in
these microparticles, sized 31.3 µm (Table 1). Similarly, microparticles incorporating
nano milled KGN presented as spherical with mean size around 30 µm and a slightly
porous outer surface (Table 1; Figure 2a3). The nano milled drug incorporation was
confirmed by UHPLC at 8.3 %, with EE % comparable to those of CXB
microparticles (Table 1). Additionally, embedded nano milled kartogenin was visible
(Figure 2b3). The distribution of drug particles in these microparticles was different
than those of embedded CXB where clear clusters with tendency to outer layers
were observed. In the KGN microparticles, no agglomeration tendency was
observable, with nano drug particles distributed throughout the polymer matrix. This
could be linked to the different solubility of the two drugs in the polymer solution (7
% w/v PDLLA in dichloromethane) that has an impact in droplet drying process.
Favorable drug precipitation or polymer drying will translate in different distribution
of nano drug particles within the microparticles. The nano size of the drug particles
is another potential factor influencing the distribution inside the dried microparticles.
As mentioned before, nano wet celecoxib has a difference of approximately 200 nm
in size from nano wet milled kartogenin (Table 1). The greater size of the nano drug
particles could have an impact on the dispersion process (magnetic stirring) when
suspending the particles prior to spray drying. Nonetheless, the optimized spray
drying process is once again validated by the fact that comparable microparticles -
In vitro evaluation of sustained release carriers for OA
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in terms of size, drug incorporation and morphology – were formulated incorporating
two different drug products.
1.2.2.2. In vitro drug release
Drug release from both formulated 10 % DL microparticles was assessed in sink
conditions, in two different PBS-based media. The cumulative release from
embedded nano wet milled celecoxib has been previously described in Chapter 3,
here presented in Fig. 3, are additional time points within the 2 months’ time frame
that match those withdrawn for the KGN microparticles. CXB microparticles with 10
% drug loading showed a slow release with no burst like effect and a steady plateau
release from 30 days. At 2 months, 33 % of celecoxib was released from these
Figure 2. SEM micrographs of unloaded and drug incorporating microparticles. Microparticles with 10 % drug loading of nano wet milled CXB, KGN or unloaded (A) and cross sectional cuts (B). Scale bars: 10 µm.
Chapter IV
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microparticles. At a later stage (data not shown), this cumulative percentage doubles
and 66 % of drug were released after 6 months. This slow and steady release was
expected and explained by the incorporation of drug particles as a suspension
(embedded), were drug solubility in the aqueous media is dependent on the drug
particle alone, and their reduced, TPGS stabilized nano size. Additionally, it was
previously demonstrated that celecoxib has high solubility in the polymer solution
[20] making the fact that the drug particles are suspended and not dissolved in the
polymer matrix, a plus in slowing down release. The same phenomena was not
observed regarding the KGN microparticles. In Figure 3, a burst release during the
first seven days can be observed where kartogenin release reaches 66 % of
cumulative release. From this point onwards, the drug release steadies and a
plateau is reached at 67 % until 2 months. Presently in the literature, there are no
extensive reports of polymeric microparticles incorporating kartogenin apart from the
ones previously developed in our laboratory [21] and kartogenin-chitosan
conjugated MPs [28]. If comparing the formulated 10 % DL kartogenin microparticles
to these systems, different hypothesis could explain this disparity in outcomes. In
the first case, the microparticles were also formulated using an ester terminated
PLA, albeit with a lower molecular weight. However, these microparticles were spray
dried using a less concentrated polymer solution in dichloromethane. Spraying of a
different concentration of polymer in an organic solvent will inherently change the
feed solution properties in terms of viscosity, which consequently alters the final dry
product in terms of drug incorporation, yield and size. Process inlet temperatures
were also different (80 °C vs 45 °C) giving different outcomes of evaporation from
sprayed droplets and shrinkage of polymer matrix. Lastly, these microparticles were
loaded at approximately 30 %, fact that could also influence drug release. In our
study, different increasing drug loadings would need to be explored in order to
comment on an effect of drug loading, drug incorporation and drug release. A
parallel between our formulated microparticles and the chitosan conjugated ones is
challenging given the fact that these were not formulated by spray drying, drug was
not incorporated as a suspension and the matrix polymer is entirely different.
Nonetheless, we can acknowledge that incorporation of commercial kartogenin with
chitosan results in a slower drug release (55 % after 50 days). Fundamentally, it is
important to adjust the polymeric drug delivery system to its desired outcome in
terms of drug delivery. In this case, it will mainly depend on the drug compounds
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activity. In OA both a fast and a slow therapeutic onset of anti-inflammatory drugs
(celecoxib) are important, whereas a slow, steady and prolonged therapeutic
window is ideal for disease modifying chondroprotective/chondrogenic drugs (such
as kartogenin) where cartilage regeneration can take up to months.
Figure 3. Cumulative in vitro drug release from both formulated microparticles over 2 months. CXB microparticles: squares, green line; KGN microparticles: circles, blue line. Symbols correspond to mean values ± S.D.; n = 3.
Chapter IV
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2. 3D IN VITRO MODEL OF OA: CHONDROCYTE PELLETS
The second part of this chapter focuses on the optimization of a 3D human
chondrocyte pellet model of OA and its role in the screening for anti-inflammatory
and chondroprotective effects of the spray dried microparticulate systems
incorporating CXB and KGN, described in the first part of the chapter.
