Chitosan Nanoparticles

(5, 6) Up to now, MC activation capacity by small molecules, like compound 48/80 (C48/40), was well characterized, and the ability of these molecules ...
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Article Cite This: Mol. Pharmaceutics 2018, 15, 72−82

Adjuvant Activity of Poly-ε-caprolactone/Chitosan Nanoparticles Characterized by Mast Cell Activation and IFN‑γ and IL-17 Production Sandra Jesus,†,‡ Edna Soares,†,‡ Gerrit Borchard,§ and Olga Borges*,†,‡ †

Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal Center for Neuroscience and Cell Biology, University of Coimbra, 3000-548 Coimbra, Portugal § School of Pharmaceutical Sciences, University of Geneva, Unssssiversity of Lausanne, 1211 Geneva, Switzerland ‡

ABSTRACT: Polymeric nanoparticles (NPs) are extremely attractive vaccine adjuvants, able to promote antigen delivery and in some instances, exert intrinsic immunostimulatory properties that enhance antigen specific humoral and cellular immune responses. The poly-ε-caprolactone (PCL)/chitosan NPs were designed with the aim of being able to combine the properties of the 2 polymers in the preparation of an adjuvant for the hepatitis B surface antigen (HBsAg). This article reports important results of an in vitro mechanistic study and immunization studies with HBsAg associated with different concentrations of the nanoparticles. The results revealed that PCL/chitosan NPs promoted mast cell (MC) activation (β-hexosaminidase release) and that its adjuvant effect is not mediated by the TNF-α secretion. Moreover, we demonstrated that HBsAg loaded PCL/chitosan NPs, administered through the subcutaneous (SC) route, were able to induce higher specific antibody titers without increasing IgE when compared to a commercial vaccine, and that the IgG titers are nanoparticle-dose dependent. The results also revealed the NPs’ capability to promote a cellular immune response against HBsAg, characterized by the production of IFN-γ and IL-17. These results demonstrated that PCL/chitosan NPs are a good hepatitis B antigen adjuvant, with direct influence on the intensity and type of the immune response generated. KEYWORDS: nanoparticles, immunological adjuvant, poly-ε-caprolactone, chitosan, HBsAg, CpG-ODN β-hexosaminidase within minutes of activation.4 Moreover, MCs release insoluble granular particles composed of heparin proteoglycans and proteases that increase the ability to present antigens to the naive T cells, once internalized by DCs.5,6 These granules are also able to reach lymph nodes and contribute their structural modification, which favors DCs interaction with lymphocytes essential for the generation of adaptive immune responses.5,6 Up to now, MC activation capacity by small molecules, like compound 48/80 (C48/40), was well characterized, and the ability of these molecules to increase vaccine immune responses was already documented.7,8 However, with the exception of a report recently published by our group on chitosan NPs,7 there are no other reports in the literature describing the ability of polymeric NPs to activate MC. In fact, chitosan NPs’ ability to induce β-hexosaminidase release from MCs was observed and this discovery has contributed to a deeper understanding of its adjuvant mechanism.7 Likewise, studies with other polymeric NPs may also help to elucidate their vaccine immunostimulant adjuvant properties. Poly-ε-caprolactone (PCL) is a biocompatible polymer and has Food and Drug Administration (FDA) approval for several biomedical applications, such as tissue repair9 or suture,10

1. INTRODUCTION Highly purified recombinant protein antigens normally display an excellent safety profile, but lack immunogenicity when compared with inactivated or live attenuated pathogens. As a result, vaccine adjuvants are an essential puzzle piece for recombinant vaccine efficacy. For a long time, adjuvants were classified in one of the two classes, antigen delivery systems or immunopotentiators. Currently, the scientific community acknowledges that many antigen delivery systems act, not only by modifying antigen bioavailability, but also by directly stimulating antigen presenting cells (APC’s). Particularly, polymeric nanoparticles (NPs), derived from synthetic or natural biodegradable polymers, have been used as vaccine adjuvants since they protect antigens, create a depot effect, and increase antigen presentation to dendritic cells (DCs), ultimately enhancing antigen specific humoral and cellular immune responses.1,2 The most studied mechanisms triggered by vaccine adjuvants rely on the activation of pathogenic pattern recognition receptors (PRRs) localized both at the surface and in the cytoplasm of immune cells, particularly DCs and macrophages.3 However, recent studies show that other cell types, like mast cells (MCs), also have an important physiological role in the modulation of appropriate immune responses. The MCs are considered the sensory arm of the immune system, capable of activating both innate and adaptive immune responses. This role is related to the ability of MCs to rapidly release stored inflammatory mediators, such as tumor necrosis factor (TNF), histamine, and © 2017 American Chemical Society

Received: Revised: Accepted: Published: 72

August 22, 2017 October 25, 2017 November 21, 2017 November 21, 2017 DOI: 10.1021/acs.molpharmaceut.7b00730 Mol. Pharmaceutics 2018, 15, 72−82

