e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /e jps

Specific and non-specific phagocytosis of ligand-graftedPLGA microspheres by macrophages

Nolwenn Brandhonneura,e, Francois Chevannea, Véronique Viéb, Benoît Frischc,Roselyne Primaultd, Marie-Frédérique Le Potier e, Pascal Le Correa,∗

a Laboratoire de Pharmacie Galénique, Biopharmacie et Pharmacie Clinique, UPRES EA 3892, 2 avenue du professeur Léon Bernard,35043 Rennes Cedex, Franceb Equipe Biophysique, Institut de Physique de Rennes, UMR UR1 - CNRS 6251, 263 avenue Général Leclerc, 35042 Rennes Cedex, Francec Laboratoire de Chimie Enzymatique et Vectorisation, Université Strasbourg 1, Institut Gilbert Laustriat, CNRS - UMR 7175,Faculté de Pharmacie, 74 rte du Rhin - B.P. 60024, 67401 Illkirch Cedex, Franced Département de Microscopie, UFR Médicales et Pharmaceutiques, 2 avenue du Professeur Léon Bernard - CS 34317,35043 Rennes Cedex, Francee Agence Francaise de Sécurité Sanitaire des Aliments (AFSSA-LERAPP), UPRES EA 3892, Unité de Virologie et Immunologie Porcines,Les Croix - B.P. 53, 22440 Ploufragan, France

a r t i c l e i n f o

Article history:

Received 31 July 2008

Received in revised form

25 November 2008

Accepted 27 November 2008

Published on line 6 December 2008

Keywords:

PLGA microspheres

Functionalization

Ligands

Macrophages

a b s t r a c t

We evaluated the influence of ligand grafting on the rate and intensity of uptake of poly(d,l-

lactide-co-glycolide) microparticles by alveolar macrophages. Microspheres with a mean

diameter of 2.5 �m were obtained by spray drying. Three ligands (WGA, an RGD containing

peptide and mannose-PEG3-NH2) and a cationic molecule (PLL) were covalently grafted on

the particle surface using the carbodiimide method. Their grafting efficiency was quanti-

fied, and WGA grafting was characterized by confocal laser scanning microscopy (CLSM) and

by atomic force microscopy (AFM). The uptake by macrophages of surface-modified micro-

spheres was quantified by CLSM. This work showed that the uptake of negatively charged

ligand-grafted microspheres (−26 to −51 mV) was increased up to two to four times accord-

ing to the ligand compared to ungrafted microspheres (−81 mV) and displayed saturation as

opposed to the cationic PLL-grafted microspheres. Moreover, a specific receptor-mediated

phagocytosis mechanism was suggested based on free ligand, cytochalasin D and +4 ◦C incu-

Phagocytosis bation that decreased the microparticle uptake. Furthermore, this work clearly showed that

the relative contribution of specific and non-specific processes to the overall uptake varied

greatly according to the ligands, and was dependent on the particle-to-cell ratio. In conclu-

sion, this work showed that ligand grafting can enhance the uptake of microparticles, with

a variable relative contribution of specific and non-specific uptake mechanism.

their ability to allow a cellular drug targeting and/or a con-

1. Introduction

Particulate systems in the micron and submicron level areincreasingly used for biomedical applications as a result of

∗ Corresponding author. Tel.: +33 2 23 23 48 72; fax: +33 2 23 23 48 46.E-mail address: pascal.le-corre@univ-rennes1.fr (P. Le Corre).

0928-0987/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.ejps.2008.11.013

© 2008 Elsevier B.V. All rights reserved.

trolled drug release (Chavanpatil et al., 2006; Panyam andLabhasetwar, 2003; O’hagan and Singh, 2003). Nanoparticlesare mainly used for their cellular targeting ability follow-

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ng systemic administration, while microparticles have beensed for many years as controlled release delivery systems forrugs and therapeutic proteins. Moreover microparticles offerhe advantage of a higher payload especially for hydrophilicrugs in particles, and a better ability to provide a controlledelease. This point is of interest for antigen delivery sincet has been suggested that the release rate influence theesponse (Waeckerle-Men et al., 2006). Among the applica-ions for microparticles, the delivery of recombinant proteins,lasmid DNA and peptides for antimicrobial vaccination isrowing (Davis, 2006; O’hagan et al., 2006; Reddy et al., 2006;iborski et al., 2006).

For vaccine application, administration via the mucosaloute has been shown to provide an advantage over systemicdministration (Mutwiri et al., 2005; Sedgmen et al., 2004; Vyasnd Gupta, 2007), namely the production of a mucosal immu-ity. In addition, compared to systemic vaccination leadingnly to systemic Ig induction, mucosal vaccination inducesoth a systemic and a mucosal immunity with the produc-ion of IgAs at the epithelia a common site of pathogenntry. Among the mucosal routes, intranasal administrationf antigen delivery systems has been largely studied (Byrdnd Cassels, 2006; Jaganathan and Vyas, 2006; Stanley et al.,004; Strindelius et al., 2004). The combination of alterna-ive immunization routes and the use of appropriate antigenelivery systems appear to be a rational approach for provid-

ng an effective mucosal immunity. Microparticles based onoly(lactide-co-glycolic acids) (PLGAs) have been studied forhis application because of their safety record, e.g. used asontrolled delivery of drugs (Jain, 2000).

