PEGylated Degradable Composite Nanoparticles Based on Mixtures ofPEG-b-Poly(γ-benzyl L-glutamate) and Poly(γ-benzyl L-glutamate)

Ma. Elisa Martınez-Barbosa,†,‡ Sandrine Cammas-Marion,§ Laurent Bouteiller,| Christine Vauthier,‡ andGilles Ponchel*,‡

UMR CNRS 8612, Laboratoire de Pharmacie et Biopharmacie, Faculte de Pharmacie, Universite Paris-Sud, 5 rue J.B. Clement,92290 Chatenay-Malabry, France, UMR 6226 CNRS-ENSCR, Equipe Chimie Organique et Supramoleculaire, Avenue duGeneral Leclerc, 35700 Rennes, France, and UMR CNRS 7610, Chimie des Polymeres, Universite Pierre et Marie Curie,4 Place Jussieu, 75252, Paris cedex 05, France. Received January 15, 2009; Revised Manuscript Received May 18, 2009

In the present work, the possibility to obtain PEGylated nanoparticles from two PBLG derivatives, PEG-b-poly(γ-benzyl L-glutamate), PBLG-PEG-60, and poly(γ-benzyl L-glutamate), PBLG-Bnz-50, by nanoprecipitation hasbeen investigated. Particles were prepared not only from one polymer (PBLG-PEG-60 or PBLG-Bnz-50), butalso from mixtures of two PBLG derivatives, PBLG-PEG-60 and PBLG-Bnz-50, in different ratios (90/10, 77/23, and 40/60 wt %). Because of the presence of PEG chains, hydrophilic particles were obtained, which wasconfirmed by � potential measurements (� from -13 mV and -21 mV) and by isothermal titration microcalorimetry(ITC). This last technique has shown no heat exchange when BSA was added to PEGylated nanoparticles. Further,complement activation has been evaluated by crossed immuno-electrophoresis demonstrating that the introductionof 77 wt % of PEGylated PBLG chains in the particles was enough to ensure a low complement activationactivity. This effect was strongly correlated to the � potential of the particles, which decreased with an increaseof PEG chains content. Interestingly, such properties are of interest for the preparation of degradable stealthnanocarriers. Moreover, it is suggested that the introduction of a reasonable amount (up to 20 wt %) of a secondcopolymer in the particle composition can be possible without modifying their stealth character. Moreover, thepresence of this second copolymer would help to introduce a second functionality to the particles.

INTRODUCTION

Over the past years, nanoparticulate carriers have beendeveloped in order to improve efficiency and site-specificity ofdrug delivery (1, 2). As a result of their small size, this firstgeneration of nanocarriers can be administrated intravenously.However, such small hydrophobic particles are rapidly (half-time of clearance about 1 min) and efficiently (about 90%uptake) cleared from the circulation due to their recognition byopsonins (3-5). Therefore, to address these limitations, a secondgeneration of nanocarriers is under development taking intoaccount the various parameters which influence carrier recogni-tion by reticuloendothelial system (3-5). It has been shownthat the coating of hydrophobic nanocarrier surfaces by hydro-philic polymers, such as poly(ethylene glycol) (PEG), inducesa significant decrease of the opsonisation process (6). Thecoating has to be dense and flexible in order to reduce all typesof interactions with hydrophobic nanocarrier surfaces. Moreover,hydrophilic polymer chains, such as PEG, have to be wellanchored on nanoparticles to avoid desorption of chains (4).Surface modification on hydrophobic nanoparticulate systemshas been carried out. Coating or covalent coupling methods (7, 8)have been proposed to introduce hydrophilic chains on nano-particle surface. Hydrophilic polymers such as poly(ethyleneglycol), poly(vinyl alcohol), poloxamers, or dextran have been