Article

Molecular Pharmaceutics

suspended PCL/chitosan NPs were either freeze-dried in a 5% (w/v) trehalose solution (FreezeZone 6, Labconco Corporation, Kansas City, MO, USA), resulting in a powder with 5.7% NPs, or centrifuged in 1.5 mL tubes (16000g, 30 min) and resuspended in a desired buffer volume for immediate utilization. For in vitro cell activation studies, NPs were produced under lipopolysaccharide (LPS) free conditions21 and tested to verify LPS absence with an endotoxin detection kit (Pyrogent gel clot Limulus Amebocyte Lysate (LAL) assay, 0.125 EU/mL, Lonza Group, Basel, CH). To perform studies requiring fluorescence analysis with PCL/chitosan NPs (FITC-NPs), chitosan was previously labeled with fluorescein isothiocyanate (FITC) (Santa Cruz Biotechnology Inc., Heidelberg, DE), accordingly to a protocol described already by us.20 HBsAg loaded PCL/chitosan NPs were prepared by incubating the antigen for 30 min with the concentrated PCL/ chitosan NPs water suspension (NPs resuspended in ultrapure water) under low stirring. All formulations were produced so that the final HBsAg concentration was 15 μg/mL and NPs concentration was either 4035 μg/mL, 8070 μg/mL or 16140 μg/mL. 2.2.1.1. Antigen Loading Efficacy. The amount of antigen adsorbed to the particles was calculated by the difference between the total HbsAg added and the HBsAg that remained free in solution (after centrifugation), as already described.12 HbsAg was quantified by Pierce Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), according to manufacturer’s instructions. 2.2.1.2. Size and Zeta Potential. Size and zeta potential of the PCL/chitosan NPs resuspended in water and of the vaccine formulations, were measured by dynamic light scattering and electrophoretic light scattering, respectively, with a Delsa Nano C (Beckman Coulter, Madrid, ES). 2.2.1.3. Transmission Electron Microscopy. Transmission electron microscopy (TEM) was used to observe the HBsAg adsorbed NPs (stored in 4% paraformaldehyde). Samples were placed in Formvar coated nickel or copper grids (300 mesh, TAAB Laboratories Equipment Ltd., Berkshire, UK), and observed under a FEI-Tecnai G2 Spirit Biotwin, a 20−120 kV transmission electron microscope (FEI Company, Hillsboro, OR, USA). 2.2.2. Studies on Human Mononuclear Cells. Peripheral blood was kindly given by IPST,IP (Coimbra, PT) and was obtained from normal donors in heparinized syringes followed by serum depletion. Peripheral blood mononuclear cells (PMBCs) were isolated on a density gradient with Lymphoprep (Axis-Shield, Dundee, SCT) according to the provider’s guidance protocol, with minor modifications. Briefly, the blood dilution performed was 1:5 (v/v) in 0.9% sodium chloride, the centrifugation step was performed at 1190g for 20 min (20 °C) and the mononuclear cell dense ring was collected and washed with PBS (pH = 7.4 at 37 °C) through consecutive centrifugations (487g, 10 min, 20 °C) until the supernatant was clear. At the end, cells were suspended in RPMI 1640 medium supplemented with 1% PenStrep and 10% heat inactivated FBS, and seeded in 12 or 48-well plates at a concentration of 1 × 106 cells/mL in 1 or 0.5 mL, respectively. After 2 h incubation, nonadherent cells were aspirated and the remaining adherent cells were washed twice with supplemented RPMI 1640, before incubation with ice-cold PBS for 20 min at 4 °C. Then, after new wash with icecold PBS, cells were incubated overnight with supplemented RPMI at 37 °C and used the next day.

among others. This highly hydrophobic polyester undergoes a slow degradation process, avoiding the generation of a harmful acidic environment at the site of administration.11 PCL’s ability to blend with other polymers to form composite nanoparticles is an important characteristic to modulate some of its mechanical, physical, or chemical properties,11 which can be particularly useful in obtaining a good adsorption matrix for the antigen of interest, as was recently demonstrated by our group.12 Accordingly, PCL/chitosan NPs were shown to combine the characteristics of both polymers, resulting in an improved delivery system for protein based vaccines.12 Recent data support the tailoring of the physicochemical properties of particulate biomaterials like polymeric NPs to modulate the immune system.13 The rationale for the inclusion of chitosan in PCL NPs was the generation of a more amphiphilic environment and therefore, more pathogen mimicking nanoparticle14 associated with the well described ability of chitosan to stimulate immune system cells, in vitro15,16 and in vivo.7,17 Additionally, chitosan NPs adjuvant effect was also demonstrated by our group for the hepatitis B surface antigen (HBsAg).18,19 The purpose of this work was to investigate the in vitro immunostimulatory properties of PCL/chitosan NPs and to evaluate their ability as a HBsAg adjuvant. To obtain a comparative knowledge, the commercially available formulation, Engerix-B was included in the immunization study. Additionally, the immunopotentiator CpG-ODN, was also included in one formulation of the vaccination studies with the aim to evaluate the possibility to modulate the immune response. In fact, the association of two or more adjuvants to better modulate the immune response has been the focus of our research group. Particularly, in past studies, we have already observed the redirection of the immune response for a more balanced Th1/ Th2 response when CpG-ODN, a Toll-like receptor 9 (TLR9) agonist, was associated with chitosan nanoparticles.18 To achieve the objectives of this work, in vitro assays using human immune system cells (a mast cell line and mononuclear cells isolated from the blood of healthy donors) were performed to investigate possible immunostimulatory properties of the PCL/chitosan NPs. Moreover, the results of the mice vaccination studies, using HBsAg loaded NPs, were reported to characterize the adaptive immune response generated.

2. MATERIALS AND METHODS 2.1. Materials. Chitosan (ChitoClear -95% DD and 8 cP viscosity measured in 1% solution in 1% acetic acid) was purchased from Primex Bio-Chemicals AS (Avaldsnes, NO) and purified according to a procedure already described.20 PCL (average Mw 14000 Da), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), D-(+)-trehalose dehydrate (≥98.5%), and 4-nitrophenyl N-acetyl-β-D-glucosaminide (NAG) were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). Cytosine-phosphate-guanine oligodeoxynucleotide (CpGODN) 1826, was acquired from InvivoGen (San Diego, CA, USA). Recombinant hepatitis B surface antigen (HBsAg), subtype adw with 25 kDa, was acquired from Aldevron (Fargo, ND, USA). Engerix-B was obtained from GlaxoSmithKline Biologicals (Rixensart, BE). All other chemicals and reagents used were of analytical grade. 2.2. Methods. 2.2.1. Nanoparticle Preparation and Characterization. PCL/chitosan NPs production was performed as previously described by us.12 Briefly, the particles were produced by a nanoprecipitation technique followed by centrifugation and dialysis of the resulting suspension. Next, water 73