For an efficient mucosal vaccination using microparticles,t is necessary to maximize their uptake by the cells of interest,.e. antigen-presenting cells (APCs), since a low level of particleptake by cells is a limiting factor. Furthermore, diffusionalarriers at the mucosal level impede the local trafficking oficroparticles throughout the mucosa, especially at the gut-

ssociated lymphoid tissue (GALT) level (Torche et al., 2006).any parameters can influence cellular uptake efficiency oficroparticles namely, diameter, nature and hydrophobicity

f the polymer and the surface properties (Carr et al., 1996;esai et al., 1997; Thiele et al., 2003, 2001; Torche et al., 2000).mong surface properties, the surface charge (i.e., zeta poten-

ial) has been focussed upon and, contrary to nanoparticles,ittle work has been undertaken on the influence of the pres-nce of ligands at the surface. For several years, many studieserformed on nanoparticles have suggested that differentolecules such as lectins, invasins and peptides can be used

s targeting molecules to increase the particles uptake by cells.ectins are glycoproteins appropriate for targeting as wheaterm agglutinin (WGA) from Triticum vulgare, because they canind to specific oligosaccharides present at the surface of dif-erent cell types, and many authors have used these moleculeso evaluate these cytoadhesive properties (Russell-Jones et al.,999; Weissenboeck et al., 2004). The use of invasin as a cellu-ar ligand has been tested using latex and PLGA nanoparticleso demonstrate an increase of particulate uptake in caco-2,

ep2 2B and MDCK cells (Dawson and Halbert, 2000; Hussainnd Florence, 1998).

Integrins constitute a family of transmembrane recep-ors involved in cell–cell and cell–matrix interactions and

l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485 475

are central players in signal transduction pathways. This ledmany groups to design selective arginine–glycine–aspartate(RGD)-containing ligands to specifically target the integrins.The RGD serves as a model ligand for the specific inter-action with integrin receptors, which are expressed on thesurface of phagocytic cells. The C-type lectins family com-prises a number of pathogen recognition receptors that bindto carbohydrate ligands. The targeting of C-type lectins recep-tors on APCs was identified as a promising strategy forimmunomodulation, as documented by several studies show-ing that immune responses were enhanced or modified bycoupling mannose type ligands (Cui et al., 2004; Jain and Vyas,2006). The study of these three ligands (WGA, RGD and man-nose) was considered as relevant approach. Indeed, the useof these ligands can lead to applications beyond macrophagestargeting and vaccination.

To our knowledge, there is only one study showing anincrease in the cellular uptake of ligand-grafted microparticles(Thiele et al., 2003), as opposed to nanoparticles for which thisis highly documented. Indeed, ligand grafting onto microparti-cles may present advantages compared to nanoparticles sincethey allow a better controlled release of the drug and a higherpayload. In the study of Thiele et al. (2003), phagocytosis of IgGcoated carboxylated polystyrene microparticles was signifi-cantly enhanced in dendritic cells and macrophages. Anotherstudy with an RGD-motif onto 4.5 �m PLL-g-PEG carboxylatedpolystyrene microparticles led to ligand-specific phagocyto-sis by APCs, but did not specifically address the influence ofligand on uptake. Indeed, they showed that the grafting ofRGD abolished the repellent nature of particles (Faraasen etal., 2003).

However, the uptake mechanisms of microspheres remainunclear, and eventhough a ligand is grafted onto micro-spheres, specific and non-specific uptake can simultaneouslyoccur and their relative contribution to overall uptake shouldbe studied. For that purpose, the comparison of differentligands in increasing the uptake should be carried out. Endo-cytosis can be divided into two categories, ‘pinocytosis’ orcell drinking (fluid and solutes) and ‘phagocytosis’ or celleating (large particles) (Conner and Schmid, 2003). Pinocy-tosis is a well described mechanism of uptake of smallparticles or nanoparticles (Dawson and Halbert, 2000; Moand Lim, 2004; Russell-Jones et al., 1999). Pinocytosis hasfour basic mechanisms of action: macropinocytosis, clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis,and clathrin- and caveolae-independent endocytosis. Themechanism of uptake of WGA-conjugated PLGA nanoparti-cles by A549 cells has been described as an endocytosis bya receptor-mediated, caveolae-dependent pathway (Mo andLim, 2004). Phagocytosis is a mechanism for uptake of largepathogens such as bacteria or yeast, and particles by spe-cialized cells including macrophages. Phagocytosis may bespecific (requiring contact with a receptor) or non-specific(Camner et al., 2002).

The aim of this study was to compare, for uptake ofPLGA microparticles, the influence of different ligands on

the rate and intensity of their phagocytosis by pig alveo-lar macrophages, and to estimate by mathematical analysisthe relative contribution of the specific and non-specificmechanisms. We selected three ligands: a lectin wheat germ

u t i c

476 e u r o p e a n j o u r n a l o f p h a r m a c e

agglutinin (WGA) which interacts with lectinic receptors, apeptide containing RGD interacting on integrins, and a carbo-hydrate moiety (mannose-PEG3-NH2) interacting on mannosereceptors. A detailed analysis of the WGA grafting was per-formed by confocal laser scanning microscopy (CLSM) and byatomic force microscopy (AFM).

