Biomacromolecules 2008, 9, 583–591
583
Towards Biocompatible Vaccine Delivery Systems: Interactions of Colloidal PECs Based on Polysaccharides with HIV-1 p24 Antigen Alexandre Drogoz,†,§ Séverine Munier,‡ Bernard Verrier,‡ Laurent David,§ Alain Domard,§ and Thierry Delair*,† Unité Mixte CNRS-BioMérieux, UMR 2714, ENS Lyon, 46 Allée d’Italie, 69364 Lyon Cedex 07, France, Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS, 7 Passage du Vercors, 69967 Lyon Cedex 07, France, and Laboratoire des Matériaux Polymères et des Biomatériaux, UMR CNRS 5223 Ingénierie des Matériaux Polymères, Université de Lyon, Université Lyon 1, Domaine Scientifique de la Doua, Bât. ISTIL, 15 Bd. A. Latarjet, 69622 Villeurbanne Cedex, France Received October 17, 2007; Revised Manuscript Received November 26, 2007
This work reports on the interactions of a model protein (p24, the capside protein of HIV-1 virus) with colloids obtained from polyelectrolyte complexes (PECs) involving two polysaccharides: chitosan and dextran sulfate (DS). The PECs were elaborated by a one-shot addition of default amounts of one counterpart to the polymer in excess. Depending on the nature of the excess polyelectrolyte, the submicrometric colloid was either positively or negatively charged. HIV-1 capsid p24 protein was chosen as antigen, the ultrapure form, lipopolysaccharidefree (endotoxin-, vaccine grade) was used in most experiments, as the level of purity of the protein had a great impact on the immobilization process. p24 sorption kinetics, isotherms, and loading capacities were investigated for positively and negatively charged particles of chitosans and dextran sulfates differing in degrees of polymerization (DP) or acetylation (DA). Compared with the positive particles, negatively charged colloids had higher binding capacities, faster kinetics, and a better stability of the adsorbed p24. Capacities up to 600 mg · g-1 (protein-colloid) were obtained, suggesting that the protein interacted within the shell of the particles. Smallangle X-rays scattering experiments confirmed this hypothesis. Finally, the immunogenicity of the p24-covered particles was assessed for vaccine purposes in mice. The antibody titers obtained with immobilized p24 was dose dependent and in the same range as for Freund’s adjuvant, a gold standard for humoral responses.
1. Introduction Bionanoparticles consisting of protein molecules immobilized on, or trapped within colloids offer a great potentiality of applications in biotechnology1,2 and biomedicine as drug3/ vaccine4 delivery systems. In particular, for the field of nanomedecine, colloids based on polyelectrolyte complexes are quite attractive because they are obtained in the absence of potentially toxic organic solvents or nonpolymeric chemical cross-linkers, simply by mixing two aqueous solutions of polyelectrolytes of opposite charges. The formation of polyelectrolyte complexes (PECs) at the colloidal scale requires high dilutions of polymer solutions and nonstoichiometric systems, as investigated by Dautzenberg.5 Other factors have an important impact on the course of the particle formation like the order of reactant addition,6 the addition rate,7 the respective molar mass of the polycation and polyanion,5–7 the salt concentration, and pH.5,8 The synthesis of particles of polyelectrolyte complexes from natural polymers for biomedical applications is of particular interest because they can be chosen for their biocompatibility and bioresorbability. Chitosan (obtained from the deacetylation of the naturally occurring chitin) is the only available polycation from biomass. The most widely used polyanion is DNA, and * Corresponding author. E-mail:
[email protected]. † Unité Mixte CNRS-BioMérieux, UMR 2714, ENS Lyon. § Laboratoire des Matériaux Polymères et des Biomatériaux, UMR CNRS 5223 Ingénierie des Matériaux Polymères, Université de Lyon. ‡ Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS.
PECs between chitosan and DNA have been largely investigated for applications in gene therapy.9 Other polyanions like poly(γ10 11 12 L-glutamic acid), carboxymethyl cellulose, alginates, carboxymethyl konjac glucomannan,8 and dextran sulfate13 were used for the formation of colloids. Alhough systematic studies are more limited than for synthetic polymers, the most relevant parameters controlling the formation of particles are the polymer concentration, the degree of polymerization (DP) of both polyelectrolytes, and the degree of acetylation for chitosan (DA). Various types of bionanoparticles were obtained with polysaccharides according to the preparation process. Complexes were produced by mixing a protein solution with a chitosan solution, hence nanocomplexes of chitosan derivatives and insulin were obtained14 via electrostatic interactions and nanogels could be prepared with chitosan and ovalbumin, provided a heating step was carried out.15 Proteins could be incorporated within chitosan gels obtained by ionic gelation with sodium tripolyphosphate,16,17 sodium sulfate,18 or within particles obtained by polyelectrolyte complexes of polysaccharides.8,11,19–22 A second approach toward bionanoparticles was to make the colloid first and then to adsorb the protein of interest. This strategy was mainly followed with chitosan particles obtained by ionic gelation with sodium sulfate23,24 or by complexation with zinc acetate.25 Following our investigations on the synthesis of colloidal PECs of dextran sulfate and chitosan,13 we report in this paper on the elaboration and characterization of bionanoparticles obtained by immobilization of p24 HIV-1 capside protein onto preformed particles. Protein sorption kinetics and isotherms were established. We also investigated the impact of the nature of
10.1021/bm701154h CCC: $40.75 2008 American Chemical Society Published on Web 01/22/2008
584 Biomacromolecules, Vol. 9, No. 2, 2008
the polymer in excess, used in the elaboration step of the colloids, and both DP and DA of chitosan on the course of the immobilization process and the stability of the bionanoparticles. Finally, the potency of the p24-coated nanoparticles as a vaccine carrier was tested in mice models, using HIV-1 p24 protein, by measurement of antibody titers.
