Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation

Preparation of PVM/MA nanoparticles.Poly(anhydride) NPs were prepared by a modification of the solvent displacement method (3). Briefly, 100 mg poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer were dissolved in 6 ml acetone. The acetone-copolymer mixture was poured into a solution containing 0.2 g mannitol in 5.8 ml of double-deionized water (H₂Odd). All solvents were evaporated under reduced pressure by spray drying (mini-spray dryer; Büchi, Switzerland).

Characterization of nanoparticles.The size and zeta potential of nanoparticles were determined by photon correlation spectroscopy and electrophoretic laser Doppler anemometry, respectively, employing a Zetamaster analyzer system (Malver Instruments Ltd., Worcestershire, United Kingdom). The diameter of the nanoparticles was determined after dispersion in ultrapure water (1:10) and measured at 25°C with a dynamic light scattering angle of 90°C. The zeta potential was determined as follows: 200 μl of the samples were diluted in 2 ml of a 0.1 mM KCl solution adjusted to pH 7.4 (25). The average particle size is expressed as the volume mean diameter (Vmd) in nanometers (nm), and the average surface charge as mV.

Differentiation of mouse dendritic cells from mouse femur bone marrow precursors.Bone marrow dendritic cells (BMDC) were generated as follows: bone marrow from BALB/c mouse femur and tibia was extracted, and erythrocytes were lysated in ammonium chloride potassium (ACK) lysing buffer for 1 min (NH₄Cl, 0.15 M; KHCO₃, 10 mM; Na₂ EDTA, 0.1 mM). Obtained cells were then washed by centrifugation in RPMI 1640 medium. The lymphocyte and granulocyte populations were then depleted by coincubation with 50 μg/ml of rabbit complement (Sigma Chem. Co.) and an antibody mixture against CD4 (GK1-5 hybridoma, 100 μg/ml), CD8 (H35.17.2 [142] hybridoma, 100 μg/ml), Ly-6G/Gr1 (10 μg/ml), and CD45R/B220 (50 μg/ml). (All reagents were from BD Pharmingen.) The mixture was incubated at 37°C for 50 min. After depletion, cells were washed, and all remaining cells were harvested in 24-well plates at 10⁶ cells/ml. The medium (RPMI 1640 with 10% fetal calf serum [FCS], 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 × 10⁻⁵ mM 2-mercapthoethanol) was supplemented with 20 ng/ml of mouse granulocyte/macrophage colony-stimulating factor and interleukin 4 (IL-4) (Prepotech, London, United Kingdom) at 37°C and in 5% CO₂. At days 2 and 5, two-thirds of the medium was replaced with fresh cytokine-supplemented medium. On the 7th day, in vitro assays for BMDC maturation markers were carried out by adding 500, 100, 20, and 4 μg/ml of poly(anhydride) NPs along with negative controls. Forty-eight hours later, supernatants were collected, and IL-12 (p70) and tumor necrosis factor alpha (TNF-α) measured by enzyme-linked immunosorbent assay (ELISA) (BD Pharmingen), according to the manufacturer's instructions. Finally, all cells were harvested and analyzed by flow cytometry by using anti-CD54 (clone 3E2), anti-CD86 (clone GL1), anti-CD11c (clone HL-3), and for the unspecific bind measurement, mouse fluorescein isothiocyanate (FITC)-labeled IgG1 (clone MOPC-21), at 4°C for 15 min. All reagents originated from BD Pharmingen. Then cells were washed, acquired on a FACScan cytometer (BD Biosciences), and analyzed using Cell Quest software (BD Biosciences). Finally, the gating was selected by comparison with positive and negative controls. The mean fluorescence intensity (MFI) of the control isotype (IgG1) was subtracted from the fluorescence data of its own group and then referred to the fluorescence of the negative control for the analyzed maturation marker, as follows: increase level = (sample MFI_marker − sample MFI_IgG1)/(untreated MFI_marker − untreated MFI_IgG1).