2. Materials and methods

2.1. Materials

Resomer RG 503H PLGA (lactide/glycolide ratio 50:50, inher-ent viscosity 0.32 dl/g, acid number 3 mg KOH/g) wasobtained from Boeringer Ingelheim (Ingelheim, Germany). 6-Coumarin, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDAC), MES buffer, sodium dodecyl sulphate (SDS), Trisbuffer, bovine serum albumin (BSA), wheat germ agglutinin(WGA), poly-l-Lysine (PLL), GRGDSPK (Gly-Arg-Gly-Asp-Ser-Pro-Lys) and GRADSPK (Gly-Arg-Ala-Asp-Ser-Pro-Lys) peptideswere purchased from Sigma (Saint-Quentin Fallavier, France).Mannose-PEG3-NH2 was synthesized according to a previouspublished method (Schneider et al., 2006). All other reagents,such as dichloromethane were of analytical grade.

2.2. Preparation and characterization of PLGAmicrospheres

PLGA microspheres were prepared by spray drying as pre-viously described (Le Corre et al., 2002). Briefly, PLGA-typepolymer was dissolved in dichloromethane (w/w, 2%) and wasspray dried with a Mini Büchi B-191 laboratory spray dryer(Büchi Laboratory AG, Switzerland) using a 0.7 mm nozzle.The process parameters were set as follows: inlet temperature(50 ◦C); aspirator setting (100%); pump setting (3 ml/min) andthe spray flow (500 NL/h). Fluorescence-labelled PLGA micro-spheres were prepared by adding 6-coumarin (5 mg/g polymer)to the polymer solution. The microspheres were kept undervacuum at 4 ◦C until characterization.

The average volume diameter, D [4,3] and the particle sizedistribution were examined by laser light scattering usinga Mastersizer S (Malvern Instruments, Orsay, France). Themicrospheres were suspended in aqueous solution containing0.05% Tween 20 as a wetting agent to minimize agglomeration,sonicated for 1 min and analysed in triplicate. The followingequation let us to know the number of microspheres per gram:

N =Dn∑

Di

6V

˘d3�

where N is the number of microspheres per g, V is the volume,d is the diameter of microspheres and � is the density.

The surface morphology of the microspheres was analysedby scanning electron microscopy (Jeol 6400, Tokyo, Japan).

2.3. Covalent binding of molecules to PLGA

microspheres

Covalent conjugation to the free carboxyl groups on the par-ticles surface was performed according to a carbodiimide

a l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485

method (data sheet #644, Polysciences, Europe). In brief, thefree carboxyl groups of the microparticles were activated byEDAC in MES buffer (50 mM, pH 6). The suspension was stirredat 30 rpm for 1 h at 20 ◦C, and then activated microsphereswere incubated for 1 h with the different ligands: WGA or BSA,RGD or RAD containing peptides, mannose-PEG3-NH2, and thecationic molecule poly-l-lysine. The amount of ligands variedaccording to their molecular weight: 100 �g for WGA, 200 �g forBSA, 10 �g for RGD, RAD and mannose-PEG3-NH2, and 60 �g forPLL. After incubation, the particles were centrifuged (3000 × g,5 min), washed twice with 10 mM Tris pH 7.5 containing 0.1%SDS and resuspended in distilled water. After freezing, themicrospheres were lyophilized.

2.3.1. Quantification methodsThe intensity of grafting was determined by measurement ofthe ungrafted ligand remaining in the reaction medium. WGAor BSA concentrations were measured by colorimetric deter-mination of proteins, with a microBCA reagent kit (Sigma) at540 nm. Separation and quantitative determination of RGDor RAD containing peptides was performed according to aRP-HPLC method (Lee and Row, 2004). The Waters chromato-graphic system was composed of a 600 gradient Pump, a 717Autosampler and a 996 Diode Array Detector controlled withthe Empower Pro software (Waters Corporation, St QuentinYvelines, France). The chromatography was performed ona 250 mm × 4 mm, 5 �m, Discovery® C18 Supelco® column(Sigma–Aldrich, St Quentin Fallavier, France) at 35 ◦C. Elutionwas performed using a gradient of solvent A (0.08% trifluo-roacetic acid in water) and solvent B (0.1% trifluoroacetic acidin acetonitrile) from 0% B to 20% B for 12 min at a flow rate of1 ml/min. UV detection was monitored from 200 to 320 nm andboth peptides were quantified at 205 nm. Mannose was quan-tified by a colorimetric determination of sugars; the resorcinolsulphuric acid micro method at 430 nm (Monsigny et al., 1988).The zeta potential of PLL-grafted microspheres was deter-mined using a Malvern nano ZS (Malvern Instruments, Orsay,France). Three samples were measured at ambient temper-ature in distilled water. The adsorption of these differentmolecules on vials or microspheres was studied without EDC.