2. Experimental Section 2.1. Materials. A weakly acetylated chitosan, produced from squid pens chitin, with a relatively high weight-average molecular weight (Mahtani chitosan PVT, batch 114, DA ∼ 5%, Mw ∼ 349 000 g · mol-1) was used in this study. Prior to use, the sample was purified as follows: dissolution in a 0.1 M acetic acid aqueous solution, filtration through Millipore membranes of decreasing porosity (from 3 to 0.22 µm), precipitation with an ammonia-methanol mixture, rinsing with deionized water until neutrality, and lyophilization. Purified high molar mass chitosans were N-acetylated in homogeneous medium at different DAs with acetic anhydride. The reaction was performed in a hydro-alcoholic mixture according to the procedure previously described by Vachoud.26 After reacetylation, chitosans were neutralized, rinsed with deionized water, and then freeze-dried. In addition, controlled nitrous deaminations yielding chain scissions13,27 were performed with chitosan samples having high molecular weight to produce low-molecular-weight polymers. Chitosans were dissolved at 0.5% (w/v) in a 0.2 M acetic acid/0.1 M sodium acetate buffer. A 0.15 M sodium nitrite solution was added to chitosan solutions to obtain a nitrite/glucosamine units molar ratio of 0.5. The reaction was performed under moderate magnetic stirring for various times (2–15 h). Low-molar-mass chitosans were recovered by precipitation with an ammonia-methanol mixture and purified by several washings with deionized water until neutrality and finally lyophilized. Dextran sulfates with weight-average molar masses ∼104 g · mol-1 (DS 10k) or 1 × 106 g · mol-1 (DS 500k) were provided by Sigma and used without further purification. The sulfur content was determined for each sample by colloidal titration. The degree of sulfation was 2.2 ( 0.2 (mol/mol) for both dextran sulfate Mw and the water content were provided by the manufacturer. 2.2. Methods. Characterization of Chitosan. Degrees of acetylation were determined on purified chitosans by 1H NMR spectroscopy (Varian, 500 MHz) according to the method developed by Hirai et al.28 The water content was determined by thermogravimetric analysis (DuPont Instrument 2950). The weight-average molecular weight (Mw), the z-average root mean square of the gyration radius (Rg,z), and the polydispersity index (Ip) were measured by size exclusion chromatography (SEC) (3000 and 6000 PW TSK gel columns, inner diameter ) 7.8 mm and length ) 300 mm) coupled on line with a differential refractometer (Waters 410) and a multiangle laser light scattering instrument (MALLS, Wyatt, Dawn DSP) equipped with a 5 mW He/ Ne laser operating at λ ) 632.8 nm. Analyses were performed in microbatch mode using the K5 flow cell. Light intensity measurements were derived following the classical Rayleigh-Debye equation, allowing us to deduce Mw, and Rg,z. A degassed 0.2 M acetic acid/0.15 M ammonium acetate buffer (pH ) 4.5) was used as eluent. The flow rate was maintained at 0.5 mL/mn. The polymers were dissolved in the same solvent at 0.1 and 0.4% (w/w) for initial and low-molar-mass samples, respectively, and injected after filtration on 0.45 µm pore size membrane (Millipore). Refractive index increments (dn/dc) were determined independently for each sample in the same solvent with an interferometer (NFT Scan Ref) operating at λ ) 632.8 nm. Preparation of Polyelectrolyte Solutions. Chitosan was dispersed in deionized water at various concentrations, taking into account the water content in the solid state. The dissolution was achieved under moderate stirring by adding a stoichiometric amount of 0.1 M hydrochloric acid, with respect to the free amino functions, for each degree of acetylation. Sodium chloride was added to vary the ionic strength. Then solutions were adjusted at pH ) 4.0 with 0.1 M sodium hydroxide or hydrochloric acid and the added amount was considered to calculate the final
Drogoz et al. concentration and the total ionic strength of 0.050 M. Before use, all solutions of chitosan were filtered on 0.22 µm pore size Millipore membranes. Dextran sulfate solutions, at different concentrations, were prepared directly in deionized water. Sodium chloride was added to obtain the required ionic strength and pH was adjusted at 4.0 with 0.1 M hydrochloric acid. Solutions were also filtered on 0.22 µm pore size Millipore membranes before use. Preparation and Purification of Recombinant p24. Recombinant HIV-1 protein p24 was prepared in Escherichia coli (E. coli) as previously described.29 The pMH-24 expression vector contains the HIV-1 p24 gene and a 6-histidine tag at its N-terminal end, allowing efficient purification by immobilized metal affinity chromatography (IMAC). E. coli strain RRE was transformed with this expression plasmid, and the extracts of soluble proteins were purified by immobilized metal ion affinity chromatography (IMAC) using a Ni-NTA activated resin. Residual endotoxin was removed by adsorption to polymixin B. The purified protein was more than 95% pure; the endotoxin content was below 10 EU/mg of p24 protein, as determined using the QCL-1000 quantitative chromogenic limulus amebocyte lysate (LAL) kit (BioWhittaker, Walkersville, Verviers, Belgium). Polyelectrolyte Complex Formation. Colloidal PECs were formed in nonstoichiometric conditions (i.e., the polymer charge molar ratio R(n+/n-) * 1) at room temperature using chitosan or dextran sulfate as starting solutions, by adding the polymer in default into that in excess.13,27 The solution containing the polymer in default was added in a single shot to the starting solution at identical ionic strength (50 mM) and pH ) 4, under a constant magnetic stirring of 1250 rpm. The final volume of particle dispersion was 30 mL with a solid content of 0.1% w/w. Positively and negatively charged particles were obtained at R ) 2 and 0.3, respectively. In all experiments, the initial amino and sulfate moieties concentrations in the starting solutions of chitosan and dextran sulfate, respectively, were set at 6×10-3 M, corresponding to a weight concentration close to 0.1% for both polymer solutions. Because of nonstoichiometric conditions, the polymer in excess was not completely consumed, thus a low amount of free polymer still remained in solution. Particles were separated from the continuous phase by centrifugation at 7800g for 30 min at 20 °C. The supernatant was discarded, the pellet resuspended in deionized water, and the centrifugation repeated. Finally the particles were redispersed in a minimum volume of deionized water to reach 2% of solid content. Protein Sorption onto Colloidal PECs. The