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Molecular Pharmaceutics Table 1. Formulations for SC Immunization of C57BL/6 Mice formulation name

PCL/chitosan NPs (μg/animal)

HBsAg (μg/animal)

403.5NPs 807NPs 1614NPs 807NPs+CpG HBsAg Engerix-B

403.5 807 1614 807

1.5 1.5 1.5 1.5 1.5 1.5a

CpG-ODN (μg/animal)

SC immunization (prime and boost) (day) 0, 0, 0, 0, 0, 0,

10

14 14 14 14 14 14

euthanasia (day) 42 42 42 42 42 42

The volume of Engerix-B given to each mouse was adjusted so that HBsAg amount was 1.5 μg/animal. Note that the commercially available vaccine contains aluminum hydroxide as adjuvant. a

CO2. The β-hexosaminidase (β-hex) release assay was performed as previously described by our group7 with slight differences. HMC-1 cells were plated at a density of 6 × 106 cells/mL (100 μL) in Tyrode’s buffer and incubated during 45 min with particles suspended in the same buffer to a final concentration of 150 μg/mL, 80 μg/mL, and 40 μg/mL. For comparison, PCL NPs prepared by the same technique as PCL/chitosan NPs but in the absence of chitosan, were tested (150 μg/mL). Controls included C48/48 (positive control), 0.5% Triton X-100 (total β-hex release) and unstimulated cells (basal β-hex release). After centrifugation (10 min, 800g), 30 μL of supernatant from each well were collected and incubated with 10 μL of NAG for 30 min at 37 °C. Afterward, 100 μL of carbonate buffer (pH = 10) induced a color change and optical density was read at 405 nm in a microplate reader (Multiskan EX Microplate, Thermo Fisher Scientific Inc., Waltham, MA, USA). β-hex release was assessed using eq 2:

2.2.2.1. NP Uptake Studies. The cellular uptake of two FITC-labeled NPs concentrations was studied by flow cytometry. The assay was performed in the 48-well plates with 1 × 106 cells/mL and 40 μg NPs/mL or 10 μg NPs/mL (final volume: 0.5 mL). After a 4 h incubation period, cells were washed with 100 μL of PBS and detached from the wells with 100 μL of 0.25% trypsin-EDTA solution (Gibco, Life Technologies Corporation, Paisley, UK) a process further inactivated by 100 μL of supplemented culture medium. Cells from 12 wells were pooled in the same tube, centrifuged for 20 min at 487g, resuspended in 200 μL of ice-cold PBS and analyzed on BD FACSCalibur Flow Cytometer (BD Biosciences, Bedford, MA, USA). Prior to the analysis, 1.5 μL of 50 μg/mL propidium iodide (PI) solution was added to samples to evaluate cytotoxicity. Duplicates of the samples were also analyzed with 0.2% trypan blue solution to exclude fluorescence at the surface of the cells. The mean fluorescence for a population of 20000 cells was collected and results were analyzed. For the confocal laser scanning microscopy assay, cells previously seeded on glass coverslips in 12-well plates were incubated with FITC-NPs (10 μg NPs/mL and 5 μg NPs/mL to 1 × 106 cells/mL, final volume: 1 mL) during 4 h and then labeled with Image-iT LIVE plasma membrane and nuclear labeling kit (Life Technologies Corporation, Paisley, UK) as previously described.20 Samples were examined under a laser scanning inverted confocal microscope Carl Zeiss LSM 510 Meta with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective (Carl Zeiss, Oberkochen, DE). 2.2.2.2. TNF-α Production and Cytotoxicity Induced by NPs. To test TNF-α secretion induced by PCL/chitosan NPs (LPS free) in PBMCs, cells plated in 48 well-plates (1 × 106 cells/mL, final volume: 0.5 mL) were incubated with different NPs concentrations ranging from 40 μg/mL to 5 μg/mL. LPS was used as positive control using a concentration of 0.2 μg/mL (0.5 EU/mL). After 24 h incubation, the supernatants were collected and stored at -80 °C until further TNF-α quantification by ELISA according to manufacturer’s instructions (Human TNF alpha ELISA Ready-SET-Go!, eBioscience, San Diego, CA, USA). To examine the cytotoxicity associated with NPs stimulation, the culture medium was replaced with new medium, and MTT cell viability assay was performed according to manufacturer’s protocol and cell viability (%) calculated according to eq 1: Cell viability(%) =

β‐hex release(%) (OD sample β‐hex release − OD control β‐hex release) = (OD Triton X‐100 β‐hex release − OD control β‐hex release) × 100

The level of granularity on HMC-1 cells was assessed by the analysis of cells stained with toluidine blue. Cells were plated on poly-L-lysine coated coverslips and incubated with or without nanoparticles under the previously mentioned experimental conditions. After incubation, cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with toluidine blue for 30 min. After staining, cells were washed repeatedly and mounted on microscope slides for further examination. To evaluate if the HMC-1 degranulation and β-hex release occurred due to NPs toxicity, MTT assay was performed. The exact β-hex release procedure described above was used and after 45 min incubation plates were centrifuged (10 min, 800g) and the medium replaced by complete IMDM. MTT assay was performed right after the medium replacement for cell viability assessment. To evaluate the NPs cellular uptake during the β-hex release assay, the protocol was repeated with FITC-NPs using 24-well plates (6 × 106 cells/mL, 80 μg NPs/mL, 1 mL/well). After 45 min incubation with the particles, HMC-1 cell samples were either analyzed on BD FACSCalibur Flow Cytometer or washed and incubated for more 24 h in fresh culture medium before cytometry analysis. In both situations, cells were collected, centrifuged for 20 min at 487g, and resuspended in 200 μL of ice-cold PBS. Cell analysis was performed as described above on Section 2.2.2. 2.2.4. Subcutaneous Vaccination with HBsAg-Loaded PCL/Chitosan Nanoparticles. Eight-week-old C57BL/6 female mice from Charles River (Saint-Germain-Nuelles, FR) were housed in filter top cages with food and water provided