2.3.2. Confocal laser scanning microscopyFor CLSM observation of peripheral grafting at the micro-spheres surface, fluorescent ligands (BSA-FITC and WGA-FITC) were used. The microspheres were suspended inaqueous solution. Preparations were mounted on coverslipsfor microscopic observation using a Confocal Leica TCS NT(Germany).

2.3.3. Atomic force microscopyMorphological and topographical modifications of micro-sphere surfaces were investigated using a Pico-plus atomicforce microscope (Molecular Imaging, Phoenix, AZ) underambient conditions, i.e., in air, using a 100 �m × 100 �mscanner. Images were acquired in tapping mode to avoid defor-mations or displacement of objects (Mu and Feng, 2001). In this

mode, the oscillating tip touches the surface once each cycle,thus minimizing the friction forces present in contact mode(Zhong et al., 1993). Silicon tips on integral cantilevers wereused with a resonant frequency F0 = 65–90 kHz and a nominal

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pring constant k = 1.2–3.5 N/m. Scanning was performed atscan speed of 1 Hz. Samples were prepared by depositingvolume of 10 �l of dispersed microsphere suspensions in

istilled water on freshly cleaved mica plates and imagingas performed after drying the samples overnight at 37 ◦C. In

he first step, we imaged a field of 50 �m × 50 �m to localiseicrospheres in samples. In the second one, we focussed on

he surface of microspheres in order to visualise the effect ofhe grafting of the surface at the nanoscale resolution.

.4. Ex vivo study

.4.1. Collection of alveolar macrophagelveolar macrophages were obtained by lung washingsnder sterile conditions from two 5-week-old pigs as previ-usly described (Baron et al., 1992). Briefly, after anesthesiand laparotomy, the lungs were removed and alveolaracrophages were harvested by broncho-alveolar washings

ia the trachea with sterile PBS. The cells were collected, cen-rifuged (280 × g, 13 min, +4 ◦C) and washed twice with PBS.he cells were subjected to a transient hypotonic shock withold sterile water to lyse contaminating erythrocytes. Theells were finally resuspended in Eagle’s Minimum Essentialedium (EMEM) for enumeration. Cell viability and enumer-

tion were evaluated by trypan blue exclusion on a Malassezell. After centrifugation, alveolar macrophages were resus-ended in a small volume of EMEM supplemented either withr without foetal bovine serum 10% (FBS), penicillin and strep-omycin (PS), fungizone (F) and dimethyl sulphoxide (DMSO)90:10) prior to freezing in liquid nitrogen.

.4.2. Phagocytosis studyhagocytosis was studied with microsphere/macrophageMS/MA) number ratios: 1, 2.5, 5, 10 and 20. It should be notedhat these evaluations were based on triplicate-independentxperiment made on different days and with three differentatches of macrophages. Alveolar macrophages were washed

n EMEM-PS to eliminate DMSO and then centrifuged. Theellet was resuspended in a small volume of EMEM-PS. Celliability and enumeration of macrophages was determineds previously. The cell concentration was adjusted at 1.5 × 106

lveolar macrophages/ml of EMEM-PS-FBS. 1 ml of cell suspen-ion was seeded into each well of a 24-well culture plate andncubated for 18 h at 37 ◦C in the presence of 5% CO2. Afterncubation, the medium was discarded and each well wasashed three times with EMEM-PS to remove non-adherent

ells. Microspheres were suspended in EMEM-PS-FBS. One mlf the microspheres suspension at the desired concentrationas added into each well and then the plates were incubated

t 37 ◦C and 5% CO2. Evaluation of phagocytosis was per-ormed after 2 h of incubation since the phagocytosis rate haslready been shown to be constant after 90 min of incubationTorche et al., 1999).

Complementary uptake experiments were performed withicrosphere/macrophage (MS/MA) number ratios of 2.5: at

◦C or with medium containing free corresponding inhibitor

WGA 100 �g/ml, RGD 10 �g/ml, mannose-PEG3-NH2 10 �g/ml)r by adding cytochalasin D (10 �g/ml) to the culture mediumor 1 h prior to the addition of particle suspensions. Evaluationf phagocytosis was realized after 2 h of incubation.

l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485 477

Uptake quantification was determined using CLSM, bycounting the mean number of microspheres internalized bycells. For each culture plate, 5 fields of 25 macrophages wereanalysed.

2.4.3. Confocal laser scanning microscopyCells were fixed with 4% paraformaldehyde in PBS for 15 minat 4 ◦C and with 15% glycin in PBS for 10 min at room tem-perature. Preparations were washed for 5 min with PBS andmounted on coverslips for microscopic evaluation using aConfocal Leica TCS NT microscope (Germany).

2.4.4. Mathematical analysisMathematical analysis was used to evaluate the relative con-tribution of specific and non-specific uptake to the overalluptake. We used a model with two sites (one saturable andone non-saturable) with the following equation which is usedfor most application in ligand binding studies (Gabrielsson andWeiner, 1997):

y = Bmax x

Kd + x+ Ns x

where y is the intensity of uptake (mean number of MS perMA), x is the MS/MA ratio, Bmax is the maximum specificuptake (mean number of MS per MA), Kd = 1/Ka where Ka is theaffinity constant of the ligand for uptake (MA per mean num-ber of MS) and NS is the non-specific uptake (mean number ofMS per MA).