2.1. Materials and Methods
2.1.1. Materials
For the cellular in vitro assays, base DMEM culture medium (#10938-025), HEPES
buffer, sodium pyruvate, DPBS-, trypsin (0.25 %) and penicillin-streptomycin
(10,000 U/mL) were purchased from Invitrogen (Gibco Life Technologies, Carlsbad,
CA, USA). Fetal bovine serum was purchased from Eurobio (Les Ulis, France) and
collagenase type II from Bioconcept (Allschwil, Switzerland). L-ascorbic acid 2
phosphate, human serum albumin, dexamethasone, ITS+ (insulin, transferrin and
selenic acid), 1,9-dimethylmethylene blue chloride, chondroitin-4-sulfate, 4′,6-
diamidino-2-phenylindole dihydrochloride and calf thymus DNA were acquired from
Sigma-Aldrich (St. Louis, MO, USA). Interleukin 1 beta (IL-1β) and prostaglandin
PGE2 quantification assay were purchased from R&D Systems (Bio-Techne,
Abingdon, UK), as well as all growth factors: transforming growth factor beta-1
(TGFβ-1), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB
(PDGF-BB). The DNA/RNA extraction kit Nucleospin™ Triprep was purchased from
Macherey-Nagel (Düren, Germany) and SuperScript™ II Reverse Transcriptase
from Invitrogen (Carlsbad, CA, USA).
2.1.2. Human articular chondrocytes
Methods of isolation, monolayer and 3D culture of human articular chondrocytes
were adapted from the literature [29,30]. All experiments were conducted under
sterile environment.
2.1.2.1. Isolation and monolayer expansion
Following a protocol approved by the local Ethics Committee (CCER, Geneva,
Switzerland, authorization number 2017-02234) with informed and consenting
patients, knee cartilage was collected from the femoral and tibial condyles of three
male adults (aged between 65 and 72 years old) with clinical osteoarthritis (OA) at
In vitro evaluation of sustained release carriers for OA
117
the time of knee replacement surgery. After collection, pieces of cartilage were
scraped and minced into small pieces using scalpel blades. Then, the cartilage was
transferred to a T75 culture flask where it was digested in a type II collagenase
solution (1 mL/100 mg sample) for 24 h in an orbital shaker (37 °C, 5 % CO2
incubation). After, the solution was filtered (100 µm) and washed twice by
centrifugation (200 x g/4 min) with 20 mL PBS- with 5 % FCS. Resulting pellet was
suspended in 10 mL of complete medium: DMEM, 1 % penicillin-streptomycin (PS),
10 % FCS, 10mM HEPES buffer and 1mM sodium pyruvate. Cells were counted
and frozen at passage 0 in 10 % dimethyl sulfoxide (DMSO). For monolayer
expansion, once removed the freezing medium, cells were seeded at 150000
cells/mL in T75 culture flasks (15 mL) in growth factor medium (complete medium
with 1ng/mL, 5ng/mL and 10 ng/mL of TGFβ-1, FGF-2 and PDGF-BB, respectively)
at 37 °C/5 % CO2. Cells were expanded to 90 % confluence with twice/week media
changes and passaged at 37500 cells/mL. No assays were performed in monolayer
culture as it was exclusively performed for cellular expansion; pellets were cultured
with cells at passage 2 and 3.
2.1.2.2. 3D pellet chondrocyte culture
Using 96 V-bottom well plates, passage 2 or 3 chondrocytes were seeded at 250000
cells/well (100 µL) in chondrogenic medium (DMEM, 10 mM HEPES buffer, 1mM
sodium pyruvate, 1 % PS, 1 % ITS+, 0.1 nM L-ascorbic acid 2-phosphate, 1.25
mg/mL human serum albumin, 10-7 M dexamethasone and 10 ng/mL TGFβ-1). In
order to form pellets, the plates were centrifuged at 400 x g for 5 min and then
incubated for 2 weeks at 37°C/ 5% CO2, changing medium twice/week.
2.1.3. Cytotoxicity assay
Cytotoxicity of tested conditions was assessed, prior to biochemical and gene
expression analysis. Grown pellets were incubated for 8 days with supernatants
containing 3 concentrations of the two drug compounds - CXB and KGN - and the
corresponding 10 % DL microparticles (Table 3). IL-1β (1 ng/mL) was added to all
wells in order to induce the inflammatory environment of OA. DMSO was used as
dead control, media alone as negative control and pellets were also incubated with
vehicle (medium with 0.01% DMSO). All conditions were tested in duplicates. After
8 days, supernatants were removed and stored at - 80 °C and remaining pellets
were incubated with cell proliferation reagent WST-1 (Roche, Basel, Switzerland),
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according to the provider instructions. Experiments were conducted on the 3 donor
samples twice (n = 6) using pellets grown from two passages (3 and 4).
2.1.4. Quantification of glycosaminoglycans (GAG)
Pellets (n = 2) were digested in 500 µL of 1 mg/mL proteinase K/Tris-EDTA buffer
solution (50 and 1 mM, respectively) for 16 h, at 56 °C. Sulfated glycosaminoglycan
(GAG) quantification was performed by 1,9-dimethylmethylene blue (DMMB), a
thiazine chromotrope that induces metachromasia when bonded with GAGs [31].
Briefly, in a 24-well plate, 40 µL of sample or standard (chondroitin-4-sulfate) were
mixed with 1 mL of dye solution (35 µM of 1,9-dimethylmethylene blue chloride in a
pH 3.5 sodium formate buffer) and absorbance was read at 535 nm. Concentration
of GAG was determined from a standard curve (µg/mL). From the same digested
sample (n=2), DNA quantification was performed using a 4′,6-diamidino-2-
phenylindole dihydrochloride (DAPI) assay [32]. Using a 96-well plate, the
fluorescence of 200 µL of DAPI buffer (10 mM Tris, 10 mM EDTA, 100 mM NaCl
and 100 ng/mL DAPI at pH 7) was read at 360 nm excitation and 450 nm of
emission. To each appropriate well, 1 µL of sample or standard (calf thymus DNA)
was added, a total of 5 times, with fluorescence readings in between. From the
consequent increased fluorescence values, a fluorescence curve was created for
every sample. From these curves, the slope was retrieved and a ratio between the
standard slopes was calculated. Concentration of DNA for each sample were
determined by multiplying the resulting ratio by the standard’s concentration (20
µg/mL). Lastly, a ratio was calculated between GAG (µg) and DNA (µg) values
(GAG/DNA). Experiments were conducted twice per donor (n =6).