sorption process consisted in mixing equal volumes of particle dispersion and protein solution with moderate end-overhead stirring. Various solid contents and protein concentrations were obtained by dilution of the initial particle dispersion (solid content 2%) and protein solution (concentration 2 g · L-1) with the same buffer. p24-coated particles were centrifuged at 3000g for 15 min. This condition allowed an easy redispersion of the dispersion with conservation of its colloidal properties. After the supernatant was separated, the pellet was resuspended in the same mixing total volume. Sorbed p24 was deduced from the titration (BCA assay, Pierce, Bezons, France, according to the instructions of the manufacturer) of free p24 in the supernatant after a second centrifugation step (10 000g, 10 min) to remove potentially residual particles. Physicochemical Characterization of the Complex Dispersions. Quasi-Elastic Light Scattering. Dynamic light scattering measurements of polyelectrolyte complexed dispersions were carried out using a Malvern Zetasizer HS3000 equipped with a 10 mW He/Ne laser beam operating at λ ) 633 nm (at 90° scattering angle). All measurements were performed at least in triplicate at 25.0 ( 0.2 °C. The selfcorrelation function was expanded in a power series (Cumulants method). The polydispersity value provided by the software is a dimensionless value defined by µ2/(Γ)2, where µ2 is the second cumulant of the correlation function and (Γ) the average decay rate. Each measurement is the average of three series of 10 measurements each. For a monodisperse colloid, the polydispersity index should be below 0.05, but values up to 0.5 can be used for comparison purposes.30
Biocompatible Vaccine Delivery Systems Small-Angle X-ray Scattering. Small Angle X-ray Scattering (SAXS) was performed at the ESRF (Grenoble, France on BM2-D2AM). The data were collected at an incident photon energy of 16 keV. We used a bidimensional detector (CCD camera from Ropper Scientific). All the data corrections were performed thanks to the software bm2img available on D2AM. The data were corrected from the dark current (i.e., nonexposed camera), flat field response (i.e., homogeneously illuminated camera), and tapper distortion. Finally, the radial average around the image center (location of the center of the incident beam) was performed. To deduce the scattering vector values (q), we used a calibration standard (silver behenate). The polyelectrolyte complex dispersions (solid content of 1% w/w), obtained as described above, were put in cylindrical sample holders (internal diameter of about 5 mm). The contribution of the cell filled with the solvent alone was subtracted from the scattering curve of the sample studied. To estimate the width of transition layer, the Porod’s assympotic behavior was studied and modeled according to: I(q) ) C/q4 - B/q2. The value of the interface of transition layer Li was calculated from the determination of the Porod’s constant C and the interface constant B, according to Li ) (12πB/C)-1/2. This expression assumes that the shape of the electron density profile is linear in the transition layer.31 Particle Solid Contents. The solid content was defined by the ratio between the weight of dried particles (after purification) at 150 °C for 30 min, to the initial weight of the solution. Electrophoretic Mobilities. Electrophoretic mobilities (µE) of the particles were determined at 25 °C with a Malvern Zetasizer HS3000 equipped with a 10 mW He/Ne laser beam operating at λ ) 633 nm. µE was expressed as the average of 15 measurements with a relative error of ∼5%. To avoid physisorption of the free polymer onto the surface, particles were separated from the medium by centrifugation for 30 min at 8000–12000 rpm depending on R ) n+/n-, the molar charge ratio. The supernatant was discarded, the pellet resuspended in deionized water, and the centrifugation repeated. Finally, the product was resuspended in a minimum volume of deionized water. Electrophoretic mobilities and size measurements of the particles as a function of pH were performed by suspending washed dispersions in 10-3 M sodium chloride solutions. Zeta potentials were derived from electrophoretic mobility measurements using Smoluchowski’s equation. 2.3. Animal Experimentations. Preparation of p24-Coated Nanoparticles for In ViVo Testing. p24 protein and PECs were diluted at a concentration of 0.9 g · L-1 and 1% solid content, respectively, with a 10 mM pH 5.7 phosphate buffer or a 10 mM pH 4.5 acetate buffer for anionic and cationic particles, respectively. The adsorption reaction was carried out with moderate end-overhead stirring at room temperature for 2 or 20 h, respectively, for negative and positive particles. Unbound p24 protein was quantified from the supernatant separated from the particles by centrifugation at 3000g for 15 min. The p24-particles pellet was resuspended with a defined volume of trehalose buffer (200 mM trehalose and 50 mM NaCl) to reach 10 or 1 µg of p24 for 100 µL of final solution. Immunization Protocol. Five- to six-week-old female Balb/C mice from Charles River Laboratories (Lyon, France), were hosted in the PBES animal care facility of Ecole Normale Supérieure de Lyon (http:// www.ifr128.prd.fr/PBES.htm) and handled following institutional guidelines. Animals (three/group) were immunized by subcutaneous injection (SC) into the flank region on days 0, 14, and 28 with 100 µL of one of the following immunogens: p24 mixed with Freund’s adjuvant (1 volume to 1 volume, complete Freund’s adjuvant for the first injection, incomplete for the following two, Sigma) or p24 adsorbed onto negative or positive nanoparticles. p24 protein, 10 and 1 µg per animal per injection, was administered, and the animals were sacrificed by cervical dislocation on day 35. The negative control consisted in both positive and negative nanoparticles without proteins. For the analysis of antibody responses, the blood samples were collected on days 14, 28, and 35. ELISA. Specific antibodies in animal sera were detected by ELISA. The 96-well plates were coated overnight at room temperature with
Biomacromolecules, Vol. 9, No. 2, 2008 585 Table 1. Physicochemical Characteristics of Chitosans Determined by SEC in an Acetic Acid/Ammonium Acetate Buffer (pH ) 4.5, µ ) 0.15 M) Mw × 105 (g · mol-1)
DA (%)
DPwa
Ipb
0.32 ( 0.08 1.32 ( 0.03 1.10 ( 0.04 1.36 ( 0.03 1.10 ( 0.02 1.58 ( 0.03
5 5 9 11.5 26 51
196 ( 5 809 ( 19 671 ( 15 820 ( 18 640 ( 15 866 ( 16
1.68 ( 0.01 1.48 ( 0.01 1.63 ( 0.03 1.43 ( 0.05 1.38 ( 0.04 1.35 ( 0.05
a Weight-average degree of polymerization. ) Mw/Mn.
