Uptake of BSA-FITC Loaded PLGA Nanoparticles by ...

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Nanoscience and Nanotechnology Letters Vol. 5, 1–7, 2013

Uptake of BSA-FITC Loaded PLGA Nanoparticles by Bone Marrow-Derived Dendritic Cells Induces Maturation But Not IL-12 or IL-10 Production E. Karagouni1 , O. Kammona3 , M. Margaroni1 , K. Kotti3 , V. Karageorgiou3 , C. Gaitanaki4 , and C. Kiparissides2 3 ∗ 1

Laboratory of Cellular Immunology, Department of Microbiology, Hellenic Pasteur Institute, Vass. Sofias 127, 11521 Athens, Greece 2 Department of Chemical Engineering, Aristotle University of Thessaloniki, P.O. Box 472, 54124 Thessaloniki, Greece 3 Chemical Process and Energy Resources Institute, Centre for Research and Technology Hellas, P.O. Box 60361, 57001 Thessaloniki, Greece 4 Department of Animal and Human Physiology, School of Biology, National and Kapodistrian University of Athens, 15721 Athens, Greece Nanoparticles prepared from biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA), represent a new approach for vaccine delivery due to their ability to be taken up by phagocytes and activate immune responses. In this study, fluorescently labelled bovine serum albumin (BSAFITC)-loaded PLGA nanoparticles, of an average size ∼ 300 nm were prepared and examined for their ability to be taken up by bone marrow-derived dendritic cells (BM-DCs) in vitro and thus to promote their maturation and activation. The synthesized nanoparticles did not exhibit any cytotoxic or hemolytic effect and were taken up by BM-DCs efficiently, in a time and dose dependent manner. The localization of BSA-FITC loaded PLGA nanoparticles both in the acidophilic cellular compartments and the cytoplasm resulted in the maturation of BM-DCs expressing higher levels of costimulatory and MHC class II molecules in comparison to empty PLGA nanoparticles. However, the absence of IL-12 or IL-10 production indicates partial activation of BM-DCs suggesting the necessity of an adjuvant addition in order to facilitate DCs functionalization.

Keywords: PLGA Nanoparticles, BSA-FITC, DCs Maturation, IL-12, IL-10.

1. INTRODUCTION Poly D,L-lactic-co-glycolic acid (PLGA) and its various derivatives have been extensively employed in the production of nano- and microcarriers and, in particular, in the encapsulation of proteins, peptides and antigens (Ag) for controlled release applications. PLGA has been approved for human use due to its excellent biocompatibility and biodegradability properties and slow drug release rates (i.e., up to several days, weeks or months).1 Furthermore, PLGA improves shelf life of the Ag by inhibiting proteolytic degradation and limit Ag distribution to professional Ag-presenting cells, such as macrophages and dendritic cells (DCs), therefore enhancing Ag immunogenicity and inducing strong humoral, CD4+ TH1 and ∗

Author to whom correspondence should be addressed.

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CTL responses.2–5 The above characteristics make PLGA a potent candidate for vaccine development. Previous studies have shown that intramuscular, subcutaneous or even oral vaccination of Ag-loaded PLGA (e.g., TT, cancer peptides, hepatitis B Ag) elicit specific cellular and humoral immune responses.4 6–8 This could be attributed to PLGA’s ability to efficiently target DCs. Adoptive transfer of DCs exposed to Ag-loaded PLGA has gained considerable attention for tumor immunotherapy.9 However, DCs-PLGA interactions need further investigation in order to develop efficient vaccine formulations in particular against intracellular pathogens. In the present study, the ability of fluorescently labelled bovine serum albumin (BSA-FITC)-loaded PLGA nanoparticles to be taken up by murine myeloid DCs (CD11c+ CD8− , and thus to promote their maturation and activation was investigated. The cytotoxicity and 1941-4900/2013/5/001/007

doi:10.1166/nnl.2013.1564

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Uptake of BSA-FITC Loaded PLGA Nanoparticles by BM-DCs Induces Maturation

hemocompatibility of the synthesized PLGA nanoparticles were also tested.