2.1.5. Gene expression analysis
RNA was extracted from pellets (n = 2) using an extraction kit (Nucleospin™
Triprep), following the recommended protocol. Pellets were vigorously disrupted in
the lysis buffer using a syringe equipped with a 21G needle, tris(2-carboxyethyl)
phosphine hydrochloride (TCEP) was added as a disulfide bonds reducing agent.
Reverse transcription was accomplished using a SuperScript™ II kit. Quantitative
PCR (qPCR) was performed using SYBR Green Power UP master mix and the
QuantStudio6Flex instrument and software (Applied Biosystems, Foster City, CA,
USA). Analyzed gene sequences can be found in Table 2. The relative gene
expression was calculated by the 2-ΔΔCT quantification method with GAPDH as
In vitro evaluation of sustained release carriers for OA
119
control (housekeeping gene). Experiments were conducted once per donor (n =3),
by pooling both passages.
GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; COL2A1: collagen type II ; ACAN: aggrecan; COX2: cyclooxygenase-2
2.1.6. Anti-inflammatory activity assay
Collected supernatants (pooled per tested condition, per passage) from pellet
incubation were tested for prostaglandin E2 release by a sandwich enzyme-linked
immunosorbent assay (ELISA). For this, a human PGE2 parameter assay kit from
R&D Systems was used according to the manufacturer’s protocol. All samples were
diluted 3 times. Experiments were conducted once per donor (n = 3), by pooling two
passages. Pellets in media alone were used as negative control, IL-1β (1 ng/mL)
was used as positive control and vehicle (0.01% DMSO) was also tested for PGE2
release.
2.1.7. Imaging and histological analysis
Monolayer and histological cuts images were acquired using a Zeiss Axioscope
(Oberkochen, Germany) optical microscope equipped at x40 magnification. Pellets
were analyzed using a Cytation 3 cell imaging multi-mode reader with the Gen5™
data analysis software (Biotek, Winooski, VT, USA). Prior to sectioning, pellets were
mounted in cryoembedding compound MCC and frozen using the PrestoCHILL
system (Milestone Medical, Sorisole, Italy). Using a CryoStar NX70 microtome
(Thermo Fisher Scientific; Waltham, MA, United States), pellets were sectioned in 6
µm cuts. These were separately stained using hematoxylin/eosin and safranin-
O/fast green dyes.
Table 2. Human primers used in real time PCR. Gene Primer Sequence
GAPDH Forward 5’-ATGGGGAAGGTGAAGGTCG-3’ Reverse 5’-TAAAAGCAGCCCTGGTGACC-3’
COL2A1 Forward 5’-GGCAATAGCAGGTTCACGTACA-3’ Reverse 5’-CGATAACAGTCTTGCCCCACTT-3’
ACAN Forward 5’-TCGAGGACAGCGAGGCC-3’ Reverse 5’-TCGAGGGTGTAGCGTGTAGAGA-3’
COX2 Forward 5’-CCCTTGGGTGTCAAAGGTAA-3’ Reverse 5’-GCCCTCGCTTATGATCTGTC-3’
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2.1.8. Statistical analysis
Data are shown in mean values ± standard deviation (s.d.). Statistical analysis was
performed using a two-way variance analysis by a multiple groups ANOVA Tukey
multiple comparisons (GraphPad Prism 8.3 software). Significance was determined
at alpha level 0.05 and p values represented are ****p < 0.0001.
2.2. Results and discussion
2.2.1. Characterization of 3D chondrocyte model
Both the isolation and propagation of cultured articular chondrocytes from human
donors were optimized for our collected samples taking into consideration previously
published methods [29,30]. Steps like freezing conditions and seeding
concentrations were adjusted. Although from the same gender, the advanced age
of the three donors (mean: 69 ± 3.6 years) affects results in terms of yield and
chondrogenic ability, where younger donors are more prone to yield higher
quantities [33]. Additionally, heterogeneity between donors was observed in terms
of quantity and quality of cartilage sourced from the knee samples. Both knee size
and OA grade (OARSI score [34]) from mild to severe, played a role in the amount
of sample collected from condyles, which influenced the cellular yield after isolation
[35]. The more severe the OA grade and the smaller the obtained condyles the least
amount of cartilage was retrieved. In order to efficiently complete all the desired
experimental plan, reaching a balance between a high enough cellular yield (higher
passages) while maintaining the adequate phenotype is crucial. However,
continuous culture and passaging in plastic vessels leads to dedifferentiation,
characterized by morphological changes, from polygonal shaped chondrocytes to
fibroblast like chondrocytes (fibrocartilage) that are typically spindle like shaped [36].