b
Polydispersity index, IP
100 µL of a 1 µg/mL p24 protein solution in PBS buffer. The plates were then blocked for 1 h at 37 °C with 200 µL of PBS containing 10% nonfat dry milk and washed three times with PBS-Tween 0.05%. Serial dilutions of mouse serum were prepared in PBS containing 1% bovine serum albumin (BSA); 100 µL were added to the plates in duplicate and incubated for 1 h at 37 °C. After three washes with PBST buffer, bound mouse antibodies were detected with peroxidaseconjugated AffiniPure goat antimouse IgG (Southern Biotech by CliniSciences, Montrouge, France) at a concentration of 0.1 µg/mL in PBS-BSA 1% for 1 h at 37 °C. Following washing, plates were developed with 3,3′,5,5′-tetramethylbenzidine (TMB) (BD Pharmingen, Le Pont de Claix, France), prepared according to the manufacturer’s instructions, for 30 min in the dark, and the reaction was stopped with 100 µL of 1 N sulfuric acid. The absorbance at 450 nm was measured in an automated plate reader. Antibody titers represent the reciprocal of the serum dilution producing an absorbance of 0.1 in the ELISA test.
3. Results and Discussion Various chitosan samples were prepared according to the method described in the Materials Section, and their physicochemical properties are reported in Table 1. Particles were prepared by complexation of polyelectrolytes of opposite charges, as described in the Methods Section. This strategy is quite attractive, as particles were formed directly from an aqueous medium in the absence of organic solvent and any other chemical cross-linker. The experimental protocol used here was selected from previous investigations13,27 and provided core–shell-like particles, the polymer in excess forming the soft shell around a hydrophobic core of neutralized polyelectrolytes.32 3.1. Preliminary Experiments: Determination of the Immobilization Conditions. A series of experimental conditions were screened, as reported in Table 2, in which the results are expressed in terms of immobilization yield of p24 protein. For the positively charged colloids, binding yields were in general higher than that for the negative particles, in the 60–75% range (Table 2a), except for the run for which the buffer contained a fraction of the size exclusion chromatography (SEC) buffer (an acetic acid/ammonium acetate buffer (pH ) 4.5, µ ) 0.15 M)) known to disrupt hydrogen bonds in solutions.33 In that case, the yield was 25%, suggesting that hydrogen bonds between the colloid and the protein molecules were necessary for the immobilization process. In these experiments with positively charged particles, the pH of the buffers had to remain slightly acidic to avoid flocculation of the particles resulting from charge neutralization of amino groups of chitosan. Hence, p24 was either weakly positively charged or close to neutrality. For the negative particles (Table 2b), a maximum yield of 93% was obtained, but in these conditions, the colloidal stability was lost. To maintain the colloidal character of the dispersion, low ionic strength buffers were used within a pH range of 4.5 (lower than
586 Biomacromolecules, Vol. 9, No. 2, 2008 Table 2. Influence of Various Buffers on Sorption of p24 Endotox+ p24 Protein for Both Positive R ) 2 (a) and Negative R ) 0.3 (b) Particles Based on Chitosan Mw ) 136 000 g · mol-1, DA ) 11.5%, and DS 500k (1.5×106 g · mol-1)a yield of sorbed p24 (mg) sorbed per g of colloid p24 (%) (mg · g-1) (a) Sorption Buffer for Positively Charged Particles 10 mM sodium acetate, pH ) 4.5 75 48 10 mM sodium acetate/SEC (3:2, v/v), 25 14 pH ) 4.5 10 mM sodium phosphate, pH ) 5 63 38 10 mM sodium phosphate, pH ) 5.7 70 42 10 mM sodium phosphate, pH ) 6 60 36 (b) Sorption Buffer for Negatively Charged Particles 10 mM sodium acetate, pH ) 4.5 31 20 10 mM sodium acetate/SEC (3:2, v/v), 93 52 pH ) 4.5 10 mM sodium phosphate, pH ) 5.7 31 20 10 mM sodium phosphate, pH ) 6 23 14 10 mM sodium phosphate, pH ) 7 30 18 10 mM sodium borate, pH ) 7 22 16 10 mM sodium phosphate, pH ) 8 40 24 a Sorption conditions: protein concentration ) 0.3 g/L in the medium after 20 h mixing. Particle solid content in media: 0.5%. Composition of the SEC buffer: acetic acid/ammonium acetate buffer (pH ) 4.5, µ ) 0.15 M).