shown to decrease significantly protein adsorption (4). In orderto obtain a high density of hydrophilic chains on the surfaceand an efficient anchoring of these chains in the carrier,core-shell type nanoparticles and macromolecular micellesformed in aqueous media by self-assembly of amphiphilic blockcopolymers have attracted considerable attention for applicationas drug delivery systems (9-14). Besides overcoming the rapidrecognition of nanoparticles by the reticuloendothelial system,research has been also focused on overcoming the many-facetedproblem of biocompatibility. In this context, efforts have beendirected toward the use of natural-like materials such as poly(R-amino acids) (15). Poly(γ-benzyl L-glutamate), PBLG, a syn-thetic polypeptide, has attracted attention for biomedicalapplications (15-21) because of the presence of a degradableamide bound in the polymer backbone (21) and a carboxylicacid function in the side chain, available after hydrolysis of thelateral benzyl ester. Poly(ethylene oxide)-block-poly(γ-benzylL-glutamate) (22, 23) allowed macromolecular micelle formationas a result of their amphiphilic nature (24, 25). Recently, inresponse to the need for targeting systems, poly(γ-benzylL-glutamate) and poly(ethylene glycol) diblock copolymers end-capped with a galactose moiety have been proposed for site-specific drug delivery (26). In this context, we focused ourinterest on the synthesis of PBLG derivatives using initiatorsallowing the preparation of biocompatible nanoparticulatesystems possessing different specific functionalities. For that,PBLG derivatives were synthesized by ring-opening polymer-ization (ROP) of γ-benzyl L-glutamate N-carboxyanhydride(BLG-NCA) using selected amine-terminated initiators (27).

In the present work, we report the possibility to obtainPEGylated nanoparticles from two PBLG derivatives, PBLG-PEG-60 and PBLG-Bnz-50, by nanoprecipitation. Compositenanoparticles with different PBLG-PEG-60/PBLG-Bnz-50 ratio

* Corresponding author. Gilles Ponchel, e-mail: gilles.ponchel@u-psud.fr, Tel: +33 (0)1 46 83 59 19, Fax: +33 (0)1 46 61 93 34.

† Present address: Departamento de Investigacion en Polımeros yMateriales (DIPM), Universidad de Sonora. Rosales y Blvd. LuisEncinas s/n. C.P. 83000, Hermosillo, Sonora, Mexico.

‡ UMR CNRS 8612.§ UMR 6226 CNRS-ENSCR.| UMR CNRS 7610.

Bioconjugate Chem. 2009, 20, 1490–14961490

10.1021/bc900017c CCC: $40.75 2009 American Chemical SocietyPublished on Web 07/13/2009

have been prepared and characterized. Interactions of thedifferent nanoparticles with bovine serum albumin (BSA) havebeen measured by ITC. Moreover, complement activation hasbeen evaluated by crossed immuno-electrophoresis.

MATERIALS AND METHODS

Materials. N,N-Dimethylformamide (DMF, Acros, 99%) andbenzylamine (Janssen Chimica) were distilled under reducedpressure over BaO and KOH, respectively, and stored underargon atmosphere. γ-Benzyl-L-glutamate N-carboxyanhydride(γ-BLG-NCA), from ISOCHEM-SNPE, was used as received.Methoxy poly(ethylene glycol) amine (mPEG-NH2), Mjw ) 5000Da from Shearwater Corporation was dried under vacuum overP2O5 at 30 °C for 24 h. Bovine serum albumin (BSA) waspurchased from Sigma. Water was purified by reverse osmosis(Milli-Q, Millipore). Poloxamer 188 (Lutrol F68), diethyl ether,and other solvents used were analytical grade, and all otherchemicals were commercially available reagent grade.