This phenotype is linked to lower productions of collagen type II and aggrecan,
decreased growth yields and high synthesis of collagen type I fibers. While
redifferentiation into polygonal morphology is possible, the full reversal of this
phenotype is not [37,38]. In our study, monolayer chondrocytes were grown until the
second and third passage (approximately 7 days until confluence) in monolayer, in
order to avoid loss of quality and the phenotype of interest. Figure 4A and B shows
differences between first passage and second passage chondrocytes. In Figure 4B
a distinct increase of spindle like chondrocytes is visible, comparing to passage 1
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chondrocytes in Figure 4A, more polygonal in shape. Additionally, the media
supplementation with growth factors (TGFβ-1, FGF-2 and PDGF-BB) increases
proliferation rates and redifferentiation ability of cultured cells [29]. A supplementary
gene expression analysis, to quantify collagen type II, aggrecan, collagen type I and
determine chondrocyte phenotype would have been advantageous, however, due
to low sample amounts this was not performed. After reaching confluence on the
second or third passage, chondrocytes were cultured in a non-adherent V-bottom
96 well plate in order to form 3D pellets (P3 and P4) aiming to accurately reproduce
cartilaginous tissue, scaffold free. In Figure 4C and D, the 1-2 mm spherical pellets
can be observed. In addition to growth factors (TGFβ-1), specific supplements
(ITS+) were added to keep physical integrity of the pellets. A clear increase in
cellular growth on the borders of the pellets in comparison with to matrix production
in the core region can be observed in Figure 4E, stained by hematoxylin/eosin. This
could be explained by the limited contact with supplemented media inside the core
of the pellets. Despite this uneven distribution, articular ECM can be observed and
classified as healthy by the blue hues, characteristic of high proteoglycan content
[39]. In Figure 4F, the cartilage specific safranin-O/fast green staining was applied
to sections of pellets. Here, in untreated pellets, intense red coloration (staining
proteoglycan content) representing healthy cartilage can be observed. Non-collagen
fibers and components would be stained green, not found in any section analyzed
of the different sections [39]. The grown 3D chondrocyte pellets were classified as
representations of healthy hyaline cartilage, where no inflammation induced loss of
cartilage is visible [30], thus as accurate models for the screening of anti-
inflammatory/chrondroprotective effects of compounds.
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2.2.2. Evaluation of anti-inflammatory and/or chondroprotective effects of CXB and
KGN polymeric microparticles
The anti-inflammatory and chondroprotective ability of the two types of polymeric
microparticles was assessed by incubating for 8 days both drug alone at three
different concentrations and microparticles (10 % DL) at drug-equivalent
concentrations (Table 3). Set concentrations were selected based on previous
literature reports, using 10 µM of CXB and 100 nM of KGN as working
concentrations. Three-fold higher and lower concentrations were used to test on a
range [17,40–43]. Simultaneously, IL-1β was added to all treated wells in order to
induce the inflammatory environment that triggers OA processes in cartilaginous
tissue [44]. The assessment of the OA environment of the tested pellets was
performed by gene expression quantification – Figure 5. When compared to non-
Figure 4. Characterization of 3D chondrocyte pellets. Monolayer pellets in passage 1 (A) and passage 2 (B), prior to pellet formation. 3D chondrocyte pellet (C and D). Histological staining: hematoxylin/eosin (E) and safranin-O/fast green (F) on cross sections of pellets (represented in whole at the superior left corner). Scale bars: 100 µm.
In vitro evaluation of sustained release carriers for OA
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treated unstimulated pellets (control -), the IL-1β stimulated pellets showed a
decrease in collagen type II and aggrecan, healthy cartilage markers, and an
increase of inflammation marker, COX-2 thus validating the 3D model for
assessment of anti-inflammatory and chondroprotective activities.
*assuming similar in vitro drug release in both release buffer and chondrocyte culture medium.
Cytotoxicity of drug compounds alone and corresponding microparticles was
assessed by WST-1, in the 3D human chondrocyte pellets (Figure 6). Assessing
cellular viability in 3D pellets was deemed more pertinent than in monolayer cultured
chondrocytes given that all other assays were performed based on the three-
dimensional aggregates. After 8 days incubation, all tested conditions exhibited a
cellular viability higher than 90 % (versus dead and alive controls). Yet, and due to
Table 3. Tested drug concentrations and final drug amount after 8 days incubation.
Drug
concentration
Corresponding MPs (10 % DL)
concentration
Drug released from MPs (8 d) [Figure 3]
Estimated* released drug concentration
[µM] [mg/ml] % [µM]
CXB 3 1.1*10-2
16 0.5
10 3.8*10-2 1.6 30 11.4*10-2 4.8
[nM] [nM]
KGN 30 9.6*10-5
66 19.9
100 3.2*10-4 66.2 300 9.5*10-4 198.6
Figure 5. Gene expression levels of collagen type II, aggrecan and cyclooxygenase 2 in IL-1β stimulated pellets versus control – (unstimulated, non-treated) pellets. Values are normalized to internal controls and represented as mean values ± s.d.; n = 3, each donor with pooled samples from passages 3 and 4 pellets.
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the practicality of the assay being performed directly in the V-bottom shape wells
and this assay being mainly used to quantify mitochondrial activity of attached
monolayer cells, this test isn’t optimized to these conditions as the reagent does
easily penetrate the entire pellets. Furthermore, commercial drug compounds were
tested in solution at working concentrations, whereas microparticulate systems were
tested as suspension in the culture medium. This fact, and the afore-mentioned
enclosed plate bottom, may have contributed to accumulation of microparticles that
sediment over time - despite media changes accompanied by gentle shaking and
pipetting of the liquid contents. In an ideal setting, a transwell setup would have been
an advantage in avoiding this phenomenon.
Figure 6. In vitro cellular viability (WST-1) of 3D human chondrocyte pellets after 8 days incubation with three different concentrations of drug alone (A: celecoxib; B: kartogenin) and drug-equivalent microparticles (10 % DL). Vehicle = culture medium with 0.01% DMSO. Bars correspond to mean values ± s.d.; n = 6, each donor (3) tested at two different passages (2).