p24 isoelectric point of 5.9) to 8. Hence, the global charge of the protein was either positive or negative. The best yield (in Table 2b) of 40%, compatible with the colloidal property of the dispersion, was obtained at pH 8, for which both the protein and the particles were negatively charged. However, the impact of pH on the sorption process was somewhat limited as yields of ca. 30% were obtained for pHs 4.5, 5.7, and 7. This preliminary set of experiments demonstrates that the interactions involved in the binding of endotox+ p24 to the colloids are various in nature. In the most efficient immobilization conditions, corresponding to the highest binding yields, both the protein and the colloid bore a similar global charge at pH 8 or 4.5, for the negative (40% yield) or positive (75% yield) particles, respectively. This would suggest that electrostatic interactions were not the only driving forces of the binding process, although one must keep in mind that electrostatic charges in proteins are not evenly distributed and that even if the global charge is negative (positive), there may remain some patches with positive (negative) charges. Hydrogen bonding also played a role in the p24-particle interaction process, as seen from the effect of the addition of a fraction of a buffer unfavorable to hydrogen bonding (identified as SEC buffer) to the sorption medium. For the cationic particles, the addition of this buffer resulted in a drastic decrease in protein binding, suggesting that hydrogen bonds, as short-range forces, stabilized the particle-protein complexes. Finally, one may also consider the involvement of hydrophobic interactions. Indeed, the particles were obtained by charge neutralization between two polyelectrolytes of opposite charges, resulting in the formation of hydrophobic domains, which could interact with hydrophobic residues of the p24 proteins. On the basis of the results described above, optimum sorption conditions were selected to allow an efficient binding of p24, with a minimal alteration of the colloidal properties of the dispersions: 10 mM acetate buffer pH 4.5 and 10 mM phosphate buffer pH 8, for the positive and negative particles, respectively. The above results were obtained with a protein, purified by immobilized metal ion affinity chromatography (IMAC), featuring an experimental molar mass (evaluated by mass spectrometry) corresponding to the expected one.34 IMAC is a very
Drogoz et al.
efficient purification method for the production of recombinant proteins (endotox+) obtained in a bacterial expression system (E. coli) for in vitro purposes but not for in vivo applications, as some traces of contaminant of bacterial lipopolysaccharide remain. To remove this contaminant, a further purification step was performed35 (see Experimental Section), but the recovered amount of endotoxin-free protein (endotox-) was rather low, which explains why the preliminary investigations were carried out with the endotox+ p24. With the positively charged particles, sorption of the endotox- protein in the 10 mM acetate buffer pH 4.5, selected above, repeatedly led to an immobilization yield of 25–35% (Table 3a) instead of 75% or 68% obtained for the endotox+ p24 (respectively Tables 2a and 3a). The impact on the sorption process of the chitosans used for the synthesis of the particles was drastic. For DA ) 5%, particles flocculated, irrespective of the purity grade of the protein and molar mass of chitosan, although no particular difference in ζ potentials of the bare particles could have allowed the prediction of such a result. For DA ) 9%, similar amounts of both quality grade p24 were sorbed, whereas for higher DAs, the endotox+ protein was bound more efficiently than the endotox-. A significant increase in the particle average diameter was observed after endotox+ protein immobilization but not for the endotox- p24, which could be related to the higher protein loads on the colloids for endotox+ than for endotox-. The great impact on the sorption process of traces of LPS was not expected, though LPS-chitosan complexes have been described in the literature.36,37 With the negatively charged particles, in the selected 10 mM phosphate buffer pH 8, a very inefficient sorption of endotoxp24 occurred at only 5% yield. Thus, the experimental conditions were changed to a phosphate buffer pH 5.7 (Table 3b), and the immobilization yields were 42–48% for DA ) 9% and above. When DA ) 5%, the sorption of the endotox- p24 increased with the molar mass of chitosan but remained lower than for DA ) 9%. Interestingly, there was a strong impact of DA on the immobilization yield of the endotox+ p24, it decreased when DA increased from 9% to 26%, contrary to the positive particles (Table 3a). On the whole, these effects of DA and molar mass of chitosan on the sorption of p24 onto the negatively charged particles are surprising if we take into account that these colloids were obtained with an excess of dextran sulfate. These results suggest that the protein may diffuse through the outer dextran sulfate shell toward the inner part of the colloid where chitosan is complexed with the polyanion. 3.2. Characterization of the Sorption Process. These experiments were carried out with the endotox- protein. As seen above, the sorption conditions selected for the endotoxprotein, considering the binding efficiency and colloidal stability, were: (i) 10 mM sodium acetate buffer pH 4.7 for the positively charged particles (experimentally pH 5.1 due to dilution, see material and methods), and (ii) 10 mM sodium phosphate buffer pH 5.7 for the anionic colloids (experimentally pH 6.2). 3.2.1. Sorption Kinetics of Endotoxin- p24. The sorption process was complete after 20 h of incubation for the cationic particles (Figure 1a), whereas 2 h only were necessary for the anionic colloids (Figure 1b). In both cases, the protein and the particles bore a surface charge of similar sign. At pH 6.2, for the anionic particles (Figure 1b), the protein was close to its isoelectric point (pI 5.9) or slightly negative, whereas at pH 5.1 (Figure 1a) for the cationic colloid, p24 had a global positive charge. Hence, electrostatic repulsive interactions with the anionic particle were less important than for the cationic ones.
Biocompatible Vaccine Delivery Systems
Biomacromolecules, Vol. 9, No. 2, 2008 587
Table 3. Comparison and Characterization between Endotox+ and Endotox- p24 (0.45 g · L-1 in media) Protein Sorption onto Positive Particle R ) 2 (a) and Negative Particle R ) 0.3 (b)a chitosan
particle
Mw (g · mol-1)
diameter (nm)
Ip
5
32 000 132 000
280 310
0.12 0.15
9
126 000
360
0.13
11.5
136 000
387
0.17
26
110 000
390
0.16
5
32 000 132 000
250 213
0.12 0.15
9
126 000
400
0.13
11.5
136 000
407
0.17
26
110 000
351
0.16
DA (%)
characterization of particle-protein complexes ζ potential (mV)
p24 grade
diameter (nm)
(a) Sorption onto Positive Particle R ) 2 41 endotoxagregated 38 endotox+/agregated endotox420 49 endotox+ 620 endotox362 37 endotox+ 710 endotox380 40 endotox+ 450 (b) Sorption onto Negative Particle R ) 0.3 -51 endotoxagregated -42 endotox240 endotox316 -45 endotox+ 374 endotox354 -43 endotox+ 385 endotox340 -43 endotox+ 357
Ip
sorbed p24 (%)
quantity of sorbed p24 (mg · g-1)
0.14 0.4 0.2 0.38 0.18 0.32
0 0 30 34 25 68 34 58
0 0 30 36 24 54 38 52
0.18 0.14 0.23 0.12 0.38 0.13 0.32
0 33 48 47 46 33 42 11
0 16 50 43 48 19 38 9
a Chitosan Mw ) 136 000 g · mol-1, DA ) 11.5%, and DS 500k (1.5×106 g · mol-1). Sorption conditions of positive particles: sodium acetate (10 mM, pH ) 4.5) buffer for 20 h; negative particles: phosphate buffer (10 mM, pH ) 5.7) for 2 h.