Synthesis and Characterization of PBLG-Derivatives.PBLG-derivatives, PBLG-Bnz-50, and PBLG-PEG-60 wereprepared by anionic ring-opening polymerization as describedpreviously (27). Briefly, PBLG-Bnz-50 was obtained by anionicring-opening polymerization of γ-benzyl-L-glutamate N-car-boxyanhydride initiated by benzylamine in DMF at 30 °C. Thereaction mixture was stirred at 30 °C until the characteristicNCA bands disappeared from FT-IR spectrum and then pouredinto a large volume of cold diethyl ether. The precipitate wasdried under vacuum at 35 °C for at least 12 h. This precipitation,purification, and drying procedure was performed twice. Themolecular weight of PBLG-Bnz-50 was estimated from intrinsicviscosity measured in DMF at 25 °C using the Mark-Houwinkequation for PBLG in DMF at 25 °C (28).The molecular weightestimated for the PBLG-Bnz-50 was Mj w ) 45 000 g ·mol-1.Similarly, PBLG-PEG-60 was obtained by anionic ring-openingpolymerization of γ-benzyl-L-glutamate N-carboxyanhydrideinitiated by methoxy poly(ethylene glycol) amine (mPEG-NH2),average Mj w ) 5000 g ·mol-1, in DMF at 30 °C. The reactionmixture was stirred at 30 °C until the characteristic NCA bandsdisappeared from FT-IR spectrum and poured into a largevolume of cold diethyl ether. The precipitate was filtered,washed with water and diethyl ether, and then dried undervacuum at 35 °C for at least 12 h. A second purificationprocedure was performed. DjPn of the PBLG segment in PBLG-PEG-60 was determined from 1H NMR spectra by the ratiobetween the peak intensities of methylene protons of the PEGchain, OCH2CH2, and the benzyl protons of PBLG chain,COOCH2C6H5. A DjPn of 231 was found.

Nanoparticles Preparation. Nanoparticles were preparedusing a slightly modified nanoprecipitation technique describedelsewhere (29), in the absence of any surfactant agent, eitherfrom one polymer (PBLG-PEG-60 or PBLG-Bnz-50) or frommixtures of these two polymers in different ratios (PBLG-PEG-60/PBLG-Bnz-50 at 90/10, 77/23, and 40/60 wt % ratio andnamed composite nanoparticles). Briefly, 15 mg of one polymeror a mixture of polymers were dissolved in 5 mL of THF at 30°C during 18 h, without stirring. The solution was thenhomogenized under gentle agitation for a few seconds andadded, by dripping to 10 mL of Milli-Q water, under magneticstirring. The mixture was left under magnetic stirring for 15min and then transferred in a Teflon-coated recipient. Thesolvent was evaporated, at 30 °C, under a light air flux. In orderto eliminate residual solvent, nanoparticles were further dilutedwith 5 mL of Milli-Q water, and evaporation was carried outto adjust the volume of the nanoparticle suspension to 10 mL.Preparations were kept at 4 °C until characterization.

Nanoparticles Characterization. Size Distribution. Meandiameter (n ) 3) was determined, after suitable dilution of bulk

suspensions in Milli-Q water, by dynamic laser light scattering(Nanosizer Coulter N4 Plus, Margency, France).

� Potential. � potential measurements (n ) 5) were carriedout using a Zetasizer 4, Malvern Instrument after adequatedilution of 200 µL of nanoparticles suspensions into 10 mL of1 mM KCl filtered through a 0.22 µm filter.

Transmission Electron Microscopy. Nanoparticles were ob-served by TEM (transmission electron microscope, Philips EM208). 7 µL of nanoparticles suspension were half-diluted in a0.125% (w/v) poloxamer solution and placed on a Formvar-carbon film previously coated on a copper grid (400 mesh). After6 min of deposition at room temperature, nonadherent nano-particles were eliminated and the sample was stained byphosphotungstic acid (1%) during 30 s. TEM bright fieldimaging was performed under a 80 kV accelerating voltage.

Isothermal Titration Microcalorimetry (ITC) Studies. ITCexperiments were carried out with an VP-ITC, MicrolCal.Interactions between bovine serum albumin (BSA), a modelglobular protein, and nanoparticles constituted by PBLG-Bnz-50 or PBLG-PEG-60 were evaluated. Solution of BSA at 5.4× 10-2 mM was placed in a 295 µL continuously rotating (270rpm) syringe. Nanoparticle suspension at 2.7 × 10-2 mM ofpolymer was placed in the sample cell of 1.43 mL. A first 2 µLaliquot was injected, without taking into account the observedheat, to remove the effect of solute diffusion across the syringetip during the equilibration period. Then, injections of 10 µLof the BSA solution were made at intervals of 10 min.Experiments were carried out at 25 °C.