2. MATERIALS AND METHODS

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The antigen encapsulation efficiency was calculated by the ratio of the antigen mass in the nanoparticles over the total mass of antigen in the recipe. Similarly, the antigen loading was calculated by the ratio of the encapsulated mass of antigen over the total mass of PLGA nanoparticles.

2.1. Nanoparticles Preparation PLGA nanoparticles containing the model antigens (i.e., BSA-FITC, OVA) were prepared by the double emulsion method. A water-in-oil (w/o) emulsion was initially formed by adding 0.3 ml of antigen solution in phosphate buffer saline (PBS) (5 mg/ml) into 3.0 ml of a PLGA chloroform solution (30 mg/ml). The emulsification was performed in an ice bath with the aid of a microtip sonicator (Sonicator Sonics Vibra Cell VC-505) at 40% amplitude for 45 sec. The primary emulsion (w/o) was further emulsified in an aqueous PVA solution (12 ml, 1% w/v) via sonication at 40% amplitude for 2 min. The resulting (w/o/w) emulsion was left overnight under magnetic stirring to allow the evaporation of the solvent(s). The PLGA nanoparticles were purified by means of four centrifugation (at 20,000 rpm for 10 min at 4 C)/redispersion cycles and were subsequently lyophilized (Thermo Electron Corp. Micro Modulyo). Empty PLGA nanoparticles were also prepared to be used as controls. For the synthesis of the empty nanoparticles the aqueous antigen solution was replaced with 0.3 ml of PBS. The surface morphology of the PLGA nanoparticles was assessed by scanning electron microscopy (JEOL JSM 6300). Accordingly, the lyophilized nanoparticles were first double coated with a gold layer under vacuum and then examined by SEM. The average particle diameter and the particle size distribution (PSD) of the PLGA nanoparticles were determined by photon correlation spectroscopy and their zeta potential ( was calculated from aqueous electrophoresis measurements (Malvern Nano ZS90, United Kingdom). The measurements were performed with aqueous dispersions of nanoparticles prior to their lyophilization. 2.2. Determination of BSA-FITC and OVA Loading and Encapsulation Efficiency A micro-bicinchoninic acid (micro-BCA) protein assay kit (Pierce Biotechnology, Rockford, IL) was employed to determine the antigen (e.g., BSA-FITC, OVA) loading (%wt) in the PLGA nanoparticles. Accordingly, 2.4 mg of lyophilized nanoparticles were dissolved in 0.4 ml of 0.1 N NaOH aqueous solution. Following the overnight incubation of nanoparticles at 4 C, the antigen concentration was determined using a micro-BCA protein assay kit according to the manufacturer’s instructions for 96-microwell plates (Corning Inc., Corning, NY). The absorbance of the samples was measured at 562 nm using a microplate reader (EL808IU-PC, BioTek Instruments, Inc., Winooski VT, USA). Empty PLGA nanoparticles were used as controls. 2