2.4.5. Statistical analysisStatistical analysis was performed by one-way analysis of vari-ance (ANOVA) followed by Student’s t-test. A p-value less than0.05 was considered as statistically significant.

3. Results and discussion

3.1. Characterization of PLGA microspheres

The granulometric analysis indicated diameters in the rangeof 0.6–15.4 �m, and an average volume diameter of 2.5 �m(data not shown) in accordance with the scanning electronmicroscopy (SEM) observations which also present a polydis-perse population. The grafting of different ligands did notmodify the diameter distribution of the particles (data notshown). Even though the size is not the only feature driv-ing the phagocytosis of microparticles by macrophages, itremains a significant factor. Indeed, the uptake of PLGA micro-spheres ranging from 2 to 3 �m by alveolar and peritoneal ratmacrophages have been shown to be significant (Jones et al.,2002). Moreover, based on the evaluation of the total volumeof particles phagocytosed, a size of 3 �m has been shown to bethe most suitable for an efficient delivery to a cell line derivedfrom rat alveolar macrophages (Hirota et al., 2007). The targetsize for macrophages is around 3 �m and the current formu-lation of microspheres had 43% of the distribution in volumebetween 2 and 4 �m.

The SEM image revealed the spherical shape and smoothnon-porous surface of the particles with some invaginationsoccurring due to the technique of spray drying (Fig. 1). Theabsence of porosity should limit the penetration of the grafting

478 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c

Fig. 1 – Scanning electron microscopy of ungrafted PLGA

microspheres prepared by spray drying.

reagent within the microsphere and consequently avoid thecontact with the encapsulated antigen preventing denatura-tion. Moreover, other factors such as polymer swelling duringincubation as well as the affinity of the grafting reagent for thepolymer may influence their penetration.

3.2. Covalent grafting to PLGA microspheres

The CLSM represents a reliable method to clearly evaluate thelocalisation of the grafting. Using fluorescent derivatives ofWGA and BSA, this method showed the presence of a flu-

Fig. 2 – Visualisation of fluorescent ligand (WGA-FITC) grafting oBSA-FITC grafted microspheres (B) and (D) ungrafted microspherslight fluorescence should correspond to ligand adsorption.

a l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485

orescent crown on the surface of microspheres (Fig. 2A–D).This method also showed that the adsorption (electrostaticor hydrophobic) of WGA-FITC and BSA-FITC was negligiblesince no fluorescent crown was evident when incubation ofmicrospheres with these derivatives was performed with-out activation of the COOH. This was also confirmed by thequantification of the grafting and adsorption of WGA-FITC(3.03 ± 0.05 and 0.39 ± 0.10 �g/mg) and of BSA-FITC (5.66 ± 0.52and 0.99 ± 0.01 �g/mg). Hence, the presence of WGA and BSAat the surface of the microspheres can be attributed to graftingand not to adsorption. The lack of penetration of the graftingreagent inside the microparticles is necessary if grafting hasto be performed on protein-encapsulated microspheres pro-duced by the WOW method to avoid a denaturation and/orgrafting of the protein inside the inner compartments of themicrosphere. Since carbodiimide reagent cannot be visualisedusing CLSM inside the microspheres, we incubated the micro-spheres with a small fluorescent molecule with a similar MW(fluorescein). After incubation for the same time used for thegrafting (i.e., 2 h), CLSM showed the lack of penetration withinthe microspheres (data not shown) suggesting that the graft-ing of a ligand on protein-containing microspheres should befeasible.

The molar grafting efficiency (pmol/mg of microsphere)varied according to the molecular weight of the ligands(WGA, RGD and mannose) and molecules (BSA, RAD and PLL)

(Table 1).

Taking into account the difference in size of the parti-cles used, the grafting efficiency of BSA was comparable tothat reported previously using the same carbodiimide method

n PLGA microspheres by CLSM. (A) WGA-FITC and (C)es: incubation performed without COOH activation. The

e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a

Table 1 – Grafting efficiency and surface charge ofdifferent surface-grafted microspheres.

Grafting efficiency Zeta potential(mV)

�g/mg pmol/mg

Ligand-grafted MSWGA-MS 1.36 ± 0.28 43 −32 ± 7RGD-MS 0.16 ± 0.01 220 −48 ± 5Mannose-MS 0.33 ± 0.03 880 −26 ± 3

Molecule-grafted MSBSA-MS 2.81 ± 0.24 43 −37 ± 10RAD-MS 0.17 ± 0.02 230 −51 ± 4PLL-MS ND ND +34 ± 6

Plain MS

(aumswouzoPootnpm

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– – – −81 ± 13

ND, not determined; –, do not apply.