In vivo killing.The system used to detect cytotoxic T lymphocyte (CTL) responses elicited after OVA-NP immunization is based on antigen presentation of OVA peptides on major histocompatibility (MHC) class I molecules. In vitro restimulation of splenic cells is performed with a specific OVA peptide characterized by its specific recognition MHC class I molecule. Thus, C57BL/6 mice were intraperitoneally (i.p.) immunized with 4 mg of empty poly(anhydride) NPs, 10 μg of OVA, or both 4 mg of NP and 10 μg OVA in different injection sites to reduce adsorptions. Due to the high ability of poly(anhydride) NPs to bind the amino-terminal part of proteins, NPs and OVA were administered in different sites of the abdomen, reducing possible OVA coating of poly(anhydride) NPs. At day 7, spleens from naïve mice were extracted and smashed, and erythrocytes lysed in ACK buffer. Resulting splenocytes were treated in the presence or in the absence of 10 μg/ml of the SIINFEKL peptide (encompassing the immunodominant H-2b-restricted CTL epitope from ovalbumin [amino acids 257 to 264], produced by Neomps, Strasbourg, France) for 30 min at 37°C. Splenic cells were then differentially labeled with 2.5 μM (SIINFEKL-pulsed targets) or 0.25 μM (nonpulsed splenocytes) of 5-(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) for 15 min at room temperature and used as target cells to detect in vivo cytotoxic activity. A dose of 10⁷ cells per mouse (50% CFSE high and 50% CFSE low) was intravenously (i.v.) injected into the naïve and immunization groups. After 24 h spleens were removed, and the high-versus-low-CFSE lymphocyte population ratio was analyzed by flow cytometry. All lysis values correspond to triplicate samples. Specific lysis was calculated as follows: % specific lysis = 100 − {[% splenocytes CFSE high (from immunized mice)/% splenocytes CFSE low (from immunized mice)]/[% splenocytes CFSE high (from nonimmunized mice)/% splenocytes CFSE low (from nonimmunized mice)]} × 100.

Specific IFN-γ production.To measure the production of gamma interferon (IFN-γ) in response to SIINFEKL, splenocytes from mice collected at day 8 after immunization were plated on 96-well plates at 8 × 10⁵ cells/well with culture medium (CM) alone or with 30 μM SIINFEKL in a final volume of 0.25 ml of CM. Supernatants (50 μl) were removed 48 h later, and IFN-γ was measured by enzyme-linked immunospot (ELISPOT) assay (BD Pharmingen) according to the manufacturer's instructions.

TLR screening.LPS-free empty NPs were shipped to Invivogen (France) to assay their capacity to stimulate TLR signaling. Briefly, samples and controls were tested on recombinant HEK293 cell lines that functionally overexpress a given TLR protein as well as a reporter gene which is a secreted alkaline phosphatase. The production of this reporter gene is driven by a NF-κβ-inducible promoter. Nanoparticles were incubated at 500 μg/ml, the highest nontoxic concentration, as recommended by Invivogen. Known TLR agonists used as positive controls include PAM2 (100 ng/ml) for TLR2; poly(I:C) (100 ng/ml) for TLR3; Escherichia coli K12 LPS (1 μg/ml) for TLR4; flagellin (1 μg/ml) for TLR5; R848 (10 μg/ml) for TLR7 and TLR8, and ODN 2006 (10 μg) for TLR9. All results are given as optical density (OD) values.

Protection studies of mice.Phosphate-buffered saline (PBS) or empty poly(anhydride) NPs were administered to groups of 10 BALB/c mice. Ten days after administration, mice were i.p. infected with a lethal dose of Salmonellaenterica serovar Enteritidis in stationary phase (1.6 × 10² CFU, in 100 μl phosphate-buffered saline). The number of dead mice after challenge was recorded daily. All mice were treated in accordance with institutional guidelines for treatment of animals (Ethical Committee for the Animal Experimentation, CEEA, of the University of Navarra).