Figure 1. Sorption kinetics of endotox- p24 (0.45 g · L-1 in media) onto positive R ) 2 (a) and negative R ) 0.3 (b) particles based on chitosan Mw ) 136 000 g · mol-1, DA ) 11.5%, and DS 500k (1.5×106 g · mol-1). Sorption condition: sodium acetate (10 mM, pH ) 4.7) buffer (positive particles); phosphate buffer (10 mM, pH ) 5.7) (negative particles).
For the cationic particles (Figure 1a), the sorption of p24 proceeded according to three steps: (i) an initial quasiinstantaneous binding of ca. 5% of the protein; (ii) a slow increase in the amount of bound proteins over 8 h, and this step could be understood as a rearrangement of the already bound p24 at the surface (or within the chitosan shell) of the colloid, accepting low amounts of protein molecules; (iii) an increase in the binding of p24, slowly first, but with an increased rate at t ) 15 h before saturation (t ) 18 h). In this last step, the initial sorbed layer had achieved its conformational adaptation and the incorporation of many more protein molecules could occur, with a rate increase suggesting a cooperative effect of the protein on the binding process. Such an effect was previously reported for the binding of proteins on hydrophobized agarose38 or hydroxyapatite.39 A similar case of a three-step kinetics was described by Calis et al. for the adsorption of salmon calcitonin
to poly(lactic-coglycolic acid) microspheres.40 In their case, the lag time, i.e., the period of time during which no (or very little) adsorption was observed, corresponding to the second step described above, could be reduced by increasing the amount of particles, at constant peptide concentration. 3.2.2. Sorption Capacity, Isotherms, and Stability. The impact of the structure of the particles on the sorption process of p24 was further illustrated by the results on the binding capacity of Figure 2. For both the positive (Figure 2a) and the negative (Figure 2b) particles, there was an optimum solid content for a maximum loading capacity. The maximal binding capacity of the cationic particles (120 mg · g-1; 8 mg · m-2) (Figure 2a) was 5-fold lower than that of the negatively charged particles (Figure 2b) (600 mg · g-1, ca. 35 mg · m-2). A capacity of 400 mg · g-1 was reported by Nagamoto et al.24 for ovalbumin and submicrometric chitosan particles obtained by sodium sulfate gelation. Using a similar kind of chitosan particles of micrometric size, Van der Lubben et al.23 could observe the penetration of FITC-ovalbumin within the core of microparticles by confocal microscopy. With colloidal PECs based on synthetic poly(diallyldimethylammonium chloride) (PDADMAC), Ouyang et al. obtained biomolecule loads in the range of 120–330 mg · g-1 according to the nature of the protein and of the polyanion used for the colloid synthesis.41 Ballauff and co-workers42–44 thoroughly investigated the interactions of various proteins, especially bovine serum albumin, onto spherical polyelectrolyte brushes (SPB) composed of a polystyrene core of ca. 100 nm in diameter with a shell thickness of poly(acrylic acid) of ca. 59 nm45 affixed by photo emulsion polymerization. These authors found that proteins could strongly bind to SPBs, irrespective of their charge at low ionic strength (10 mM), whereas almost no binding occurred at higher ionic strength (100 mM). They explained this result by considering the proteins as multivalent counter-ions whose incorporation in the brushes would lead to a concomitant release of monovalent co- or counter-ions. Therefore, this entropic gain could counterbalance the repulsive Coulombic interactions and the steric hindrance from the brushes. Kabanov et al.46 had already mentioned this kind of cooperative process for the sorption of proteins within slightly cross-linked polyelectrolyte hydrogels. With small-angle X-ray scattering experiments,43 Ballauff and co-workers dem-
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Figure 2. Sorption capacity and sorption yield of endotox- p24 (0.45 g · L-1 in the medium) onto positive R ) 2 (a) and negative R ) 0.3 (b) particles based on chitosan Mw ) 136 000 g · mol-1, DA ) 11.5%, as a function of solid content. Sorption condition: sodium acetate (10 mM, pH ) 4.7) buffer for 20 h (positive particles); phosphate buffer (10 mM, pH ) 5.7) for 2 h (negative particles).
onstrated that the protein was effectively accommodated within the brushes of the particles, a result confirmed by cryo-TEM.47 In the view of the existing work described above, the high amount of protein we observed at saturation could be explained by the diffusion of p24 within the shell of the particles brought by the polymer in excess during the formation of the colloid. It is worth comparing our loading capacities with the surface concentration for a close-packed monolayer of bovine serum albumin (Mw 69 000 g · mol-1) of 2.5 mg · m-2, according to Fair et al.48 Some of our experimental values for p24 (Mw 24 000 g · mol-1), around 20 mg · m-2 (Table 4), are well above this monolayer limit. The investigation of the variations of the binding capacity and immobilization yield with the particle solid content, at constant p24 concentration, can provide further details on the binding mechanism. For negatively charged particles (Figure 2b), while the capacity was between 500 and 600 mg · g-1, the sorption yield increased from 15% to 50% with increasing solid content, up to 0.05%, as a result of the increased area available for binding. Above this critical value, increasing the amount of particles in the medium resulted in a capacity decrease as a consequence of the leveling off of the immobilization yield at 50% of the total protein input. This means that the immobilization process was governed by the protein/particle ratio and the protein partition coefficient between the solution and the colloid. At low solid contents, the protein being in excess with regard to the particles, high capacities were obtained until the equilibrium concentration of free protein in the medium was reached (corresponding to a sorption yield of 50%). Particles in excess, for solids above 0.05%, could not displace this equilibrium beyond 50%, hence the binding capacity dropped. In a subse-
Drogoz et al.