2.3. L929 Cytotoxicity Assay The biocompatibility of synthesized PLGA nanoparticles was evaluated by means of viability and hemocompatibility assays. For viability assessment, conventional L929 bioassay was performed as previously described.10 by incubating 3 × 105 /ml L929 fibroblasts with 1 g/ml Actinomycin D and PLGA nanoparticles at a concentration range of 25–1600 g/ml for 24 h. Then, cells were fixed with methanol and viable cells were stained with crystal violet. Cell lysis was achieved using 33% acetic acid and released crystal violet was measured in 570 nm. L929 cell line (ATCC® Number: CCL-1, LGC Promochem, London, UK) was maintained in DMEM culture medium (Biochrom AG, Berlin, Germany) supplemented with 24 mM NaHCO3 , 100 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, 0.05 mM 2-mercaptoethanol, 100 u/ml penicillin, 100 g/ml streptomycin and 10% (v/v) heat inactivated fetal bovine serum (FBS; Gibco, Paisley, UK) at 37 C in 5% CO2 and 95% humidified air. Viability (Vi) was expressed as a percent value determined by the formula: Vi = 1001 − a − b/a, where a and b are the mean absorbance of triplicate wells with culture medium and test sample, respectively. 2.4. Hemocompatibility Assessment Regarding hemocompatibilty, whole blood cell lysis and in vitro agglutination tests were performed. For that reason, blood was collected in heparinized tubes from Wistar rats, maintained in Hellenic Pasteur Institute breeding unit (HPI, Athens, Greece), and centrifuged for 10 min at 800× g at 4 C. The cell pellet was resuspended in 0.9% (w/v) NaCl solution in order to prepare a 2% (v/v) cell suspension. One hundred l of this suspension was plated in each well of a 96-well round bottom plate. Empty PLGA nanoparticles solutions of different concentrations (4.5– 600 g/ml) in 0.9% (w/v) NaCl were added to the plated cells and incubated for 1 h and 24 h at 37 C in 5% CO2 . The release of hemoglobin, as an index of red blood cell (RBC) lysis (hemolysis), was determined by spectrophotometric analysis of the supernatant at 570 nm (MRX, Dynatech Laboratories, Guernsey, UK). Complete hemolysis (positive control) was achieved by adding 1% (v/v) Triton X-100 (Fluka, Germany), while cells in 0.9% (w/v) NaCl solution served as negative control. Otherwise, for the agglutination test, 25 l of whole blood cell suspension was plated in each well of a 24-well tissue culture plate and empty PLGA nanoparticles of different concentrations (0.06–600 g/ml) in 0.9% (w/v) NaCl solution Nanosci. Nanotechnol. Lett. 5, 1–7, 2013

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were added at a final volume of 500 l. Cells were incubated for 1 h at room temperature and at the end of the incubation RBC morphology was examined under a Leica bright light microscope (Leica DM IL, Leica Microsystems, Germany) equipped with a camera (Leica DC 100, Leica Microsystems, Germany). 2.5. Generation of BM-DCs and Nanoparticles Uptake and Localization To determine the uptake of thePLGA nanoparticles by DCs, murine BM-DCs generated from normal BALB/c mice in the presence of rGM-CSF (20 ng/ml) following the method described by Agallou et al.11 were used. On day 8, non adherent cells were collected and characterized; cell viability was > 95% as determined by trypan blue exclusion and the percentage of CD11c+ CD8− cells was > 75% as determined by Flow Cytometry. BM-DCs were incubated with PLGA nanoparticles loaded with a low (PLGA-BSA-FITCL or a high (PLGA-BSA-FITCH BSA-FITC concentration at a dose range of 25–600 g/ml. Further experiments were conducted in order to examine the fate of PLGA nanoparticles after internalization by BM-DCs. Cells were pulsed with PLGA-BSA-FITC nanoparticles loaded with high amount of BSA-FITC at the optimal concentration of 600 g/ml, for 30 min at 4 C. After pulsing, PLGA nanoparticles that had not been attached to BM-DCs surface were washed gently with cold PBS and cells were incubated in fresh complete RPMI1640 medium for 1, 2 and 6 h at 37 C in 5% CO2 in order to allow synchronous nanoparticles internalization. To identify endolysosomal localization, cells were incubated with the fluorescent acidotropic probe LysoTracker Red (Molecular Probes, Eugene, Oregon, USA), which is highly selective for acidic organelles.12 Cells were examined using a confocal microscope (Leica TCS-SP; Leica Microsystems, Wetzlar, Germany) and the acquisition and processing of images was carried out using the aid of Leica Confocal Software. 2.6. Effect of Nanoparticles Uptake on BM-DCs Functionalization The effect of PLGA nanoparticles internalization on BMDCs phenotypic maturation was evaluated based on the surface expression of CD40, CD80, CD86 and MHC class II molecules. For this purpose, BM-DCs were exposed to empty PLGA, PLGA-BSA-FITCH , PLGA-BSA-FITCL or OVA-PLGA nanoparticles (600 g/ml) for 24 h at 37 C in 5% CO2 and stained with R-PE-conjugated rat antimouse CD40 (clone 3/23; BD Biosciences), CD80 (clone 16-10A1; BD Biosciences), CD86 (clone GL1; BD Biosciences), anti-MHC class II and anti-CD11c monoclonal antibodies. Untreated BM-DCs (medium alone) served as negative control, whereas LPS (1 g/ml)-stimulated BMDCs served as positive control. Nanosci. Nanotechnol. Lett. 5, 1–7, 2013