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The anti-inflammatory activity of the CXB microparticles was assessed by
quantification of prostanglandin E2 (PGE2) release by the chondrocytes into the
culture media from the recovered supernatants (Figure 7). Celecoxib is a specific
COX-2 inhibitor, of which PGE2 is a byproduct. Albeit non-inflammatory cells, the 3D
human chondrocyte pellets are, in this study, stimulated by IL-1β and representative
of the inflammatory conditions clinically observed in OA. Moreover, different pro-
inflammatory cytokines are secreted into the cultured medium and have been
quantified previously [45]. Tested concentrations (3, 10 and 30 µM) of drug alone
exhibited significant high inhibition of prostaglandin release (70, 72 and 80 %,
respectively – Figure S1). CXB microparticles comparably and significantly inhibited
the release of PGE2 from stimulated chondrocytes (70, 73 and 81 % of inhibition at
3, 10 and 30 µM, respectively). A slight dose effect with a 10-fold increase from 3 to
30 µM was observed. The same dose effect can be found in Table 3, where the
supposed concentrations after 8 days drug release were calculated from in vitro
release results (Figure 3). Though the release medium and culture medium are not
comparable, we can infer the behavior of the microparticulate systems in aqueous
media. The concentrations – 0.5, 1.6 and 4.8 µM - correspond to 16 % drug release
from microparticles at 3, 10 and 30 µM and take into account their 10 % drug loading
and confirm the anti-inflammatory effect of CXB in this model, even at low dose.
Figure 7. Quantification of PGE2 release in culture medium by ELISA from drug alone vs CXB 10 % DL microparticles. Controls: CTRL- refers to non-stimulated, non-treated pellets; IL-1β refers to stimulated, non-treated pellets. Bars correspond to mean values ± s.d.; n=3, each donor with two passages pooled. **** p < 0.0001 (treatment vs stimulated non treated - IL-1β).
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In order to further assess the anti-inflammatory and chondroprotective activities of
the microparticles, the anabolic effects were assessed by quantification of
glycosaminoglycan (GAG) content (Figure 8). GAGs are polysaccharides related to
protein (proteoglycans) binding functions in cartilage. The three main GAGs found
are keratan sulphate, chondroitin sulphate and hyaluronic acid with healthy adult
cartilage being mainly composed of chondroitin sulphate [46,47]. Assessing GAG
concentration gives an insight on cartilage matrix production. In our study, a DMMB
assay was performed. Compared to control (stimulated, non-treated pellets) results
suggest no effect of CXB alone (Figure 8A), however, a clear increase in GAG levels
can be observed at 30 µM drug-equivalent microparticles. For KGN (Figure 8B),
drug alone presented an increase in GAG production at 100 nM yet no effect at 30
and 300 nM. Drug eluted from the microparticles, once again, in no correlation to
drug alone, showed an effect at the highest drug-equivalent concentration – 300 nM.
Additionally, a slight effect could be observed at 30 nM, however none was present
at 100 nM so dose effect cannot be estimated. Colorimetric assays are commonly
used to quantify these structures but are considered crude and unspecific, often
requiring high amounts of sample. Immunohistochemistry and mass quantification
assays can give higher quality results but require specific equipment, are costly and
time consuming [48]. The GAG increase at highest drug-equivalent concentrations
of both microparticulate systems but not the same concentration of drug alone
deserves further investigation, looking into cell/particles interactions or developing
an effective transwell model to avoid any interactions. Issues in quantification using
the DMMB method could also explain the disparity of results. As discussed above,
sample amounts play an important role in colorimetric assays of GAG detection, and
in this study the 3D pellets are of approximately 1-2 mm (Figure 4) which translates
to a minimal amount of chondrocyte sample to quantify.
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Further analysis to assess cartilage matrix production either from an anti-
inflammatory or a chondroprotective/restorative effect of the studied drug
compounds was needed. For that, a gene expression analysis of collagen type II,
aggrecan and cyclooxygenase 2 was performed by real time PCR. Results are
detailed in Supplementary Material - Figure S2. Although the establishment of the
3D human chondrocyte pellets model was successful (Figure 5), outcomes of
cartilage production were not in line with the expected results. Indeed, collagen II
and aggrecan does not reflect any increase thus no cartilage matrix production and
COX-2 gene expression shows inexplicable results in regards to CXB alone and
corresponding microparticles - for KGN, no effect was expected as no anti-
inflammatory effect is expected from this compound. In general, no effect in blocking
gene expression of COX-2 was observed though a clear anti-inflammatory effect
was determined by immunoassay in the quantification of a product of COX-2
inflammation - PGE2 (Figure 7). This questions the assay itself, rather than the 3D
chondrocyte pellet model. Effectively, in an in vivo mice OA model both these drug
compounds have shown positive outcomes in decreasing inflammation environment
and increasing cartilaginous matrix [51]. In the present study, gene expression
analysis was performed once for each donor, by pooling the two tested passages
(P3 and P4). A higher number, of at least 3 repetitions would be needed in order to
confirm the obtained results. Additionally, though this model has been established
in other studies [30], sample size had an influence in our assay battery, where larger
pellets would have translated into higher genetic material making both biochemical
Figure 8. GAG production of 3D human chondrocyte pellets after 8 days incubation with (A) CXB and (B) KGN (drug alone and drug equivalent 10 % DL microparticles). Results are normalized to DNA content in samples. Symbols correspond to mean values ± s.d.; n=6, each donor (3) tested at two different passages (2).
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and gene expression analysis more robust. As mentioned before, the earlier and the
less chondrocytes are passaged, the lower the risk of dedifferentiation into
undesired phenotypes. In our study, pellets were cultured at both passage 3 and
passage 4, which might have negatively influenced the results in terms of phenotype
change and thus different responses to treatment. Lastly, incubation time may play
a role in the outcomes of certain treatments. Although inflammation is nearly
immediate after IL-1β stimulation, production of hyaline cartilage is a long process.
In order to avoid further dedifferentiation and cellular hypoxia at the core of the
pellets, this model in this current setup cannot withstand incubation times longer
than two weeks, which could hinder the development of quantifiable cartilage
markers and thus effect of treatments.