quent experiment with anionic particles, we fixed the solid content at 0.5% (Figure 3a) and varied the protein concentration. The amount of particle-bound protein increased linearly with the protein concentration, but saturation was not reached, as the protein was never in excess with regard to the colloid. On the other hand, for a much lower solid content of 0.04% (Figure 3b), saturation was reached at 600 mg · g-1 on increasing the protein concentration in the medium. With the cationic particles (Figure 2a), a maximum binding capacity was obtained for a solid content of 0.05%, followed by a continuous decrease as in the case of their anionic counterparts, suggesting that an excess of particles could not enhance the sorption process toward a consumption of the free protein. The binding yield increased and reached a maximum for a solid content of 0.15%, but conversely to the anionic colloid, it decreased with increasing particle concentrations. This result was repeatedly observed and can be better understood considering the following experiments on protein release. After protein sorption onto the cationic particles, removal of the unbound proteins by centrifugation, and redispersion of the pellet, a second centrifugation process induced the desorption of 40% of the total amount of proteins bound at high particle concentration (0.5%), whereas only 10% desorbed when proteins were immobilized at low particle concentration 0.05%). These results showed that the binding energy of the protein to the colloid was higher at low particle concentration than at elevated particle content. The strong binding of the protein in the first case might be explained by the entrapment of p24 within the colloidal structure via a cooperative multivalent mechanism similar to that mentioned above. In the second case, at high particle content, the protein was not in sufficient excess to diffuse within the particle structure and thus remained weakly bound on the surface of the colloids. The more p24 was surfacebound, the more could be desorbed during centrifugation. With these results in mind, the decrease in binding efficiency, observed in Figure 2a at high particle content, could be understood from a release upon centrifugation of the p24 protein molecules weakly bound on the surface but not entrapped within the colloidal carrier. Interestingly, for both types of colloids, low concentrations of particles were necessary to reach a maximum binding capacity. But for the positive particles, a lower limit solid content was observed under which the binding was not efficient. This was not the case for the negatively charged particles for which high capacities were always obtained, irrespective of the particle concentration, provided the protein was in excess. p24 sorption onto the PEC-based colloids could be regarded as an equilibrated process on the basis of the following stability experiments. Cationic particles (obtained with chitosan of DA 11.5% and dextran sulfate DS 500k), after p24 sorption (conditions p24: 0.45 g · L-1, solid content 0.5%), were centrifuged (3000g, 15 min). The supernatant was removed (S0) and the pellet redispersed in a 200 mM trehalose, 50 mM sodium chloride buffer. Then 5 h or 12 days later, the colloidal dispersion was centrifuged and the supernatants collected (S1 and S1′ for the 5 h and 12 day storage, respectively). The pellet of the sample stored 5 h was redispersed as above (200 mM trehalose, 50 mM sodium chloride buffer), and after 2 h, the same cycle was repeated and S2 recovered. For each supernatant, a protein assay was performed and the results showed that after 5 h of storage, 40% of the total amount of sorbed protein (determined from S0) had desorbed and this amount remained constant if the storage lasted 12 days. After the second cycle, 45% of the remaining amount of protein was again desorbed
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Table 4. Variations of the Diffuse Interface Width of Positive R ) 2 (a) and negative R ) 0.3 (b) Particle-p24 Complexes for Various Particle Solid Contentsa length of the particle solid naked particle sorption capacity sorption capacity diffuse interface -1 -2 (mg · m ) (Å) contents (wt %) diameters (nm) p24 grade (mg · g )
chitosan
-1
DA ) 11.5% Mw ) 1.36×10 g · mol 5
DA ) 26% Mw ) 1.10×105 g · mol-1 DA ) 51% Mw ) 1.58×105 g · mol-1
DA ) 11.5% Mw ) 1.36×105 g · mol-1 DA ) 26% Mw ) 1.10×105 g · mol-1
(a) Diffuse Interface Width of Positive R ) 2 Complexes 0.5 endotox24 0.5 390 endotox+ 50 0.05 endotox240 0.5 endotox+ 52 400 0.5 endotox31 0.5 endotox+ 17 0.05 endotox+ 172 750 0.5 endotox17 0.05 endotox175 (b) Diffuse Interface Width of Negative R ) 0.3 Complexes 0.5 endotox42 0.05 400 endotox415 0.5 endotox+ 20 0.5 350 endotox38
1.5 3 9 3.5 2 2 21 2 21
0 0 0 0 0 91 140 56 110
2.5 27 1.3 2.2
0 0 0 0
a Particles with chitosan DA 51% were elaborated with DS 10k (104 g · mol-1), whereas others were made with DS 500k (1.5×106 g · mol-1). Sorption conditions: positive particles, sodium acetate (10 mM, pH ) 4.7) buffer for 20 h; negative particles, phosphate buffer (10 mM, pH ) 5.7) for 2 h.
Table 5. Characteristics of p24- Particles for In Vivo Assays (Bioparticles Were Prepared Prior to Each Injection (a) Cationic, (b) Anionic) (a) CPL+/p24 1st injection 2nd injection 3rd injection (b) CPL-/p24 1st injection 2nd injection 3rd injection
Figure 3. Endotox- p24 sorption isotherm of negative particules based on chitosan Mw ) 136 000 g · mol-1, DA ) 11.5%, and DS 500k (1.5×106 g · mol-1). Sorption conditions: phosphate buffer (10 mM, pH ) 5.7) 2 h at room temperature. (a) Particle solid content in the medium ) 0.5%. (b) Particle solid content in the medium ) 0.04%.