Thiele et al., 2001). The molar grafting efficiency of BSAnd WGA was comparable, which is essential since BSA wassed as a control of the cellular uptake of WGA-graftedicrospheres. RGD and RAD containing peptides also had a

imilar molar grafting efficiency. The grafting of PLL, whichas not directly determined, was verified by the measuref the zeta potential of microspheres in comparison withngrafted microspheres. We noticed a significant increase ofeta potential from −81 to +34 mV, indicating a cationisationf microspheres (Table 1). Such a degree of cationisation withLL is usually obtained (Thiele et al., 2003). The zeta potentialf microspheres was influenced by the grafting of moleculesr ligands. Indeed, their presence increased the zeta poten-ial compared to ungrafted microspheres but remained highlyegative (ranging from −26 to −51 mV). Such a negative zetaotential should not favour a significant interaction with cellembranes.If the CLSM gave evidence of the presence of WGA on

he surface of the microspheres, the use of the atomicorce microscopy allowed us to visualise this surface with

resolution well beyond that of an optical microscope. Forxample the detection of discontinuity of protein film smallerhan 400 nm can be revealed by topographic AFM imagesVie et al., 1998). At larger scan (50 �m × 50 �m), the AFMbservation showed the polydispersity of microspheres pre-iously presented in SEM (data not shown). Because of theirphericity and large diameter, the microspheres saturated themage contrast, and only partial part of the microspheres ishown. The surface topography of 800 nm × 800 nm imagesf ungrafted microspheres and WGA-grafted microspheresere compared (Fig. 3). In both cases, invaginations observed

y SEM appeared clearly at the surface (Fig. 3A and D) andheir depth was of several tens of nanometers. During thecan, the amplitude and phase variations of the signal wereimultaneously recorded (Fig. 3B, E and C, F). In the phaseode, the signal variations were related to the composition,

riction, viscoelasticity and adhesion of the sample surfaceithout topographic dependence (Bar et al., 1998; Martinez

nd Garcia, 2006). The topographic, amplitude and phasemages between both samples clearly showed differences.omparison between the topographic images (Fig. 3A and) revealed the presence of a discontinuous monomolecular

l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485 479

layer on the surface of WGA-grafted microsphere. The sectionson these topographic images presented in Fig. 3G showed amean apparent height of this layer equal to 1.8 nm ± 0.2 nm.The mean apparent height is consistent with the formationof a monomolecular layer film of WGA given its relatively lowmolar weight (36 kDa), and the fact that dehydration of thesample reduces even more the film height (Dubreil et al., 2003;Mackie et al., 1999).

This monomolecular layer also appeared in the amplitudeimage (Fig. 3B and E) as well as in phase image (Fig. 3C and F)demonstrating that the surface properties had changed as aresult of the protein grafting.

3.3. Uptake

In order to study cellular uptake of microparticles ex vivo,the use of fluorescently labelled microparticles is the mostcommon experimental approach (Thiele et al., 2003, 2001).However, the polydispersity of the microsphere populationcan influence the result when the intensity of fluorescenceis measured (e.g., by FACS or fluorimetry). Indeed, the fluores-cence of one particle may be equivalent to the fluorescenceof several smaller particles, and consequently the quantifica-tion of the cellular uptake can be biased. For that reason weused CLSM for the quantification of the cellular uptake thatshowed the localisation of microspheres, and thus allowedus to distinguish between internalized microspheres fromadsorbed microspheres at the surface of cells. Moreover apreliminary experiment with labelling of the actin cytoskele-ton of the macrophage membrane with methylrhodamineisothiocyanate-phalloidine showed that particles at the sur-face of macrophages and not within the cells, and these werenot considered as internalized for our evaluation. Evaluationsin triplicate of culture plates by counting 5 fields containing25 cells per field allowed a precise quantification of the meannumber of internalized microspheres.

The uptake of microspheres by the macrophages wasdifferent according to the type of surface modification. What-ever the ligands (WGA, RGD and mannose) or molecule(PLL) grafted, the uptake significantly increased with theMS/MA number ratio (Fig. 4). The uptake of BSA- andof RAD-grafted microspheres was not significantly differ-ent from ungrafted microspheres, and was not significantlyincrease with the MS/MA ratio. This was not surprisinggiven the lack of receptors for BSA and RAD (involving anon-specific mechanism of uptake), and the fact that thesesurface-modified microspheres remained highly negative andthus have a low interaction potential with cell membranes(Table 1). However, a much higher uptake was observed formannose-, WGA-, and RGD-grafted microspheres (Fig. 4). Ata microsphere/macrophage ratio of 20, the cellular uptake ofmannose-, WGA-, and RGD-grafted microspheres were aroundfour-times, three-times and twice higher than for ungraftedmicrospheres, respectively (Fig. 4A–C).