EValuation of Complement ActiVation by Crossed Immuno-Electrophoresis. The specific activation of the C3 componentof the complement from human serum and induced by thenanoparticle surface was evaluated by comparative measure-ments of C3 cleavage into C3b. Nanoparticle suspensions (1.5mg mL-1) were first concentrated by evaporation of part of thewater to reach a nanoparticle concentration of 9 mg mL-1 or 6mg mL-1 in order to obtain a surface area of 500 cm2 in avolume V e 100 µL. The amount of C3 and C3b was measuredby crossed immunoelectrophoresis by an adaptation of themethod described elsewhere (7, 30) after incubation of thenanoparticles with serum. Briefly, human serum obtained fromhealthy donors was stored at -80 °C until use. Equivalentvolumes of nanoparticle suspensions, corresponding to a surfacearea of 500 cm2 in 100 µL, were incubated with 50 µL of humanserum and 50 µL of veronal-buffered saline containing 0.15 mMCa2+ and 0.5 mM Mg2+ in ions (VBS2+), under gentle agitation,for 1 h at 37 °C. Nanoparticle surface area was calculated,assuming that particles were spherical and using the followingequations:

where S and V are the surface area and volume, respectively,of n spherical nanoparticles of average radius r. S(sp) is thespecific surface area of 1 g of spherical nanoparticles of averagediameter d and density F (taken as F ) 1.2 g/cm3 relative to Ffor PBLG).

After incubation with human serum, samples (7 µL) weresubjected to a first electrophoresis in 1% agarose gel for about45 min. The second-dimension electrophoresis was carried outduring 18 h with 1% agarose gel on Gelbond films in thepresence of a polyclonal antibody to human C3, recognizingboth C3 and C3b (complement C3 antiserum developed in goat,Sigma). Buffer used for electrophoresis migration was Trisveronal buffer diluted 1:5 in water and prepared as follow: 44.3 g

S ) n4πr2 (1)

V ) n4/3πr3 (2)

leading to S(sp) ) S/V ) 6/dF (3)

PEGylated Degradable Composite NPs Bioconjugate Chem., Vol. 20, No. 8, 2009 1491

of Tris(hydroxy-methyl-amino-methane), 22.4 g of phenobar-bital, and 0.53 g of lactate calcium were dissolved in 1 L ofwater at 50 °C, sodium azide was added to avoid bacterialdevelopment, pH was adjusted to pH ) 8.6, if necessary. C3and C3b migration was followed by Coomasie blue staining.The height of the peaks given by the immunoprecipitation ofC3 and C3b gave a semiquantitative estimation of the amountof each component in the serum. The complement activationfactor (CAF) was expressed as the ratio of the peaks height asfollows:

RESULTS

Nanoparticle Preparation and Characterization. Possibili-ties to prepare, by nanoprecipitation, nanoparticles, either fromone polymer (PBLG-Bnz-50 or PBLG-PEG-60) or from mix-tures of these two polymers (composite nanoparticles) indifferent ratios, have been evaluated. As shown in Table 1,nanoparticles could be easily and reproducibly obtained withoutany surfactant agents by a slightly modified method describedelsewhere (29). THF was selected, being both a good solventof the polymer and miscible to water. Nanoparticle diameter,determined by dynamic laser light scattering, was typically lowerthan 100 nm. From transmission electron microscopy (Figure1), it could be seen that nanoparticles prepared from bothpolymers exhibited ellipsoidal geometries, whose dimensionswere compatible with dynamic light scattering measurements.Moreover, it can be seen that particles issued from mixtures ofthe two polymers were quite homogeneous, suggesting that theyformed a single population.