The ability of BM-DCs that have internalized empty or Ag-loaded PLGA nanoparticles to produce IL-12 or IL-10 was assessed by intracellular staining and flow cytometry. The cells were stained with PE-conjugated anti-IL-12p70/p40 (clone C15.6; BD Biosciences) or antiIL-10 (clone JES5-16E3; BD Biosciences) monoclonal antibodies. Where appropriate, data are expressed as the mean values ± SD. For comparison of the mean of two experimental groups, statistical significance was assessed by two-tailed Student’s t-test. The probability (P ) of ≤ 0 05 ≤ as considered to indicate statistical significance.

3. RESULTS 3.1. BSA-FITC and OVA Containing PLGA Nanoparticles In all cases (empty and antigen loaded nanoparticles), perfectly spherical PLGA nanoparticles were obtained with an average diameter in the range of 289 to 298 nm and a negative zeta potential value, varying from −17 5 to −24 2 mV (Table I, Fig. 1(A)). The negative zeta potential values of the PLGA nanoparticles are due to the carboxyl groups residing on the surface of the nanoparticles.13 (A)

(B)

Fig. 1. SEM photomicrograph of PLGA-BSA-FITCH (A) and size distributions of PLGA, PLGA-OVA and PLGA-BSA-FITCH (B) nanoparticles.

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Uptake of BSA-FITC Loaded PLGA Nanoparticles by BM-DCs Induces Maturation Table I. Characteristic properties of PLGA nanoparticles. Antigen in Av. Zeta Antigen enc. Antigen recipe diam. potential eff. loading (mg) (nm) (mV) (%) (wt %)

Nanoparticles PLGA PLGA-OVA PLGA-BSA-FITCH PLGA-BSA-FITCH PLGA-BSA-FITCH PLGA-BSA-FITCL

– 1 5 5 5 5 1 5

297 296 296 289 291 298

−22 0 −17 5 −17 6 −24 2 −22 0 −19 5

– 78.43 86.87 84.78 88.74 80.70

– 1.307 4.826 4.710 4.930 1.345

experiments (Table I, PLGA-BSA-FITCH . As can be seen, there was an excellent reproducibility with respect to nanoparticles characteristics and BSA-FITC loadings. The particle size distributions of empty and antigen loaded PLGA nanoparticles are presented in Figure 1(B). It is apparent that the incorporation of antigen does not affect the particle size distribution of the PLGA nanoparticles. 3.2. Effect of PLGA Nanoparticles on Cell Viability

The respective values for BSA-FITC and OVA loadings are also reported in Table I. High encapsulation efficiencies for the antigens were obtained independently of the initial antigen concentration in the initial solution. The increased encapsulation efficiencies of BSA-FITC and OVA could be attributed to the ionic interactions between their positively charged amino groups and the negatively charged uncapped carboxylic end groups of PLGA. Furthermore, it was apparent that PLGA nanoparticles containing various amounts of antigen (1.3–4.9% wt) could be successfully prepared by varying the initial antigen concentration. It should be mentioned that high encapsulation efficiency of BSA-FITC in PLGA nanoparticles has been also observed in the literature but only for larger nanoparticles (i.e., 350–400 nm).4 14 The reproducibility of the double emulsion method for preparing different batches of nanoparticles was tested in three independent (A)