(OD sample(540 nm) − OD sample(630 nm)) (OD control(540 nm) − OD control(630 nm)) × 100

(2)

(1)

2.2.3. Mast Cell Activation. The human mast cell line HMC-1 was kindly provided by Dr. Butterfield, Mayo Clinic (Rochester, MN, USA), and maintained in Iscove’s modified Dulbecco’s medium (IMDM) at 37 °C, 96% relative humidity (RH), 5% 74

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Figure 1. Characterization of formulations for vaccination studies. (A) Particle mean size distribution, zeta potential, and HBsAg loading efficacy of the 403.5NPs, 807NPs, 1614NPs, and 807NPs+CpG formulations resuspended in ultrapure water. Data are presented as mean ± SD, n = 3. (B) Representative TEM images of HBsAg virus-like particles (1) and HBsAg-loaded PCL/chitosan NPs after fixing, contrasting, and embedding into Formvar coated nickel electron microscope grids (2).

2.2.4.3. Cytokine Production by Vaccinated Mice Spleen Cells. At day 42, spleens were aseptically collected from euthanized mice. Individual mice spleen cell suspensions were prepared as previously described by our group7 without erythrocytes lysis. Spleen cell suspensions (0.25 × 106 cells/well) were cultured in 96-well plates, for incubation with and without stimulation by concanavalin A (con A) (1.25 μg/well) and HBsAg (1 μg/well). Duplicates of each condition were incubated at 37 °C with 96% relative humidity (RH) in the presence of 5% CO2 for 48 h (IL-17, IFN-γ) and 72 h (IL-4). After that period plates were centrifuged and the clear supernatants collected and stored at −80 °C until further analysis. Cytokines were measured by ELISA technique according to kit manufacturer’s instructions (Murine IL-4, IFN-γ, and IL-17 standard ABTS ELISA development kits, PeproTech, Rocky Hill, NJ, USA). 2.2.5. Statistical Analysis. Results were expressed as mean ± standard error of the mean (SEM) or standard error (SD), as indicated in each figure. Flow cytometry data were analyzed with CellQuest Modfit LT analysis software (BD Biosciences, Bedford, MA, USA). All other data were analyzed with GraphPad Prism v 5.03 (GraphPad Software Inc., La Jolla, CA, USA) and tested for statistical significance by ANOVA, followed by a Tukeýs post-test for multiple comparisons. P < 0.05 was considered statistically significant difference).

ad libitum. All experiments were approved by the Ethical Animal Care Committee of the Center for Neuroscience and Cell Biology of the University of Coimbra (ORBEA_50_2013/ 27092013) and carried out in accordance with FELASA and institutional ethical guidelines and with National (Dec. No. 113/2013) and International (2010/63/EU Directive) legislation. Groups of 5 mice were used to test different subcutaneous (SC) vaccine formulations whose composition and immunization schedules are shown in Table 1. Immunizations were performed under light isoflurane anesthesia by SC injection of 100 μL formulations between the shoulders (back neck region). 2.2.4.1. Blood Sample Collection. Blood was collected by submandibular lancet method at day 14 and 42 from mice under light isoflurane anesthesia. After coagulating, blood was centrifuged at 4500g for 10 min for serum collection. Samples were stored at −20 °C until further analysis. 2.2.4.2. Determination of Immunoglobulin Titers. The quantification of serum HBsAg-specific IgG, IgG1, IgG2c, IgG3, and IgE was performed by enzyme-linked immunosorbent assay (ELISA). High-binding 96-well plates (Nunc MaxiSorp, Thermo Fisher Scientific Inc., Waltham, MA, USA) were incubated overnight at 4 °C with 0.1 μg/well HBsAg in 50 mM sodium carbonate/bicarbonate pH 9.6. Plates were washed 5 times with PBS-T 20 and blocked with the same buffer for 1 h at 37 °C. After intensive washing, serial dilutions of serum (starting on 1:128 for IgG and starting on 1:64 for IgG1, IgG2c, IgG3, and IgE) were applied and incubated for 2 h at 37 °C. Following extensive washing, specific antibodies were detected using horseradish peroxidase (HRP) conjugated goat antimouse IgG (Bethyl Laboratories, Montgomery, TX, USA), IgG1 (Rockland Immunochemicals Inc., Limerick, PA, USA), IgG2c (GeneTex, Irvine, CA, USA), IgG3 (SouthernBiotech, Birmingham, AL, USA), or IgE (Nordic Immunological Laboratories, Susteren, NL), respectively, for 30 min at 37 °C according to the antibody dilution recommended by the suppliers. Next, o-phenylenediamine (OPD) solution (5 mg OPD to 10 mL citrate buffer and 10 μL H2O2) was incubated for 10 min at room temperature. Reaction was stopped with 50 μL of 1 M H2SO4 and absorbance was read at 492 nm with a microplate reader (Multiskan EX Microplate, Thermo Fisher Scientific Inc., Waltham, MA, USA).