The � potential (Table 1) was similar for nanoparticles at100/0, 90/10, and 77/23 wt % ratio. A weak decrease in the �potential was observed for a 40/60 wt % ratio. As expected,the stronger � potential value was found for non-PEGylatednanoparticles (0/100 wt % ratio).

Isothermal Titration Microcalorimetry (ITC) Studies. Inorder to evaluate the capacity of nanoparticles to avoid proteinadsorption and, therefore, to confirm the presence of PEG chainsat the surface of nanoparticles, isothermal titration microcalo-rimetry (ITC) has been applied. PEGylated (PBLG-PEG-60) andnon-PEGylated (PBLG-Bnz-50) nanoparticle-protein interac-tions were measured using bovine serum albumin (BSA) asmodel globular protein. As shown by Figure 2, the interactionof BSA with the nanoparticle surface is an exothermic phe-nomenon. The area underneath each injection peak is equal tothe total heat released for that injection. In the case ofnanoparticles constituted by PBLG-Bnz-50 (Figure 2a), signalsafter each BSA injection were much stronger than signals ofnanoparticles constituted by PBLG-PEG-60 (Figure 2b). Thecorresponding control dilution experiment consisted of BSAinjected in water and is shown by Figure 2c.

Evaluation of Complement Activation by Crossed Im-muno-Electrophoresis. Figure 3 presents the immuno-electro-phoresis patterns of the complement proteins C3 and C3b inserum incubated with the different nanoparticles prepared and

of control experiments. The control experiments performedunder experimental conditions in the absence of nanoparticles(Figure 3a) showed a major peak of protein C3 and a smallpeak of protein C3b. It gives the complement activationbackground under experimental conditions. The CAF valuedetermined for the background activation was 0.16. In theabsence of divalent ions (Figure 3b), no C3b peak can be seenon the immuno-electrophoregram in agreement with the factthat activation of the complement system requires divalentcations.

The immuno-electrophoregram of serum incubated withnanoparticles presented both C3 and C3b peaks. The height ofthe C3b peak was less pronounced for the serum incubated withPEGylated PBLG-PEG-60 nanoparticles (Figure 3d) than theone observed for serum incubated with non-PEGylated PBLG-Bnz-50 nanoparticles (Figure 3c). As reported in Table 2, theCAF was higher for serum incubated with the non-PEGylatednanoparticles (0.40) than the CAF measured for serum incubatedwith the PEGylated nanoparticles (0.21), indicating that non-PEGylated particles could be rapidly opsonized by serumproteins. The CAF value of the serum incubated with thePEGylated nanoparticles was close to the CAF value of thebackground activation of the complement under experimentalconditions.

The immuno-electrophoresis patterns of the serum incubatedwith the different composite nanoparticles (Figure 3e,f,g) werevery similar to the immuno-electrophoresis pattern shown forPBLG-PEG-60 nanoparticles (Figure 3d). The CAF valuesreported in Table 2 only showed a slight shift toward a highervalue for the serum incubated with the composite nanoparticlescontaining the lower percentage (40 wt %) of PBLG-PEG-60polymer. The CAF values obtained for serum incubated withthe two other composite nanoparticles were the same than theCAF of serum incubated with nanoparticles prepared with 100wt % PBLG-PEG-60 polymer.

DISCUSSION

The development of efficient targeting systems necessitatesthe simultaneous introduction of a set of functionalities withinthe system itself, regardless of its physical structure. Variouspolymers have been used to prepare nanoparticles for pharma-ceutical applications, and different strategies have been proposedto introduce specific functionalities, e.g., optimized drugencapsulation, controlled release properties, stealthiness in thebloodstream, organ or cell targeting by ligands. It has beenshown previously (27) that copolymers of poly(γ-benzyl L-glutamate), PBLG, bearing pharmaceutically interesting moietiescould be quite easily prepared. Interestingly, mixtures of thesecopolymers can easily self-associate, which offers a convenientmeans for introducing by a soft technique desired functionalitieswithin the particles.