IFN-γ and IL-4 release from splenic cells.In order to determine the degree of activation of the two subset Th1/Th2 populations in immunized mice, IFN-γ and IL-4 release from splenic cells was determined. Three animals immunized with PBS or poly(anhydride) NPs were sacrificed at day 10 by cervical dislocation, and spleens were removed and placed in RPMI 1640 media (Gibco-BRL, Paisley, United Kingdom) under sterile conditions. Spleens were smashed in tissue culture dishes and centrifuged (400 × g, 10 min), the supernatant was discarded, and the sediment was washed twice in phosphate-buffered saline. The cellular pellet was resuspended in 2 ml RPMI 1640-HEPES medium (Gibco-BRL) supplemented with 500 μl of antibiotic-antimycotic solution (Sigma) and 10% (vol/vol) fetal bovine serum. In a 24-well microtiter plate (Costar, NY), cell suspensions were added at 800,000 cells/well, along with Salmonella serovar Enteritidis heat extracts (HE) at 100 μg/well (36). Negative controls without HE were also included. All wells were set up in duplicate. Cells were incubated (37°C, 48 h, 5% CO₂), supernatants were collected, and the levels of IFN-γ and IL-4 released determined with a commercial sandwich ELISA kit (Biosource, Camrillo, CA).

Statistical analyses.When required, the nonparametric Kruskal-Wallis test, followed by the Mann-Whitney U test, was used. Statistically significant differences were considered when P was <0.05. All data processing was performed using the GraphPadPrism 5 software.

Dendritic cell costimulatory molecule increase triggered by nanoparticles.Nanoparticle formulations displayed a homogeneous volume mean diameter of 230 ± 5 nm, with a low polydispersity index. In addition, poly(anhydride) NPs displayed an electronegative surface charge with a Zeta potential value of −36 ± 2 mV.

After the differentiation of BMDC, an analysis of CD54 and CD86 markers was performed in order to confirm the induction of positive maturation triggered by NPs. Flow cytometry studies showed that incubation of 4 μg/ml of poly(anhydride) NPs induced 2.3- and 2.4-fold increases of expression of both CD54 and CD86, respectively, compared with BMDC incubated with medium alone (Fig. 1). When the concentration of empty NP was raised to 20 μg/ml, the expression of the costimulatory molecule CD54 reached a 3.6-fold increase relative to the negative control.

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FIG. 1.

Flow cytometric analysis of BMDC maturation markers. Bars show the fold-increase of mean relative fluorescence over negative control of CD86 and CD54 after coincubation of BMDC with 500 μg of NP, 100 μg of NP, 20 μg of NP, and 4 μg of NP.

Induction of cytokine production of BMDC by NP stimulation.We then examined whether the NP stimulation was able to stimulate bone marrow-derived DCs to produce the proinflammatory cytokine IL-12 or TNF-α. It was found that NP stimulation induced TNF-α release in a dose-dependent manner (Fig. 2A), varying from 27.8 pg/ml at 4 μg/ml of NP to 478.7 pg/ml at 500 μg/ml and being in all cases significantly higher (P < 0.001) than those found in supernatant from nonstimulated cells. The analysis of IL-12 production revealed also a dose-response pattern, being significant from 20 to 100 μg/ml. A higher concentration of NP (500 μg/ml) reduced the IL-12 release to basal levels (Fig. 2B).

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FIG. 2.

Proinflammatory cytokine detection in supernatants of BMDC. Levels of TNF-α (A) and IL-12 (B) released to the BMDC culture supernatant after 24 h of coincubation with 500 μg of NP, 100 μg of NP, 20 μg of NP, and 4 μg of NP. Nonstimulated controls are also included. All values are shown as arithmetic means ± standard errors of the means (SEM) (n = 3).

Cytotoxicity induced by NP uptake: in vivo killing.An in vivo CTL assay was conducted to measure the capacity of NPs to activate OVA-specific CD8⁺ T cells using the specific SIINFEKL OVA peptide. C57BL/6 mice were immunized i.p. with OVA (10 μg), NP (4 mg), or a mixture of OVA and NP. At day 7 after immunization, mice were injected i.v. with a mixed population of CFSE-labeled splenocytes. Twenty-four hours after injection of the CFSE-labeled cells, splenocytes were analyzed by flow cytometry for the presence of the two populations of CFSE-labeled cells. Results indicate that the specific T-cell lysis response to SIINFEKL treatment was negligible in the absence of the NP adjuvant. However, when lymphocytes from mice immunized with NPs plus OVA were tested, the in vivo specific CTL response against SIINFEKL was 41.2% ± 16.5% (Fig. 3).

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FIG. 3.