3. RESULTS 3.1. Characterization of the PCL/Chitosan NPs-Based Formulations. In the present work, particles with a mean diameter of 208.1 ± 28.7 nm and a low polydispersity index (0.18 ± 0.06) were produced by a simple nanoprecipitation process. In previous studies we have observed that PCL NPs had a zeta potential of −1.4 ± 4.6 mV while chitosan NPs presented +26.0 ± 0.4 mV.12 Therefore, the zeta potential of +25.7 ± 2.9 mV (Figure 1A) observed for PCL/chitosan NPs suggests that there was chitosan located at the surface of the nanoparticles. The adsorption of HBsAg antigen on PCL/chitosan NPs surface was performed in water, through simple incubation, resulting in high loading efficacies (>96%). The characterization of the different formulations according to its NPs:HBsAg (w/w) ratios, is illustrated in Figure 1A. Similar to unloaded PCL/chitosan 75

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Figure 2. In vitro studies with PBMCs. (A) Flow cytometry setting regions used to identify PBMCs subpopulation: lymphocytes (R1 region) and monocytes (R2 region) were defined according to cell size and granularity by nonspecific fluorescence from a FSC vs SSC dot plot using free draw regions. (B) Flow cytometry analysis of NPs uptake by monocytes after 4 h incubation with PBMCs. NPs uptake (black) and cell surface interaction (white) were evaluated and the results expressed as mean fluorescence intensity (MFI) ratio (left Y axis) between the geometric mean of the sample and the geometric mean of the background. For cytotoxicity evaluation (right Y axis), the percentage of cells expressing PI were evaluated by the threshold criterion. Data were expressed as mean ± SEM, n = 3. (C) Confocal microscopy images of FITC labeled PCL/chitosan NPs uptake by monocytes. Images represent the merging of z-stack slides. The nucleus was stained white, the cell membrane red and the NPs green. The image presented illustrates some NPs efficiently inside the cells (solid line arrows) while others are attached to the membrane (dotted line arrows). (D) TNF-α released by PBMCs after stimulation with different concentrations of PCL/chitosan NPs during 24 h. LPS was used as positive control. Basal TNF-α production was considered the negative control. Data points correspond to replicates of 3 independent assays. (E) PBMCs were assessed for cell viability (%) under the conditions of TNF-α release experiment through MTT cell viability assay. Data were expressed as mean ± SEM, n = 5.

The trypan blue was used to quench the fluorescence of the particles outside the cells. Despite the analyzed cells having been washed with PBS before collection, results showed that some particles remained adsorbed on the cell surface (white part of the graph bars on Figure 2B). To assess whether PCL/chitosan NPs had pro-inflammatory activity through the TNF-α pathway, as a possible consequence of the described interaction of PCL/chitosan NPs with the cells, PBMCs were stimulated during 24 h with different concentrations of LPS-free-NPs. The LPS was used in this experiment as a positive control for TNF-α production and as was previously anticipated, LPS induced elevated concentrations of this cytokine (Figure 2D). In contrast, PCL/chitosan NPs did not induce TNF-α production at the concentration range from 5 μg/mL to 40 μg/mL, even with cell viability decreasing to values near 50% with the highest NP concentration (Figure 2E). 3.3. PCL/Chitosan NPs Induce Mast Cell Activation. Another type of cells that can be recruited to the local site of injection during vaccination are mast cells. To evaluate the capability of PCL/chitosan NPs (endotoxin free) to induce mast cell activation, several NP concentrations were tested with HMC-1 cells, a human mast cell line. The C48/80, a wellknown mast cell activator,23 was used in this experiment as a positive control. For all conditions tested, no cytotoxic effect was observed and cell viability results were above 100% (Figure 3A). Although some vaccine adjuvants may mediate their activity through a controlled cell death mechanism,23 in vitro studies suggested an adjuvant activity of PCL/chitosan NPs through MC activation not dependent on cytotoxicity. The percentage of β-hex released from HMC-1 cells was PCL/chitosan NP dose-dependent (Figure 3A, left Y axis). In particular, the lowest NP concentrations (40 μg/mL and 80 μg/mL) were

NPs, HBsAg loaded NPs were monodisperse (polydispersity index ≤0.18) with a mean size of around 200 nm and a zeta potential superior to +18 mV. The exception was the 807NPs +CpG formulation, since the adsorption of the coadjuvant CpG-ODN shifted the overall surface charge of the NPs to highly negative values (−35.45 mV). Importantly, these differences in the zeta potential of nanoparticulate formulations (with and without CpG-ODN) might influence the adjuvant ability of each system, by affecting the degree of interaction of the particles with the immune cells. In fact, it has been widely described that cell interaction and uptake via endocytosis is higher for positively charged delivery systems.22 Representative TEM images illustrate free HBsAg VLPs (Figure 1B.1) and HBsAg VLPs adsorbed on PCL/chitosan NPs surface (Figure 1B.2). 3.2. PCL/Chitosan NP Internalization Does Not Induce TNF-α Production by Human Monocytes. To explore the intrinsic immunostimulatory activity of PCL/chitosan NPs (endotoxin free), we studied the interaction of PCL/chitosan NPs with PBMCs. After 4 h-cell incubation with 2 different concentrations of FITC-NPs, PBMCs were analyzed using flow cytometry. Lymphocytes are smaller and less granular than monocytes and therefore were distinguished using nonspecific fluorescence from forward scatter (FSC) and side scatter (SSC) (Figure 2A, R1 and R2 gate, respectively). The cytotoxicity of the NPs was evaluated with PI. Less than 2% of monocyte death was observed (Figure 2B, right Y axis), demonstrating that NP concentrations used during the assay were not cytotoxic. The results of particle uptake suggested that the NPs were efficiently internalized in a dose-dependent way, as the fluorescence resulting from the uptake of 10 μg/mL and 20 μg/mL of NPs in the presence of trypan blue was 4-fold and 7-fold higher than the controls, respectively (Figure 2B, left Y axis). 76