In the present work, we investigated the possibility toprecisely tune nanoparticle surfaces by introducing an optimalamount of a PEGylated copolymer of PBLG within the structureof nanoparticles. The nanoprecipitation method could be usedconveniently to prepare nanoparticles either from one polymer(PBLG-Bnz-50 or PBLG- PEG-60) or from mixtures of thesetwo polymers (composite nanoparticles) in different ratios.Nanoparticles could be easily and reproducibly obtained withoutany surfactants by a slightly modified method describedelsewhere (29) as a result of the hydrophobic nature of PBLG.Moreover, DLS and TEM measurements showed that very smallparticles (lower than 100 nm) could be prepared. In addition,the homogeneous aspect of the particle shapes for the differentpreparations made from the polymer mixtures suggested thatin this case the suspensions were composed of a singlepopulation of composite nanoparticles.

Table 1. Physicochemical Characteristics of NanoparticlesConstituted by PBLG-PEG-60 and PBLG-Bnz-50

polymer ratio (wt %) mean size (nm)b � potential (mV)c

100/0a 58 ( 20 -13.3 ( 0.690/10a 62 ( 20 -12.6 ( 0.377/23a 60 ( 21 -13.6 ( 0.340/60a 54 ( 17 -16.1 ( 0.60/100a 85 ( 30 -21.1 ( 0.2

a PBLG-PEG-60/PBLG-Bnz-50 ratio (in wt %) in the formulation.b Measured using dynamic laser light scattering, n ) 3. c Measuredusing a Zetasizer 4 Instrument, n ) 5.

CAF ) C3b/(C3 + C3b) (4)

1492 Bioconjugate Chem., Vol. 20, No. 8, 2009 Martınez-Barbosa et al.

Characterization by ITC of PBLG-PEG-60 or PBLG-Bnz-50 nanoparticle-protein interactions using bovin serum albumin(BSA), a model globular protein, permitted confirmation of thedifference in surface properties of PEGylated and non-PEGy-lated nanoparticle suspensions. Theoretically, PEG chains ofPEGylated nanoparticles should avoid or, at least, reduce proteinadsorption on the nanoparticle surface as a result of stericrepulsions and hydrophilization induced by PEG chains. In thecase of nanoparticles constituted by PBLG-Bnz-50 (Figure 2a),signals after each BSA injection were higher than signalsobserved for nanoparticles constituted by PBLG-PEG-60 (Figure2b). From this result, it could be concluded that interactions ofBSA with nanoparticle surfaces were stronger in the absenceof PEG chains.

Quantitative interpretation of observed enthalpy effect afteran adsorption process (∆Ηads) could be difficult because various

factors can contribute to this effect. For example, Norde andLyklema (8, 31) took into account several factors while studyingadsorption of human plasma albumin and bovin pancreasribonuclease on negatively charged polystyrene surfaces. Someof these factors were as follows: the structural changes that theprotein molecule and other involved molecules (for example,water and electrolyte) can undergo during the adsorptionprocess; the alterations in the hydration state, disruption of thesurface/solvent contacts, and simultaneous formation of protein/surface contacts; the modification of protein/solvent interactionsdue to differences of the chain density between dissolved andadsorbed protein molecules; and the effect of partners dilution.

Because such factors have not yet been elucidated for PBLG-Bnz-50 and PBLG-PEG-60 systems titrated with BSA, inter-pretation of ∆Ηads remains difficult. However, as shown inFigure 2, these studies made possible a qualitative surfacecharacterization of tested nanoparticles. From these results,enthalpies of interactions could be obtained after integration ofinjection signals and subtraction of control (Figure 2c). Valuesof ∆Ηads ) 414 kCal/mol and 83 kCal/mol were obtained forthe interaction of BSA with PBLG-Bnz-50 and PBLG-PEG-60nanoparticles, respectively. Thus, it could be concluded fromthe existence of this 5-fold factor between adsorption enthalpiesthat interactions of BSA with non-PEGylated nanoparticles weremuch important than those observed for PEGylated nanoparticles.