(S2). A similar type of experiment was run with the negatively charged particles and only 20% of the initial sorbed amount was released after 5 h or 12 days of storage. Moreover, no further protein was desorbed after the second cycle. These stability results show that the protein binding on the cationic particles was an equilibrium that could be displaced by removal of the free p24 from the continuous phase. Moreover, the protein was immobilized more efficiently (less protein release) on the negative colloids than on the positive ones, suggesting a more efficient trapping of the protein within these particles. 3.2.3. Morphology Characterization. In the preceding sections, we showed that the incorporation of the p24 protein depended on the chemical nature of the polyelectrolyte in excess used for the PEC synthesis. Hence, the protein-colloid interactions should depend on the structural organization and morphology of the particles before and after p24 sorption. The organization of the protein layer was investigated by smallangle X-ray scattering experiments. Among all investigated
size of sorption sorbed p24 p24/CPL+ yield (%) (mg · mL-1) (mg · g-1) p24/CPL+ (nm) 25 22 24
0.112 0.099 0.108
23 21 22
479 ( 18 440 ( 8 420 ( 10
size of sorption sorbed p24 p24/CPLyield (%) (mg · mL-1) (mg · g-1) p24/CPL- (nm) 48 46 44
0.108 0.103 0.099
21 20 19
362 ( 15 345 ( 14 318 ( 5
colloids, only cationic samples with a diameter of 750 nm (Ip ) 0.2) obtained with chitosan of DA ) 51% and DS 10k, initially featured a diffuse interface of about 45 Å in a 10 mM pH ) 5.7 phosphate buffer. From Table 4, the addition of proteins maintained the initial surface structure of the particles: when no interface was detected without protein, none was observed in the presence of p24, even at capacities of more than 200 mg · g-1. In other words, the protein layer maintained the sharp variation of the scattering power at the surface of the particles. Conversely, when the structure of the polyelectrolyte complex was indeed characterized by a transition layer (i.e., for positively charged particles formed with chitosan at DA ) 51%), the addition of protein increased the size of the interface. Moreover, this interface increase was related to the amount of incorporated protein. This illustrates the evolution of the PEC organization during the incorporation of p24 within the structure of the positively charged colloids. 3.3. In Vivo Testing. The potential of anionic and cationic nanoparticles as delivery system and/or adjuvant for p24 was tested in the BALB/c mouse model. Animals were immunized with p24-coated nanoparticles by subcutaneous injection on days 0, 14, and 28. Two doses, 10 or 1 µg of p24 per injection, were used. The 10 µg p24-coated nanoparticles were prepared as described in the Experimental Section, and the lowest doses were obtained by a 10-fold dilution. Different batches of nanoparticles based on chitosan of DA 11.5%, Mw ) 136 000 g/mol and DS 500k were prepared for each injection, and their size and characteristics are reported in Table 5. No significant variation in size or in sorption yields for cationic (Table 5a) or anionic (Table 5b) particles were observed as a proof of the
590 Biomacromolecules, Vol. 9, No. 2, 2008
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titers as Freund’s adjuvant, with dose-dependent kinetics. Though this promising potential must be confirmed with further experiments (in particular a more detailed immunomonitoring), our work shows that we have achieved an efficient and safe protein delivery system for vaccination purposes, based on the self-assembly of biocompatible polysaccharides, in the absence of any toxic surfactant, solvent, or cross-linker.
Figure 4. Antibody titers in mice immunized with p24-particles and comparison with Freund’s adjuvant. Animals, three per group, received three injections of 10 or 1 µg of p24 protein mixed with Freund’s adjuvant or sorbed onto cationic or anionic particles based on chitosan Mw ) 136 000 g · mol-1, DA ) 11.5%, and DS 500k (1.5×106 g · mol-1). For each animal, the IgG titers of serum sample taken on days 14, 28, and 35 was determined individually.
Acknowledgment. This work was financially supported by a grant from the Fondation Mérieux (A.D.), Sidaction (S.M.), and Agence Nationale de Recheche contre le Sida (ANRS) (T.D., B.V.). We thank, Jean-Michel Lucas for SEC data, Caroline Weber and Marie-Hélène Charles for their technical assistance in the animal evaluation, and Cyrille Rochas for small-angle X-ray scattering and fruitful discussions. A part of this work was performed in the European Program NanoBiosaccharide.
References and Notes good reproducibility of the sorption process with an identical batch of protein. Both types of particles carried similar amounts of p24, close to 20 mg · g-1 (protein-particles). The well-known Freund adjuvant was used for comparison purposes, and specific antibody titers were determined by ELISA. The results (Figure 4) indicate that the kinetics of the humoral response depended on the amount of injected p24. With the higher dose (10 µg) for both anionic and cationic particles, the kinetics were similar to Freund’s adjuvant, and only 2 injections were sufficient to achieve the maximum antibody titers (∼106). With the lower dose of 1 µg, antibodies were only produced after the second injection. However, the booster effect of the last injection was clearly seen and the antibody titer increased to a value only one decade below that obtained with the highest protein dose.
4. Conclusion In this work, we studied the immobilization of p24, the VIH-1 capsid protein, onto colloidal PECs obtained from dextran sulfate and chitosan. The level of purity of the protein had a great impact on the sorption process as well as the global net charge of the particles. The highest immobilization yields were obtained when both the colloids and the protein bore a global charge of similar sign. Experiments with endotox- p24 showed that the negatively charged colloids had higher binding capacities and faster kinetics than the positive ones. Immobilization capacities up to 600 mg of protein per gram of colloids suggested that a fraction of protein could be trapped within the shell of the particles. For both the positive and negative particles, the binding of the p24 protein was more efficient when the protein was in excess with regards to the colloid: higher capacities and higher binding stabilities were obtained. For negatively charged particles, the process was also controlled by the protein partition coefficient between the dispersed and continuous phases, limiting the sorption to 50% of the original input. With positive particles, at high solid content, the p24 protein was weakly bound and could be released by centrifugation, thus decreasing the binding yields with increasing solid contents. To obtain a high binding stability, low solid contents were required, to allow the diffusion of p24 within the core of the colloids. The morphological effect of this diffusion at the nanoscale could be evidenced in one case by small-angle X-ray scattering experiments. The immunogenicity of p24-particles complex was assessed in mice. Colloidal PECs were able to induce similar antibody
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