CONCLUSIONS
OA is a complex multifactorial chronic disease. Currently, there is an unmet need
for both disease modifying drugs to slow progression and more effective treatments
in terms of administration route. IA seems to answers these demands as it can
deliver directly to targets a high drug payload in a controlled and sustained manner.
To this extent, in this study, spray dried microparticles of approximately 30 µm have
been developed, with comparable features, incorporating both nano wet milled anti-
inflammatory drug - CXB - and a disease modifying drug - KGN. In an attempt to
accurately assess treatments, different clinically relevant in vitro models of OA have
been developed. In the current study, three-dimensional human chondrocyte pellets
have been successfully established to evaluate the anti-inflammatory and/or
chondroprotective activities of both CXB and KGN polymeric microparticles versus
drug compound alone (in solution). In our findings, a significant anti-inflammatory
effect from CXB eluting microparticles was observed in an immunoassay, at all
tested concentrations. However, this effect was not confirmed by GAG quantification
and gene expression analysis. The biological effects of KGN microparticles were not
quantifiable using the chosen setup and assays although these have been reported
in the literature for other type of 3D cellular structures. In the future, a clear
optimization and further development of the quantification assays as well as the 3D
pellet model – lower passages and increased pellet size - is imperative to adequately
infer effects of disease modifying drugs that require long incubation periods.
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Supplementary material
Figure S1. Inhibition percentage of PGE2 release normalized to 100 % stimulation with IL-1β, as a function of celecoxib concentration. Bars correspond to mean values ± s.d.; n = 3.
Figure S2. Gene expression levels of collagen type II (COLL2), aggrecan (ACAN) and cyclooxygenase 2 (COX2) in IL-1β stimulated pellets versus control (stimulated, non-treated) pellets. Values are normalized to internal controls and represented as mean values ± s.d.; n = 3, each donor with pooled samples from passages 3 and 4 pellets.
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CONCLUSIONS AND PERSPECTIVES
As a leading cause of disability worldwide, osteoarthritis (OA) is a chronic disease
that poses a severe threat both for healthcare and socio-economic scenarios.
Several approaches have been explored in the search for a cure, a current unmet
need. Local delivery, intra-articular (IA) of therapeutic molecules, has risen in recent
years as an alternative to oral systemic treatment by circumventing drawbacks like
low bioavailability of molecules in joint capsules and off-target adverse effects.
This thesis aimed at taking a more in-depth look at the development of intra-articular
drug delivery systems (IA DDS) for OA treatment, in an attempt to push this research
further.
A deep understanding of OA in vitro models was deemed crucial for better design
and formulation of IA DDS for OA treatment and, as one of the aims of this thesis.
As so, in Chapter I, current OA in vitro models were extensively reviewed.
Advantages and drawbacks of 2D, 3D cellular models, and explants were explored
regarding outcome testing for different IA DDS like hydrogels, polymeric
microparticles, and nanoparticles. To this extent, IA DDS developed in the past five
years for OA treatment were listed and reviewed regarding type of formulation,
tissue target, and in vitro model applied. The different models - cell lines, type of
scaffold or explant tissue - were compiled and linked to the outcomes evaluated in
these studies. Monolayer cellular models, typically human synoviocytes or murine
macrophages, are more frequently used in screening of viability and/or bioactivity of
IA DDS carrying anti-inflammatory molecules. Conversely, for IA DDS eluting
chondroprotective molecules, studies in cartilage-mimicking models or actual tissue
like chondrocyte 3D cellular models or explants are preferred. Still, as IA DDS
represent an appealing option for OA treatment, and no gold standard is set for OA
in vitro models, further research efforts should focus on better tailoring models to
suit outcome evaluation of this type of formulation.
Knowledge gathered in in vitro OA models allowed the establishment of model and
outcome evaluation applied in Chapter II. In this study, the viability and anti-
inflammatory activity of A. brachypoda root extracts, a shrub used in traditional
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Brazilian medicine, were evaluated in human fibroblast-like synoviocytes (HFLS).
The main goal of this first study was to assess the potential of two root extracts, one
hydroethanolic and the other in dichloromethane, and three compounds isolated
from the latter, as IA deliverable molecules. Another goal was to deepen the
understanding of the use of this plant in OA settings. The last task was to isolate
and quantify the three compounds in the dichloromethane extract. The
hydroethanolic extract was found to be safe but not active in terms of suppressing
inflammation in HFLS. Conversely, and due to the presence of the three
compounds, the dichloromethane extract showed increased bioactivity against OA
HFLS. This anti-inflammatory activity, however, was accompanied by an increase in
cytotoxicity. It was determined that the most abundant in the dichloromethane
extract was Compound 3, followed by Compound 2 and Compound 1. Their anti-
inflammatory strength and cytotoxicity were found to be directly correlated to their
amounts in the extract. Overall, the therapeutic window of the isolated compounds
was considered too narrow for a local administration in OA treatment. Nonetheless,
and seemingly due to the protective effects of the mixture of these isolated
compounds, the dichloromethane extract remains attractive for the development of
IA formulations.