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detected was mostly quenched by trypan blue (Figure 3B, left Y axis). Therefore, it was proven that NPs activated cells most probably by interaction with a cell surface receptor. This observation is supported by the fact that mast cells possess high-affinity binding receptors at their surface (FcεRI, FcγRIIB and FcγRIIIA), which may be responsible for the cell activation24 observed when in contact with NPs. When using PI to assess cell death, the results demonstrated elevated percentage of cell death (Figure 3B, right Y axis). This result contradicts the MTT cell viability results previously presented (Figure 3A, right Y axis) where the cell viability was near 100%. In fact, the test with PI showed that the cell permeability is compromised and so the hypothesis presented was that this permeability increase was most likely associated with the β-hex release and was not long-lasting. To confirm this, after the 45 min incubation with particles, cells were washed and incubated with complete culture medium for 24 h before the flow cytometry analysis. While, right after the 45 min incubation with particles, more than 80% of cells presented a PI permeable membrane, after 24 h recovery at normal cell culture conditions, the permeability was restored to less than 20%. On the other hand, the overall MFI ratio only showed a slight decrease. With these results, it was possible to conclude that PCL/chitosan NPs are effective mast cell activators, enhancing mast cell membrane permeability and consequently β-hex release, apparently without causing irreversible damage to cells. 3.4. Strong and NP-Dose Dependent Humoral Immune Response Induced after SC Vaccination. The in vitro mast cell activation studies suggested that PCL/chitosan NPs could act as a vaccine adjuvant, and therefore, vaccination studies were performed with different formulations (see Table 1). After the first immunization, the group vaccinated with nonadjuvanted HBsAg was not able to induce detectable anti-HBsAg IgG end point-titers (1:128 serum dilution) (Figure 4). However, we are aware that HBsAg VLPs have intrinsic immunogenicity.25,26 Contrasting with this result, both, NPs formulations and the Engerix-B generated good anti-HBsAg IgG titers. In fact, no statistical difference was found between the IgG titers generated by the four NP-based formulations and the Engerix-B formulation, which contains aluminum hydroxide as adjuvant. At day 42, all mice efficiently presented detectable anti-HBsAg IgG titers and as expected, nonadjuvanted HBsAg induced lower levels of antibodies compared to other formulations (Figure 4B). Among the NPs-based formulations, the 1614NPs one was particularly important, since the titers induced by it were also statistically higher than the ones induced by Engerix-B (p < 0.05). 3.5. HBsAg-Loaded PCL/Chitosan NPs Do Not Induce Anti-HBsAg IgE Production. We evaluated our formulations regarding the presence of IgE in mice serum at day 42, 3 weeks after the vaccine boost. None of the formulations containing only HBsAg loaded PCL/chitosan NPs induced detectable IgE titers (Figure 5A) which supports the absence of the generation of an allergic response to the delivery system, even though it presents MC ability in vitro. Similarly, the SC administration of Engerix-B to mice did not induce detectable specific IgE titers at day 42. On the other hand, HBsAg loaded PCL/chitosan NPs adjuvanted with GpG-ODN, induced IgE titers in all immunized mice. Considering the nonadjuvanted HBsAg group, 2 mice developed anti-HBsAg IgE low titers, which may be justified by the intrinsic immunostimulatory properties of HBsAg VLPs, expected to induce a Th2 type antibodies as IgE.

Figure 3. Evaluation of HMC-1 cellular activation. (A) Percentage of cell viability (right Y axis) and β-hex release (left Y axis) induced by C48/80, PCL/chitosan NPs, and PCL NPs at different concentrations in contact with cells during 45 min. Data are presented as mean ± SEM, n ≥ 3. ** p < 0.01 refers to NPs concentrations that differ from C48/80 at 80 μg/mL. All other comparisons to positive controls present no statistical differences. (B) Evaluation of HMC-1 cellular interaction with FITC-labeled PCL/chitosan NPs at two different time-points: after 45 min incubation with the particles and 24 h after the 4 h incubation and medium replacement. Uptake (black) or cell surface interaction (white) were evaluated by the mean fluorescence intensity (MFI) ratio between the geometric mean of the sample and the geometric mean of the background (left Y axis). At the same time intervals, the percentage of cells expressing PI was evaluated by the threshold criterion (right Y axis). Data are expressed as mean ± SEM, n = 3. (C) HMC-1 cell activation illustrated by the toluidine blue dye assay. From top to bottom: untreated cells, cells submitted to incubation with 80 μg/mL C48/80 and cells submitted to 80 μg/mL PCL/chitosan NPs. Their granularity evidenced by toluidine blue staining after cell fixation was observed by microscopy on a Zeiss Axioskop 2 plus with a PlanNeofluar 20× objective (Carl Zeiss AG, Oberkochen, DE) and representative areas are presented.

able to induce as much β-hex release as 40 μg/mL C48/80, the positive control. On the other hand, the highest NP concentration (150 μg/mL) induced a β-hex release concentration that was statistically similar to 80 μg/mL C48/80. To understand the role of the chitosan included into PCL/chitosan NPs in the mast cell activation, a control with PCL NPs (without chitosan) was tested at a concentration of 150 μg/mL. The results showed that PCL NPs were themselves, like chitosan NPs,7 mast cell activators and the inclusion of the chitosan into PCL NPs increased its activation activity (Figure 3A). In fact, contrary to PCL/chitosan NPs, the value for β-hex release induced by PCL NPs was statistically lower than 80 μg/mL C48/80, indicating that the chitosan included in NPs, had an important role on mast cell activation. To better explain HMC-1 activation by PCL/chitosan NPs flow cytometry studies were performed on cells after 45 min incubation with FITC labeled NPs, or 24 h after the incubation period. After 45 min incubation, results showed a major interaction of the NPs with the cell surface, since the fluorescence 77

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Figure 4. Immune response of SC immunized mice. Serum antiHBsAg IgG levels of mice immunized on days 0 and 14 (↑) with formulations containing increasing amounts of PCL/chitosan NPs (403.5, 807, and 1614 μg/dose) and 1.5 μg/dose of HBsAg. One group was immunized with 807 μg NPs + 1.5 μg HBsAg + 10 μg CPG per dose. Sera were evaluated on day 14 and day 42. Control corresponds to nonadjuvanted HBsAg and Engerix-B, both dosed to 1.5 μg of antigen. Antibody levels were determined by ELISA as described in the Materials and Methods Section. The end-point titer presented in the results represents the antilog of the last log 2 dilution for which the OD were at least 2-fold higher than the value of the naive mice sample equally diluted. Numbers in the graphic were presented only when groups presented nonresponder mice and represent the number of mice on which antibody levels were detected; Data are presented as mean ± SEM, n = 5. Titers from 403.5NPs, 807NPs, 1614NPs, and 807NPs+CPG group at day 42 were significantly different from HBsAg control group (p < 0.001). Titers from 1614NPs group at day 42 were significantly different from Engerix-B control group (p < 0.05).