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PLGA nanoparticles in high concentrations may cause cell death due to toxicity of PLGA metabolic products if present in high concentrations inside cells. It is also known that proteins adsorbed on nanoparticles influence interactions with erythrocytes and may lead to cells aggregation which may prove detrimental for the cardiovascular system.15 As shown in Figure 2(A), exposure of L929 cells to BSA-FITC-loaded PLGA nanoparticles did not affect cell viability even at the highest concentration (1.6 mg/ml) used irrespective of the antigen loading and its amount compared to L929 cells left in medium alone. Also, empty PLGA nanoparticles were not associated with any cytotoxicity at any of the concentrations tested. 3.3. PLGA Nanoparticles Hemocompatibility Incubation of whole blood cells with PLGA nanoparticles at the concentration range of 0.3–600 g/ml indicated no detectable release of hemoglobin compared to whole

(B)

(C)

Fig. 2. Cell viability of L929 fibroblasts (A) exposed to various concentrations of empty PLGA, PLGA-BSA-FITCH or PLGA-OVA ranging from 25 to 1600 g/ml nanoparticles for 24 h, at 37 C, in 5% CO2 . Each point represents the mean ± SD obtained from two independent experiments in triplicates. Hemolytic effect (B) and agglutination (C) of rat RBCs exposed to various concentrations of empty PLGA ranging from 4.5 to 600 g/ml NPs for 24 h, at 37 C, in 5% CO2 . RBCs incubated with medium alone or 1% TritonX100 served as negative or positive control, respectively. Figures 2(B), (C) are representative from two independent experiments.

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Uptake of BSA-FITC Loaded PLGA Nanoparticles by BM-DCs Induces Maturation (A)

(C)

(B)

Fig. 3. Concentration—(A) and time-dependent (B) uptake of PLGA-BSA-FITCH and PLGA-BSA-FITCL by BM-DCs following exposure to various concentrations of nanoparticles for different time-points at 37 C in 5% CO2 . Each point represents the mean ± SD obtained from three independent experiments in duplicates. Localization of PLGA-BSA-FITCH nanoparticles (C) in BM-DCs pulsed with 600 g/ml nanoparticles and incubated at 37 C in 5% CO2 for 1, 2 and 6 h. Thirty min prior to culture termination, cells were stained with LysoTracker, specific for acidic organelles staining. Lysosomes and nanoparticles are illustrated in red and green, respectively; co-localization is indicated in yellow. Figure 3 is representative from two independent experiments.

blood cells incubated with Triton X-100 which served as lysis positive control (Fig. 2(B)). In addition, whole blood cells exposure to PLGA nanoparticles did not induce any change in their morphology or their agglutination as revealed by light microscopy (Fig. 2(C)). The above results show that PLGA nanoparticles are biocompatible confirming previous observations that suggest PLGA as safe carriers for antigen delivery.16 3.4. Effect of BSA-FITC Loading on Nanoparticles’ Uptake Optimization of PLGA nanoparticles for use in vaccination presupposes their efficient uptake by professional Ag-presenting cells, DCs. The extent of nanoparticle uptake by CD11c+ CD8− cells was independent from the amount of Ag loaded on the PLGA nanoparticles, since similar internalization curves were observed for Nanosci. Nanotechnol. Lett. 5, 1–7, 2013

both nanoparticles formulations (Fig. 3(A)). However, internalization was concentration dependent reaching 49 86 ± 5 6% for PLGA-BSA-FITCH and 51 8 ± 6 9% for PLGA-BSA-FITCL at the dose of 600 g/ml after 24 h of exposure. No enhancement in the total number of cells taking up PLGA nanoparticles was observed when incubated with higher concentrations up to 1600 g/ml. As far as time of exposure is concerned, the number of CD11c+ CD8− cells internalizing PLGA nanoparticles increased exponentially until 4 h of exposure for both PLGA-BSA-FITCH and PLGA-BSA-FITCL (38 06 ± 5 9% and 36 63 ± 4 5%, respectively) (Fig. 3(B)). Further but smaller increase was observed till 24 h of exposure (PLGA-BSA-FITCH : 49 89 ± 3 3%, PLGA-BSA-FITCL : 52 52 ± 4 4%. Probably, PLGA-BSA-FITC nanoparticles internalization is mediated through ionic interactions between cell membrane and PLGA nanoparticles, as reported by other groups regarding similar sized 5