After the investigation of potential IA drug delivery molecules in studies of natural
products, research efforts were shifted in the direction of known drug molecules
used in OA treatment. Celecoxib is a non-steroidal anti-inflammatory drug (NSAID),
frequently taken prescribed for symptomatic management of pain and inflammation
in OA. Chapter III describes the formulation and characterization of celecoxib
polymeric microparticles for IA treatment of OA. In this study, celecoxib was
incorporated either as drug in solution or as nano wet-milled drug particles, using a
spray-drying method. The main goal was to assess the effects on drug release
profiles of drug incorporation as nano wet-milled particles vs. drug in solution, and
of increasing drug loadings. Resulting microparticles were characterized in terms of
size, morphology, drug incorporation, crystallinity, and their 90 days celecoxib
release profiles were compared. The microparticles were tested for viability and anti-
inflammatory activity against free drug, using the same in vitro OA model described
in the previous chapter. The combination of high drug loading and incorporation of
celecoxib as nano-milled drug in polylactic acid (PLA) translated into 30 µm
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microparticles with a long-lasting steady drug release profile. In the local treatment
of OA (IA), it is advantageous for a drug delivery system to have a sustained,
controlled drug release profile to ensure a therapeutic window over long periods of
time, thus avoiding repeated IA injections. However, it is also viewed as favorable
to have an early burst-like release of drug from the delivery systems, to achieve an
almost instant relief of symptoms like pain and inflammation. The developed
celecoxib microparticles cover both these desirable features of an IA drug delivery
system as their tunable drug loading, both in type and concentration, enables
tailoring of the drug release profile. This sustained release, confirmed by the HFLS
in vitro OA model, could also be interesting to explore using other OA molecules
with chondroprotective effects. This developed IA DDS is highly versatile as different
microparticles, with different drug loadings, of molecules in different forms and even
different molecules, with different drug release profiles could be combined in a single
formulation.
The potential of this IA DDS in incorporating other molecules and as a combination
treatment of OA is further reported in Chapter IV. In this part of the thesis, the wet-
milling and spray-drying methods from the previous chapter were used to
incorporate kartogenin. This molecule induces chondrogenesis, thus cartilage
repair, making it an attractive therapeutic molecule for OA. PLA microparticles with
embedded nano-milled kartogenin were characterized and compared to celecoxib
microparticles with the same drug loading (10 %). Comparable results in terms of
size, morphology and drug incorporation, between the tested microparticles
contributed to the versatility, robustness, and reproducibility of the developed
formulation methods. Considering the mode of action of kartogenin and knowledge
gathered in Chapter I, in this study, the drug delivery systems were tested for
viability and bioactivity in a 3D chondrocyte OA model. Pellet characterization
allowed for confirmation of the selected model as an accurate OA in vitro model.
Based on this belief, cytotoxicity was assessed over eight days, as the
microparticles exhibited a sustained controlled release over a long period of time.
During the same one-week incubation period, the bioactivity of both drugs eluted
from the microparticles systems was evaluated. The anti-inflammatory effects of
celecoxib and chondrogenic activity of kartogenin, were assessed by ELISA, gene
expression analysis, and glycosaminoglycan (GAG) content. However, the chosen
Conclusions and perspectives
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model was not capable to fully characterize the bioactivity of the established drug
delivery systems.
The different studies presented in this thesis allowed to face and tried to reply to a
number of challenges encountered in research of IA DDS for OA treatment. Firstly,
the potential of therapeutic molecules for local delivery. Natural products are rich
sources of therapeutic molecules; potentially disease-modifying osteoarthritis drugs
(DMOADs) and molecular pathways deserve further exploration by applying
accurate OA pre-clinical models. Then, the development of polymeric carrier drug
delivery systems and lastly, the establishment of accurate OA in vitro models for
outcome evaluation of this type of formulations. Polymeric microparticles with nano
wet-milled drug particles and tailorable drug loadings seem to answer to more than
one demand of IA OA treatment. For improved design and development of these IA
DDS, better and more predictable OA in vitro models need to be explored, with
particular regards to features, like long-lasting drug release, of these formulations.
All the explored angles of IA DDS research deserve, and will benefit, from further
improvement with the ultimate goal of better OA treatment options.
French summary
French summary
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FRENCH SUMMARY
L'arthrose est une maladie chronique et dégénérative. Cette forme d'arthrite la plus
courante a une prévalence élevée et toujours croissante dans la population
mondiale vieillissante (> 65 ans). Étant l'une des principales causes d'invalidité chez
les personnes âgées, la charge socio-économique causée par l'arthrose est
considérable Actuellement, les traitements pharmacologiques sont basés sur la
gestion des symptômes par des analgésiques et des anti-inflammatoires oraux.
Cependant, il existe toujours un besoin pour un traitement curatif, soit utilisant des
formulations ou voies d'administration plus efficaces, soit par des médicaments
modificateurs de la maladie (DMOAD) qui aident à ralentir sa progression ou même
inverser son cours. Ces dernières années, des efforts importants ont été déployés
dans la recherche et le développement de nouvelles molécules et de meilleurs
systèmes d'administration des médicaments. Parmi ces études, l'administration
locale intra-articulaire (IA) de molécules thérapeutiques a été explorée comme une
alternative pour contourner les problèmes de la thérapie orale systémique, tels que
la faible biodisponibilité articulaire et les effets indésirables. Une libération contrôlée
et prolongée des molécules directement dans la capsule articulaire a été envisagée
avec le développement de systèmes de délivrance de médicaments. Dans la
recherche sur les DMOADs et les systèmes d'administration de médicaments par
voie IA, les essais précliniques constituent une étape essentielle et cruciale. Dans
cette phase de développement, il est crucial d'envisager des modèles d'arthrose in
vitro et in vivo subtiles et hautement prédictifs pour une meilleure translation vers
une application clinique. De meilleures conception et prédiction pour une application
chez l'homme sont essentielles pour faire progresser la recherche sur l'arthrose.
Cette thèse s'articule autour de trois axes différents : recherche de nouvelles
molécules ayant un potentiel en tant que DMOADs; développement de systèmes
d'administration de médicaments IA (IA DDS) pour la délivrance locale de
traitements contre l'arthrose et finalement, conception et établissement de modèles
in vitro précis d'arthrose. L'objectif principal était le développement d'options de
traitement de l'arthrose par l'exploration de nouvelles molécules et la mise au point
de systèmes d'administration de médicaments appropriés. Tout au long de ces
French summary
142
travaux, un autre objectif était de modéliser précisément l'arthrose in vitro, avec
l'établissement de modèles cellulaires et une évaluation adéquate des résultats.