Figure 5. Immune response profile of SC immunized mice with formulations containing increasing amounts of PCL/chitosan NPs (403.5, 807, and 1614 μg/dose) and 1.5 μg/dose of HBsAg, on days 0 and 14. Serum anti-HBsAg IgE (A), IgG1 (B), IgG2c (C), and IgG3 (D) levels are presented. Control corresponds to free HBsAg and Engerix-B, both dosed to 1.5 μg of antigen as for the NPs. Blood was collected on days 14 and 42. IgGs antibody levels at day 42 were determined by ELISA as described in the Materials and Methods Section. The end-point titer presented in the results represents the antilog of the last log 2 dilution for which the OD were at least 2-fold higher than the value of the naive sample equally diluted. Numbers above bars represent the number of mice on which antibody levels were detected, if not detected on all mice. Data are presented as mean ± SEM, n = 5.

3.6. Coadjuvant Activity of CpG-ODN Results in a More Balanced Th1/Th2 Immune Response. The antiHBsAg IgG subtypes IgG1, IgG2c, and IgG3, were evaluated to understand the profile of immune response generated by each formulation. High serum specific IgG1 titers were found in all mice from all groups (Figure 5B). Calculating the ratios IgG3/ IgG1 (from 4 out of 5 animals) or IgG2c/IgG1 (from 1 out of 5 animals) we reach a mean value of 0.76 which indicates that CpG-ODN was efficiently helping the formulation by contributing to a more balanced Th1/Th2 immune response. On the other hand, the generation of Th1 antibodies (IgG2c and IgG3) was low or inexistent in all immunized mice (Figure 5C and D) with the exception of the group vaccinated with the adjuvant CpG, where IgG3 was detected in 4 out of 5 animals. Animals immunized with nonadjuvanted HBsAg did not develop Th1-type antibodies. Only one mouse from Engerix-B presented a low IgG2c titer, which was expected as the aluminum hydroxide adjuvant is typically responsible for Th2 type immune responses.27 Regarding the animals immunized with the PCL/ chitosan NPs-HBsAg formulations, one animal from each group presented serum specific IgG2c. 3.7. Adjuvant Effect of PCL/Chitosan NPs Is Characterized by a Th1/Th17 Cellular Immune Response. Cytokines have a major role in cell mediated immune responses. In this report, cytokine production by immunized mice spleen cells after stimulation with HBsAg were analyzed for better understanding the adjuvant mechanism of PCL/chitosan NPs. Regarding specific IL-4, there was no production of this cytokine upon restimulation with HBsAg (data not shown). On the other hand, specific IFN-γ production showed interesting differences (Figure 6A). Mice immunized with nonadjuvanted

HBsAg did not produce IFN-γ upon restimulation with HBsAg. Conversely, IFN-γ increased production seemed dependent on the amount of NPs used in the formulations (Figure 6A). As a matter of fact, the IFN-γ concentration observed in mice immunized with the 1614NPs formulation was significantly different ̈ mice and in (p < 0.05) from the production observed in naive mice immunized with nonadjuvanted HBsAg and was similar to that obtained in mice vaccinated with 807NPs+CpG formulation, with CpG-ODN being a well-known Th1 cytokine inducer. While nonadjuvanted HBsAg formulation was not able to induce antigen specific IL-17, HBsAg-loaded PCL/chitosan NPs formulations induced IL-17 production and the concentrations of the cytokine appeared to be NP dose-dependent (Figure 6B). However, the statistical treatment of the results showed that 807NPs and 1614NPs formulations induced IL-17 ̈ mice (p ≤ concentrations statistically different from the naive 0.05) and the 807NPs formulation induced IL-17 concentrations statistically different from the nonadjuvanted HBsAg immunized mice. On the other hand, the association of the CpG to the NPs formulation prevented stimulation of IL-17, previously seen in the NPs’ containing formulations. That interesting result supports the idea that the modulation of the immune response would be possible by the association of two or more adjuvants.28−30 78

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̈ C57/BL6 Figure 6. In vitro cytokine production after restimulation with HBsAg. Spleen cells (0.25 × 106 cells/well) from both immunized and naive mice were restimulated with 1 μg HBsAg as described in the Materials and Methods Section. Supernatants were collected 72 h later and tested for IFN-γ and IL-17 by ELISA. Results were expressed as mean cytokine concentrations ± SEM, resulting from HBsAg stimulation (basal concentration ̈ mice. *p < 0.05 and ***p < 0.001 indicates values that differ significantly from naive subtracted) for each group of 5 immunized mice and 10 naive group. +p < 0.05 and ++p < 0.01 indicates values that differ significantly from HBsAg control group. ##p ≤ 0.01 indicates values that differ significantly for the group containing CpG-ODN.