In Vitro evaluation of complement activation induced bynanoparticles is a very convenient method that can be used topredict their fate after intravenous injection in ViVo (4, 32).Indeed, most of the nanoparticles inducing a strong activationof the complement systems are generally rapidly taken up bymacrophages of the mononuclear phagocyte systems, whilenanoparticles appearing as low or non-activators of the comple-ment systems are not recognized by the macrophages and remainin the bloodstream. In the present work, we have investigatedthe activation of the complement system induced by both PBLG-PEG-60 nanoparticles and PBLG-Bnz-50 nanoparticles, as wellas by composite nanoparticles made from blends of both typesof polymers at different composition. The CAF value of PBLG-Bnz-50 nanoparticles was much higher than the CAF value of

Figure 1. Transmission electron microscope micrographs of the nanoparticles. Simple nanoparticles constituted by (a) PBLG-PEG-60 (100/0 wt %)and (b) PBLG-Bnz-50 (0/100 wt %); Composite nanoparticles constituted by PBLG-PEG-60/PBLG-Bnz-50 at (c) 90/10, (d) 77/23, and (e) 40/60(wt %), respectively.

Figure 2. Plots of heat flow vs time of microcalorimetric titration of(a) PBLG-Bnz-50 nanoparticles and (b) PBLG-PEG-60 nanoparticles,by BSA solution. Injections of 10 µL, at 25 °C, of 5.4 × 10-2 mM ofBSA solution were made on nanoparticle suspensions (2.7 × 10-2 mM).Control of BSA dilution (c) was carried out by injections of BSAsolution in water.

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the PBLG-PEG-60 nanoparticles. This result indicated thetendency of uncoated nanoparticles to activate the complementsystem contrarily to nanoparticles bearing PEG chains. Thesenanoparticles which gave a CAF value close to the backgroundactivation of the serum under experimental conditions appearedas low complement activating nanoparticles. The results ob-served here are in good agreement with the literature, whichreports that nanoparticles prepared with PEG-containing poly-mers are generally low complement activators due to thepresence of PEG chains at the nanoparticle surface (4, 5). Thus,the results also suggested that the organization of PBLG-PEG-60 polymer chains to form the particle was highly efficient toexpose PEG moieties at the surface of nanoparticles, ensuringa coating layer able to avoid complement activation. All these

results were also in good agreement with the results obtainedfrom the evaluation of PEGylated and non-PEGylated nano-particle interactions with BSA. Indeed, the nanoparticles show-ing the lowest adsorption of BSA were also the lowestcomplement activators in agreement with the fact that surfacesof such nanoparticles are generally less opsonized as a resultof the presence of hydrophilic PEG chains. In contrast, non-PEGylated nanoparticles, which adsorbed higher amounts ofBSA, appeared as the highest complement activators in agree-ment with the fact that they are generally strongly opsonizedby seric proteins.

Composite nanoparticles also displayed a low capacity toinduce activation of the complement system. It is noteworthythat composite nanoparticles containing 90 and 77 wt % ofPBLG-PEG-60 showed identical behavior to nanoparticlescomposed of 100 wt % of this polymer. The PEG coatingformed at the surface of these nanoparticles seemed to haveidentical properties to the one formed on the surfaces of 100wt % of PBLG-PEG-60 based nanoparticles. Interestingly, thethree types of nanoparticles giving the lowest CAF value alsoshowed identical � potential suggesting that they showed similarproperties (Figure 4).