Les différentes études présentées dans cette thèse ont tenté de répondre à un
certain nombre de défis rencontrés dans la recherche des systèmes de libération
intra-articulaires pour le traitement de l'arthrose. Tout d'abord, concernant le
potentiel de molécules thérapeutiques pour l'administration locale. Les produits
naturels sont de riches sources de molécules thérapeutiques ; les médicaments
contre l'arthrose potentiellement modificateurs de la maladie (DMOAD) et les voies
moléculaires méritent d'être explorées plus avant en appliquant des modèles
précliniques précis de l'arthrose. Les études ont porté dans un deuxième temps sur
le développement de systèmes d'administration de médicaments par des vecteurs
polymériques et enfin, sur l'établissement de modèles in vitro de l'arthrose
pertinents, pour l'évaluation des résultats de ce type de formulations. Les
microparticules polymères avec des nanoparticules de médicament broyées par
broyage en milieu humide (wet-milling) et des taux de chargement de médicament
élevé, semblent répondre à plus d'une demande de traitement de l'arthrose par IA.
Pour améliorer la conception et le développement de ces systèmes de libération
intra-articulaires, il est nécessaire d'explorer des modèles in vitro d'arthrose
améliorés et plus prédictifs, en accordant une attention particulière aux
caractéristiques de ces formulations, comme la libération prolongée du
médicament.
Tous ces aspects de la recherche sur les systèmes de libération intra-articulaires
méritent et bénéficieront d'améliorations futures dans le but ultime d'offrir des
options efficaces de traitement permettant de modifier de façon significative
l’évolution des patients souffrant de l'arthrose.
143
Acknowledgements
Firstly, I would like to thank Prof. Eric Allémann for allowing me to join his lab and
mostly, the osteoarthritis group. Thank you for let me engage and follow this PhD
journey. Thank you for your help, guidance and endless discussions. For putting up
with my very opinionated and, at times, stubborn self and my never-ending
comments on all lab matters. A big thank you to Dr. Olivier Jordan, for the same
reasons. For your always poignant and important comments and reviews of
manuscripts, endless hours of discussion, help and guidance. Mostly for putting up
with my non-mathematical brain and solving all my statistical dilemmas and the
goiabada cascão, a must! A special thanks to Profs. Norbert Lange and Gerrit
Borchard for always being available to lend me an ear and discussions on my future
and career prospects.
My warmest thank you to everyone with whom I have collaborated in the making of
this thesis. The first and most important, Dr. Laurence Neff. Thank you Laurence for
your dedication, patience, time and endless help with all matters of biology and
cellular work. This thesis would have not been possible without your valuable help.
Thank you to Dr. Emerson Ferreira Queiroz for always being available to help,
pushing our research efforts further and our always interesting discussions. Thank
you to Nayara, Hugo and Thanise for their valuable contributions, help and good
spirits. To Prof. Jean-Luc Wolfender for the nice discussions and Dr. Laurence
Marcourt for all the help and reviews of the manuscript. A very big thank you to Prof.
Radovan Černý and Dr. Laure Guénée for their valuable contributions, many great
discussions and never ending availability. Thank you to Luca, for his assistance. It
has been a true pleasure working with you all! In this category, my biggest thank
you to my two master students, Alexandre and Benjamin. You have taught me more
than you could imagine and I am thankful to have you as good friends. And a big
thank you to Archie, without whom this manuscript would look less colorful and oh
so drab.
They say it takes a village, well in this case, it very well did. Thank you to the FATEC-
FABIO teams for all the great moments. For allowing my party planner, costume
obsessed persona to soar, always saying yes to my crazy ideas. Nathalie and
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Marco, two people who have been crucial and the pillars of this village. Thank you
Nathalie for all your help and endless hours of therapy sessions at the SEM. For
always being available to listen to my many many dilemmas both scientific and
personal. And mostly, for your good mood and positive energies. Marco Paulo, thank
you. For always, without fail, being there for me. In and out of lab matters, you made
it possible. My biggest thank you to past lab members who have guided and helped
me in this long hard PhD journey: Imène, Yanna, Jordan, Viorica, Floriane and
Tiziana. Thank you to my dear friends, Karolina and Vassily. My day ones, here’s to
many more memories together! An immense, deep thank you to all my work wives,
who have literally carried me through these past 5 years and to whom I own
everything: Thais, Bettina, Sara, Ester, Kenza and Cíntia. Thank you to some very
important people who have been more than a support system in this lab: Souad,
Joel, Allegra, Adèle, Martin (my successor, big hopes!) and Franck. May all our
futures shine bright! Thank you to some other elements of this village, always ready
to lend a helping hand: Aymeric, Micaela, Julia, Marta, Hisham and Barbara. Thank
you to George and Ghali, you keep me forever young and always on my toes.
Thank you to my friends, both in Geneva and back home. You have kept me sane
and motivated to always carry on: Sara, Jasmin, Karin, Arjan, Vanessa, Ana, Pedro,
André, Zé Diogo, Diogo, Inês, Joana, Rebekah, Teresa, Filipa, Francisca and many
more not named here.
My biggest thank you goes out to my family. To my GVA family, without whom none
of this would have been possible, Tia Matu and Tio Zé, my cousins João, Mariana,
Zeca and Matilde. To all my cousins, may we always be this united. To my
grandparents, thank you for keeping me rooted and always showing me the way.
Especially to Avô João, to whom this thesis is dedicated to. Last but never ever
least, the other 5 pieces of my heart. Thank you to my brothers Zé Maria, Henrique
and Francisco who are my pride and joy, keep me on my toes, are there for me
through thick and thin and until the end of times. To my parents, my biggest
examples in life. Thank you for everything, no words can express how thankful I am
for all you have given me and just wish one day to be able to give back. This thesis
is as much yours as it is mine. Obrigada!