PLGA nanoparticles uptake by DCs and macrophages,17 since similar trend of uptake for both nanoparticles was observed. Microscopic observation showed that PLGA nanoparticles internalization in cytoplasm was achieved as early as 15 min of incubation (data not shown). After 1 h of incubation there was a partial co-localization of PLGABSA-FITCH with lysosomes, while after 2 h of incubation most of PLGA-BSA-FITCH was co-localized with lysosomes indicating their possible processing by lysosomes (Fig. 3(C)). Interestingly, at later time points (6 h), although nanoparticles were still observed in the lysosomes, labeled dots were also observed in the cytoplasm indicating possible cross-presentation of the encapsulated antigen, as proposed by Han et al.18 It is nevertheless difficult to differentiate whether green fluorescence indeed depicts PLGA-BSA-FITC nanoparticles or BSAFITC fragments alone.19

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(A)

(B)

3.5. PLGA Nanoparticles’ Uptake Induces BM-DCs Maturation But Not IL-12 or IL-10 Production Subsequently, the effect of PLGA internalization on BM-DCs maturation was investigated, since, efficient DCs maturation, characterized by increased co-stimulatory (e.g., CD40, CD86) and MHC class I and II molecules expression, is necessary for efficient T -cell priming.20 Compared to untreated cells, internalization of BSAFITCH -loaded PLGA nanoparticles conferred significant increase of cells expressing CD40, CD80, CD86 and MHC class II molecules in contrast to BM-DCs exposure to PLGA-BSA-FITCL that resulted in significant increase of cells expressing CD40 molecules only (P < 0 05) (Fig. 4(A)). However, the frequency of cells expressing surface molecules was lower than that detected after LPS stimulation. Interestingly, exposure to empty or PLGA-OVA nanoparticles did not induce increase of cells expressing the surface molecules comparing to negative control. Whether PLGA nanoparticles induce DCs maturation needs further investigation, since there are controversial results in literature. Some researchers have found that PLGA biofilms induce surface molecules levels upregulation in immature hDCs,21 while others claim that stable cationic PLGA microparticles do not induce DCs maturation.22 Concerning PLGA particles of nanoscale size, Elamanchili et al.16 as well as Clawson et al. have shown that empty PLGA nanoparticles induce moderate increase in expression of maturation molecules.23 On the contrary, there are results indicating that PLGA nanoparticles do not have any effect on DCs maturation.24 25 This discrepancy may be partially explained by differences in nanoparticles preparation and size as well as in different proportion of Ag loading. Enhancement in the expression of maturation molecules on DCs surface by itself is insufficient in generating an 6

Fig. 4. Effect of PLGA nanoparticles on the expression of CD40, CD80, CD86 and MHc class II molecules (A) and on IL-12 and IL10 intracellular production (B) by BM-DCs. Cells were exposed to 600 g/ml nanoparticles at 37 C in 5% CO2 for 24 h. Medium alone or LPS (1 g/ml) stimulated cells served as negative or positive control, respectively. Bars represent the mean ± SD obtained from three independent experiments. Significant differences from empty PLGA nanoparticles are indicated by ∗ P < 0 05 ∗∗ P < 0 01.

effective T -cell response. Specifically, the outcome of T -cell immune responses elicited is dictated by the type and quantities of cytokines secreted by fully mature DCs.26 Compared to LPS-stimulated BM-DCs which elicited significant increase of the frequency of IL-12+ or IL-10+ BM-DCs (14 ± 2 5%, P = 0 01 and 17 ± 1 6%, P = 0 002, respectively) compared to untreated cells, exposure to Ag-loaded PLGA nanoparticles did not induce IL-12 or IL-10 production (Fig. 4(B)). In addition, internalization of empty PLGA nanoparticles did not induce cytokine production. Our results indicate that despite the induction of phenotypic maturation, exposure of BMDCs to either PLGA-BSA-FITC or PLGA-OVA nanoparticles did not trigger the production of IL-12 or IL-10 Nanosci. Nanotechnol. Lett. 5, 1–7, 2013

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cytokines indicating the need for an adjuvant encapsulation in the PLGA nanoparticles used in order to achieve BM-DCs functionalization. The ability of DCs exposed to Ag-loaded PLGA nanoparticles to induce T cell priming in vitro has been reported suggesting that PLGA nanoparticles promote protein antigenicity.16 27 For that reason, the ability of DCs to produce cytokines after exposure to PLGA nanoparticles formulations must be further investigated in in vivo models.