As described previously, the influence of surface mod-ification on uptake has been largely documented with

nanoparticles, and this has been shown more specifically withthe ligands used in the current study. An increase in theuptake of PLGA nanoparticles grafted with WGA by caco-2(Weissenboeck et al., 2004) or A549 malignant pulmonary cell

480 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485

Fig. 3 – Atomic force microscopy of ungrafted and WGA-grafted microspheres. (A, B, and C) images in the topographic,amplitude and phase mode of a ungrafted microsphere, (D, E, and F) images in the topographic, amplitude and phase modeof a WGA-grafted microsphere. (G) Section analysis on topographic images.

e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485 481

Fig. 4 – Uptake of different surface-grafted microspheres (MS) as a function of microsphere/macrophage ratio in comparisonto ungrafted MS at 37 ◦C. (A) WGA- and BSA-grafted MS, (B) RGD- and RAD-grafted MS, (C) mannose-PEG3-NH2-grafted MS or(

lgo2n5aamspeTmopHntowdttGn

D) PLL-grafted MS. Data show the mean ± S.D. (n = 3).

ines (Mo and Lim, 2004, 2005) has been demonstrated. RGD-rafted pegylated PLGA nanoparticles had a higher uptaken follicle associated epithelium co-culture (Garinot et al.,007; Gullberg et al., 2006). The uptake of mannan-coatedanoparticles on mouse macrophage cell line (J774E) was0% higher than that of the uncoated nanoparticles (Cui etl., 2003). The influence of surface modification with lig-nd on the uptake of larger particles (i.e., microspheres) isuch less well documented. An increase in the uptake PLL

urface-modified microspheres (1.0 and 4.5 �m carboxylatedolystyrene microspheres) has been shown on APCs (Thielet al., 2001) compared to BSA surface-modified microspheres.his surface modifier is not implicated in a specific (ligand-ediated) uptake mechanism. An increase in the uptake

f IgG grafted surface microspheres (1.0 �m carboxylatedolystyrene) has also been shown on APCs (Thiele et al., 2003).owever, since specific and non-specific uptake can simulta-eously occur, the evaluation of their relative contribution tohe overall uptake should be studied. Such results should bef interest for biological applications requiring microparticleshich allow a high loading of hydrophilic entities and/or arug controlled release. In vivo antimicrobial vaccination by

he mucosal route, and ex vivo loading of APCs for cellularherapy in oncology and haematology (Waeckerle-Men androettrup, 2005) for which the optimal strategy of loading hasot yet been established, could be the most promising appli-

cations. For these applications, the optimization of the uptakeof the microparticles by cells of interest (i.e., APCs) is manda-tory since a low level of particle uptake by cells is a limitingfactor.

Furthermore, a non-linearity of the uptake of these threeligand-grafted microspheres was observed (Fig. 4A–C). Accord-ing to the ligand used, the non-linearity was more or lesspronounced. For RGD, the non-linearity was greater than forWGA and for mannose, respectively. Conversely, in the caseof cationic PLL-grafted microspheres, the uptake was notsaturated but was linear (Fig. 4D), while the uptake was signif-icantly greater than for the ungrafted microspheres.

To explain the mechanism of uptake of these ligand-graftedmicrospheres, we have studied the influence of the tempera-ture (4 ◦C) and cytochalasin D which are non-specific inhibitorsof phagocytosis. For the three ligands (Fig. 5), we observed asignificant decrease of the cellular uptake under these twoexperimental conditions. A minimal uptake remained thatwas not significantly different under the experimental con-ditions. At 37 ◦C, the cells are metabolically active so that anenergy-consuming uptake of microspheres can take place. Incontrast, at 4 ◦C, the fluidity of the cell membrane and the

metabolism is reduced so that the uptake of microspheres isdecreased. The influence of the temperature is a criterion ofanalysis for the study of the cellular uptake; many authors useit to discriminate a passive phenomenon. The inhibition of

482 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485

Fig. 5 – Uptake of ligand-grafted microspheres bymacrophages at a microsphere/macrophage ratio of 2.5 indifferent conditions. (Black bar) control conditioncorresponding to initial condition (2 h of incubation, 37 ◦Cand without specified medium), (blank bar) incubation witha free correspondent ligand, (hatched bar) incubation at 4 ◦C

Fig. 6 – Specific and non-specific uptake of ligand-graftedmicrospheres as a function of microsphere/macrophageratio. (A) WGA-grafted MS, (B) RGD-grafted MS and (C)mannose-PEG3-NH2-grafted MS. Data obtained with atwo-sites mathematical model: one saturable and onenon-saturable uptake from data presented in Fig. 4. Data

affinity of the ligands for their receptors. The mathematical

and (grey bar) incubation with cytochalasin D.

the uptake observed with cytochalasin D suggested a phago-cytosis, which is an actin-dependent mechanism. Similarly,a complete inhibition of phagocytosis by macrophages anddendritic cells has been achieved by the addition of cytocha-lasin D (Thiele et al., 2001). Furthermore, the influence of thefree ligand allowed evidencing a phagocytosis using a spe-cific pathway. Such influence of free ligand has been used forligand-modified nanoparticles which are captured by specificendocytosis. In a study showing a significant increase in theuptake of WGA-conjugated PLGA, Mo and Lim (Mo and Lim,2004) showed a decrease of the uptake of their nanoparticlesby A 549 cells (in vitro model of the Type II alveolar epithelialcells) in the presence of free WGA. In a study showing that thedegree of uptake was influenced by the presence of lectin onnanoparticle, the effect of exogenous free lectins on the inhibi-tion of the uptake of WGA, LTB or ConA-coated nanoparticlesby caco-2 and OK cells (opossum kidney carcinoma cell) hasalso been shown (Russell-Jones et al., 1999).