The composite nanoparticles containing the lowest amountof PBLG-PEG-60 (40 wt %) showed a slightly higher capacityto induce complement activation as suggested by their CAF,

Figure 3. Crossed immunoelectrophoresis of C3 antigens in human serum diluted 1:4 after incubation in the presence of nanoparticles (500 cm2).(A) Serum/VBS2+ (control of complement activation), background. (B) Serum/VBS2+-EDTA (negative control). (C) Serum/VBS2+/PBLG-Bnz-50nanoparticles. (D) Serum/VBS2+/PBLG-PEG-60 nanoparticles. (E) Serum/VBS2+/(PBLG-PEG-60/PBLG-Bnz-50, 90/10 wt %) nanoparticles. (F)Serum/VBS2+/(PBLG-PEG-60/PBLG-Bnz-50, 77/23 wt %) nanoparticles. (G) Serum/VBS2+/(PBLG-PEG-60/PBLG-Bnz-50, 40/60 wt %)nanoparticles.

Table 2. Relationship between PBLG-PEG-60 Content into thePBLG-PEG-60/PBLG-Bnz-50 Nanoparticles and the ComplementActivation Factor

sample CAF

controls background activation 0.16serum + EDTA 0.05

nanoparticles polymer ratio (wt%) 100/0 0.2190/10 0.2177/23 0.2140/60 0.250/100 0.40

1494 Bioconjugate Chem., Vol. 20, No. 8, 2009 Martınez-Barbosa et al.

which was shifted toward higher values. When combining thisresult with the fact that the � potential was also shifted towardthe value of the � potential measured for nanoparticles composedof 100 wt % non-PEG polymer, it can be suggested that thePEG coating characteristics of the surface of these nanoparticleswas modified in such a way that it reduced the effectiveness toprevent complement activation. The lower value of the �potential shown by these nanoparticles was in favor of amodification of the surface composition of the nanoparticles,which can be attributed to a decrease in the density of the PEGchains at the nanoparticle surface. Thus, a higher contributionof the core polymer can be associated with the measured �potential. This may also contribute to increasing the capacityof inducing complement activation, hence increasing the chanceto be taken up by macrophages after intravenous administrationin animals.

Finally, from a practical point of view, it is interesting tonote that low complement activation characteristics could beobtained with only 77% of PBLG-PEG copolymer chains inthe particles, making possible to imagine composite particlescontaining not only PBLG-PEG copolymer (for ensuring stealthproperties in the body), but also other copolymers bearing otherchemical moieties, such as recognition ligands, in order to confersimultaneously other properties to the particles.

CONCLUSIONS

The results reported in this contribution permit one todemonstrate that nanoparticles based on PEGylated derivativesof PBLG with a low complement activation pattern can beobtained by nanoprecipitation, an easy and reproducible method.Because of this property, it is strongly expected that thesenanoparticles will behave as stealth nanoparticles after in ViVoadministration by the intravenous route. Isothermal titrationmicrocalorimetry and complement activation measurementsallowed us to conclude that recognition of PEGylated PBLG-based nanoparticles by proteins is significantly lower thanrecognition of non-PEGylated nanoparticles. It is worth notingthat composite nanoparticles based on a mixture of PBLG-PEG-60 and PBLG polymers have been prepared by nanoprecipitationand present similar properties to PBLG-PEG-60 based nano-particles, suggesting that it may prove possible to introduce inthe particles reasonable amounts of other structurally similarcopolymers chains bearing other ligands, without modifyingtheir complement activation properties. This would help toadjunct on demand additional functionalities to the particles.

ACKNOWLEDGMENT

Pr. Denis Labarre is warmly acknowledged for fruitfuldiscussions concerning the discussion of the complementactivation experiments. M.EMB would like to thank the

CONACyT (Mexico) for the financial support, which enabledher to conduct this study. TEM experiments where performedat the CCME (U. Paris-Sud) facility and authors acknowledgeJeril Degrouard for technical support and fruitful help. Theauthors would like to acknowledge Laurent Fontaine andVeronique Montembault for their advice and support in thesynthesis of the polymers used in the formulation of thenanoparticles.

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Figure 4. Evolution of � potential and complement activation factoras a function of PBLG-PEG-60 ratio (wt %) in nanoparticles constitutedof PBLG-PEG-60 and PBLG-Bnz-50. Circle: Complement activationfactor. Square symbols: � potential.

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