4. CONCLUSIONS In summary, PLGA nanoparticles with an average size in the range of 289 to 298 nm and antigen (i.e., BSAFITC, OVA) loadings in the range of 1.3 to 4.9 wt% were successfully prepared by the double emulsion method. The PLGA nanoparticles were found to be non-cytotoxic and hemocompatible. Furthermore, the PLGA nanoparticles were efficiently taken up by BM-DCs irrespective of their Ag loading and they were localized in the lysosomes. However, they were not able to induce functional BM-DCs. These findings show that with a further encapsulation of an adjuvant the synthesized nanoparticles can be employed as potential delivery systems for vaccination against intracellular pathogens. Acknowledgment: We gratefully acknowledge EC for supporting this research under the FP6-2004-NMP-NI4 026723-2 Project.

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3. J. Ma, et al., J. Pharm. Sci. 87, 1375 (1998). 4. M. Diwan, M. Tafaghodi, and J. Samuel, J. Control Release 85, 247 (2002). 5. C. D. Partidos, A. Delmas, and M. W. Steward, Mol. Immunol. 33, 1223 (1996). 6. J. Ma, J. Samuel, G. S. Kwon, A. A. Noujaim, and R. Madiyalakan, Cancer Immunol Immunother 47, 13 (1998). 7. C. S. Chong, et al., J. Control Release 102, 85 (2005). 8. F. Sarti, et al., Biomaterials 32, 4052 (2011). 9. D. W. O’Neill, S. Adams, and N. Bhardwaj, Blood 104, 2235 (2004). 10. K. Saotome, H. Morita, and M. Umeda, Toxicol In Vitro 3, 317 (1989). 11. M. Agallou, M. Margaroni, and E. Karagouni, Vaccine 29, 5053 (2011). 12. S. Klingel, G. Rothe, W. Kellermann, and G. Valet, Methods Cell Biol. 41, 449 (1994). 13. O. Molavi, Z. Ma, S. Hamdy, A. Lavasanifar, and J. Samuel, Immunopharmacol Immunotoxicol. 31, 214 ( 2009). 14. U. Bilati, E. Allemann, and E. Doelker, J. Microencapsul. 22, 205 (2005). 15. J. K. Armstrong, R. B. Wenby, H. J. Meiselman, and T. C. Fisher, Biophys. J. 87, 4259 (2004). 16. P. Elamanchili, C. M. Lutsiak, S. Hamdy, M. Diwan, and J. Samuel, J. Immunother. 30, 378 (2007). 17. L. Thiele, et al., J. Control Release. 76, 59 (2001). 18. R. Han, J. Zhu, X. Yang, and H. Xu, J. Biomed. Mater. Res. A 96, 142 (2010). 19. G. Romero, et al., Biomacromolecules 2993 (2010). 20. J. Banchereau and R. M. Steinman, Nature 392, 245 (1998). 21. J. E. Babensee and A. Paranjpe, J. Biomed. Mater. Res. A 74, 503 (2005). 22. C. Wischke, H. H. Borchert, J. Zimmermann, I. Siebenbrodt, and D. R. Lorenzen, J. Control Release 114, 329 (2006). 23. C. Clawson, et al., Nanomedicine 6, 651 (2010). 24. S. Hamdy, A. Haddadi, V. Somayaji, D. Ruan, and J. Samuel, J. Pharm. Biomed. Anal. 44, 914 (2007). 25. W. Ma, et al., J. Transl. Med. 9, 34 (2011). 26. M. Moser and K. M. Murphy, Nat. Immunol. 1, 199 (2000). 27. K. D. Newman, J. Samuel, and G. Kwon, J. Control Release 54, 49 (1998).

Received: 6 September 2011. Accepted: 26 July 2012.

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