The non-linearity can be ascribed to the simultaneouspresence of two phenomena, i.e., a linear non-specific uptake,and a non-linear specific saturable uptake. However, depend-ing on the relative contribution of each phenomenon, thesaturation can be more or less obvious according to theligands (Fig. 4). Indeed, if the non-specific (linear) processpredominates over the specific process, the saturation is lessobvious. Since the uptake of microparticles may result fromboth specific and non-specific mechanisms, we performeda mathematical analysis according to a model incorporatingthe two simultaneous phenomena to estimate the relativecontribution of the specific and non-specific mechanismsthat should be saturable and non-saturable, respectively.Such a mathematical approach is rather unusual since weapplied it to particles as it is used usually for soluble ligandbinding studies. We showed that the specific and non-specificuptake varied according to the ligands (Fig. 6). For WGA, thespecific uptake was higher than the non-specific uptake forthe first four ratios tested (1, 2, 5 and 10). In the case of RGD,the specific cellular uptake was more significant than the

non-specific uptake whatever the particle-to-cell ratio used.For mannose, the specific uptake was only slightly higherthan the non-specific uptake for the first three ratios. Hence,

show the mean ± S.D. (n = 3).

according to the ligand, the contribution of the specific uptaketo the overall uptake was more or less significant, dependenton the MS/MA ratio used. Such feature indicates that thedemonstration of the influence of the ligand grafting onmicroparticle uptake is highly dependent on the experimen-tal conditions. Hence, from a methodological point of view,a range of microspheres-to-cell ratios should be used to fullycharacterize its potential without any bias. This differencein the relative contribution of the specific and non-specificmechanisms according to ligands may result from the numberof receptors present on macrophages and/or from the relative

study allowed the calculation of the maximal specific uptake,and of the affinity for each ligand-modified microsphere, aswell as a value representing the non-specific uptake (Table 2).

e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a

Table 2 – Uptake parameters of surface-graftedmicrospheres. Data obtained with a two-sitesmathematical model: one saturable and onenon-saturable uptake for WGA-, RGD-, mannose-graftedMS, and from a one-site non-saturable model forPLL-grafted MS. Bmax is the maximum specific uptake(mean number of MS par MA), Kd = 1/Ka where Ka is theaffinity constant of the ligand for the specific uptake (MAper mean number of MS), and NS is the non-specificuptake (mean number of MS par MA). Data show themean ± S.D. (n = 3).

Bmax Ka Ns

WGA-MS 5.52 ± 1.48 0.97 ± 0.51 0.31 ± 0.07RGD-MS 5.04 ± 0.90 0.64 ± 0.28 0.16 ± 0.05Mannose-MS 4.63 ± 0.31 0.51 ± 0.06 0.44 ± 0.03PLL-MS ND ND 0.49 ± 0.01

TndmlbaKoppamairtStrsa

4

TcufpItvttessd

r

ND, not determined.

he differences were not statistically significant for Bmaxor for Ka but only for the non-specific uptake. The lack ofifference in Bmax for the three types of ligand-modifiedicrospheres may suggest that the specific phagocytosis was

imited by the membrane cellular activity although the num-er of each receptor on the cell membrane may be differentccording to the ligands. There was only a trend for a highera for WGA which was 1.5- to 2-fold higher to the valuesbtained with RGD and mannose (Table 2). RGD was the ligandresenting the lowest non-specific uptake while the mannoseresented a significant non-specific uptake close to that ofnon-specific phenomenon (Table 2). The uptake of cationicicrospheres was linear, characterizing a non-specific mech-

nism (Table 2). These results could have implications for then vivo use of this strategy. Indeed, the microsphere-to-cellatio should be rather low in vivo, as a result of the dissemina-ion of the particles on the epithelium after administration.ince, the contribution of the specific uptake was higher thanhe non-specific uptake at low microsphere-to-cell ratio, theseesults demonstrate that ligand grafting can be a suitabletrategy for improving the cellular uptake of microparticles,s shown previously for nanoparticles.

. Conclusion

his work has shown that surface modification by graftingell-specific ligands on PLGA microparticles increased theirptake by phagocytosis by macrophages. Such uptake resulted

rom the simultaneous occurrence of a linear non-specificrocess and of a non-linear specific and saturable process.

n addition we have shown that the relative contribution ofhe specific and non-specific processes to the overall uptakearies greatly according to the ligands, and is dependent onhe particle-to-cell ratio. Hence, the decision to choose a ligand

o increase the cell uptake of microparticles needs a carefulvaluation to be sure that its contribution to the total uptake isignificant. The development of surface-modified PLGA micro-pheres with suitable ligands could be exploited for increasingrug and antigen targeting to specific cells.

l s c i e n c e s 3 6 ( 2 0 0 9 ) 474–485 483

Acknowledgments

This research was partially funded by Region Bretagne (PRIR- ZVNPX 337) and NoE EPIZONE (EUFP6). The authors thankEvelyne Hutet for her skillfull technical assistance in the col-lection of macrophages and Philip Wakeley.

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