In vivo induction of CTL activity specific for SIINFEKL peptide. C57BL/6 mice were immunized with OVA, poly(anhydride) NPs, or OVA plus NP; 7 days after immunization, naïve mice were sacrificed, and splenocytes coincubated in the presence or absence of SIINFEKL. Pulsed cells were incubated in the presence of high levels of CFSE, while nonpulsed cells were incubated with low levels of CFSE. After administration of CFSE-high and CFSE-low splenocytes to immunized mice, the specific cytotoxic activity against the SIINFEKL peptide was measured by an in vivo killing assay. The data represent the mean percentage values from triplicate samples.

In addition to measuring the in vivo cytotoxicity, we also measured by ELISPOT the number of IFN-γ-producing cells specific for the SIINFEKL peptide induced by immunization. Thus, 7 days after immunization, splenic cells were restimulated in vitro in the presence or absence of the SIINFEKL peptide. The number of IFN-γ spots was counted to evaluate the frequency of SIINFEKL-specific cytotoxic T cells. The ELISPOT assay revealed IFN-γ-producing OVA-specific cells from mice injected with OVA-NP (129.25 ± 56.20 spot-forming cells [SFC]/10⁶ cells compared with 27.5 ± 11.1 SFC/10⁶ cells in the absence of peptide, P < 0.05) (Fig. 4). In contrast, IFN-γ-producing splenocytes specific for the SIINFEKL peptide were not detected in mice immunized with NPs or OVA alone.

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FIG. 4.

In vivo induction of OVA-specific IFN-γ-secreting CD8⁺ T cells. Mice were immunized with OVA, poly(anhydride) NPs, and OVA plus NP and 8 days after immunization sacrificed. Splenocytes were cultured in triplicate in the presence of SIINFEKL or culture medium alone (negative control). Each bar represents the mean value of SFC/10⁶ cells.

Nanoparticle-induced TLRs.In order to understand the adjuvant capacities shown by the poly(anhydride) NPs, their ability to trigger different TLR responses was measured. As shown in Fig. 5, nanoparticles significantly activated hTLR2-, hTLR4-, and hTLR5-expressing cell lines. In addition the hTLR3 cell line was also weakly activated. The TLR⁻ cell line was not stimulated at all. This cell line does not express any TLR but still has an NF-κβ-inducible promoter. This result confirmed that all measured OD values were strictly a consequence of TLR stimulation.

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FIG. 5.

Effects of nanoparticles on the activation of TLR signaling. Bars represent engagement to TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 after incubation with positive controls and poly(anhydride) NPs. Names inside the bars of positive controls represent the specific agonist for each TLR used in the study: PAM2 (100 ng/ml) for TLR2, poly(I:C) (100 ng/ml) for TLR3, E. coli K12 LPS (1 μg/ml) for TLR4, flagellin (1 μg/ml) for TLR5, R848 (10 μg/ml) for TLR7 and TLR8, and ODN 2006 (10 μg) for TLR9. A TLR nonexpressing recombinant cell line is also included (TLR⁻). Results are given in OD values.

Immunization and protection studies of mice against Salmonella serovar Enteritidis.Ten days after the intraperitoneal administration of poly(anhydride) NPs, splenocytes showed a basal expression of IFN-γ (20 pg/ml). Restimulation with an antigenic complex containing LPS (HE, from S. Enteritidis) resulted in a significative (P > 0.001) increase in detected IFN-γ (577 pg/ml). Nonimmunized animals showed no IFN-γ release before restimulation and a very small amount (40 pg/ml) after incubation with HE containing LPS. When IL-4 production was measured, a very weak basal secretion was detected for restimulated splenocytes in both the inoculated and control groups (10 pg/ml).

Nanoparticle inoculation elicited a 30% protection against lethal intraperitoneal challenge with S. Enteritidis (1.6 × 10² CFU) (Fig. 6). In addition, the end survival date was significantly delayed (P < 0.001) from day 7 for the control group until day 23 for the treated animals.

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FIG. 6.

Comparative protection against the virulent serovar of S. Enteritidis. Groups of 10 BALB/c mice were i.p. immunized with poly(anhydride) NPs (♦) or PBS (○). Ten days later, mice were challenged i.p. with 1.6 × 10² CFU of the virulent strain 3934. Data are expressed as percentage of survivals after challenge.