4. DISCUSSION An ideal technique for nanoparticle (NP) production should be simple to execute and generate reproducible batches of particles such as the nanoprecipitation method presented herein. Furthermore, PCL/chitosan NPs show great antigen adsorption, explained by the electrostatic forces between chitosan’s positive amino groups and negative charges from HBsAg, together with hydrophobic interactions between this antigen and PCL, a phenomenon also observed for ovalbumin.12 The first objective of the present work was to study the intrinsic immunostimulatory activity of PCL/chitosan NPs, particularly to produce mechanistic data aiming at unraveling the key factors for the initiation of the innate and adaptive immune response, since to date, and to the best of author knowledge, nothing was reported by other research groups. Following the cellular interactions between pathogens or particles with phagocytic cells, these entities can be recognized as invaders and induce the generation of signals from innate immune system like the production of inflammatory mediators by diverse cell types including monocytes. In fact, some particulate adjuvants have shown to create a local pro-inflammatory environment to recruit immune cells by the up-regulation of cytokines and chemokines,31 like TNF-α, a pro-inflammatory cytokine generally viewed as an early mediator of inflammatory responses.32 Therefore, PBMCs collected from human blood were chosen to test the ability of the PCL/chitosan NPs to stimulate TNF-α, since they are, among others, normally recruited to the local of vaccine injection. Results showed that PBMCs efficiently interacted and internalized PCL/chitosan NPs, but no production of TNF-α was observed. Notably, no previous reports tested this cytokine release from PBMCs after stimulation with PCL/chitosan NPs. Moreover, some reports regarding the stimulatory activity of the isolated polymers are not aware of endotoxin contamination (LPS contamination) and results presented may be misleading. For instance, a report about the in vitro cellular responses induced by polyesters, showed some inflammatory response of J774 macrophages, after 48 h incubation with pure PCL polymer,33 but no reference to the evaluation of endotoxin contamination of the material was made. On the other hand, a recent report acknowledging LPS free conditions, suggested chitosan oligosaccharides

were found to stimulate TNF-α production by RAW264.7 macrophages.34 Furthermore, an extended discussion of the literature can be performed taking into consideration the nanoparticulated nature and the size of PCL/chitosan delivery system. Xiong and co-workers35 found that endotoxin free PLGA NPs with size greater than 100 nm, did not induce TNF-α production on RAW264.7 macrophages after 24 h incubation, in contrast to 60 nm NPs, highlighting the fact that smaller sizes may trigger this inflammatory pathway. PCL/chitosan NPs had a mean size around 200 nm, which is in line with this observation. Another type of cells that can be recruited for the local of injection during vaccination are mast cells. Their role in inflammation or in allergic immune response is still not completely clarified but in recent years, mast cell activators have been described as good vaccine adjuvants.23,36 Mast cell degranulation occurs after cell stimulation by specific molecules and is characterized by the release of small vesicles containing inflammatory mediators. According to literature, these vesicles have the ability to drain to local lymph nodes via lymphatics or activate APCs nearby which may result in the initiation of adaptive immune responses.6,37 Although PCL/chitosan NPs had not been evaluated before regarding its ability to induce mast cell activation, previous studies have reported the effects of chitosan oligosaccharides,38 chitosan polysaccharide,39 and chitosan NPs.7 Using a rat basophilic leukemia cell line (RBL2H3), a cell model physiologically similar to human mast cells chitosan oligosaccharides were shown to have an inhibitory effect on mast cell activation through downregulation of the high-affinity receptor for IgE (FcεRI) expression.38 In another study, contrasting with this result, chitosan polysaccharide induced the activation on the same cell line.39 Concerning chitosan NPs, they were found to activate human mast cells (HMC-1 cell line) apparently due to the extensive adsorption on cell surface immediately after being added to the cells.7 Considering this literature data, the mast cell activation induced by PCL/ chitosan NPs may be higher than the activation induced by PCL NPs due to chitosan presence at the particle’s surface, which also influences NPs adsorption on the mast cell surface. Further, Aridor and co-workers40 suggested that the capacity to induce mast cell degranulation demonstrated by peptides and 79

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Molecular Pharmaceutics other polybasic molecules, involves the insertion of their hydrophobic moiety into the membrane, enabling its positively charged domain to interact and directly activate G proteins in a receptorindependent manner. Since PCL/chitosan NPs present the hydrophobic properties of PCL associated with the positive chitosan charges, we may assume a similar activation mechanism. Although there is no evidence of the advantages of mast cell activation on the immune response to HBV, positive hexosaminidase results suggested that PCL/chitosan NPs could act as a vaccine adjuvant. Therefore, subcutaneous vaccination studies using the recombinant HBV surface antigen, were performed primarily to test the NPs dose effect on the adjuvant ability of the delivery system. The results showed a dose dependent adjuvant ability, with the formulation containing the higher amount of NP (1614 μg) able to induce antibody titers statistically higher than the ones induced by Engerix-B (p < 0.05). Importantly, potent adjuvant action is often correlated with increased toxicity. Regulatory requirements to approve new adjuvants are very demanding since vaccine formulations are to be administered to healthy people, especially children. Therefore, one of the immunologic parameters frequently required is the titer of the antigen-specific IgE, as it may be related to allergic or anaphylactic reactions upon antigen challenge.41 In this case, results of the 403.5NPs, 807NPs, and 1614NPs formulations showed no induction of antigen-specific IgE antibody, despite the high IgG titers induced. Similarly, Engerix-B formulation did not induce this antigen-specific IgE. Nonetheless, in the literature is widely reported that when aluminum salts are used as vaccine adjuvants (as in the case of Engerix-B), the induction of IgE antibodies is normally observed3,42 and together with IgG, these antibodies are key factors for the persistent HBV memory after immunization.43 The disagreement to our results may be related to the mouse strain used or with the low amount of aluminum hydroxide per dose (37.5 μg/dose). In fact, Yanase et al.44 showed elevated IgE titers at day 35, on BALB/c mice subcutaneously vaccinated with 10 μg ovalbumin and 200 μg aluminum hydroxide (vaccines at day 0 and day 28). However, at day 42, they reported that the IgE titers had diminished comparing to day 35, meaning the stronger IgE response may be transient. Our adjuvant dose was 5-fold less, and the evaluation of the IgE titer was performed 28 days after the last immunization. Considering the results of the evaluation of the IgG antibody subsets, we may hypothesize that the PCL/chitosan NPs adjuvant effect was not able to shift the antibody immune response to a more Th1-type. Nevertheless, in the 1614NPs group, the animal that presented IgG2c titers possessed a more balanced Th1/Th2 immune response with a ratio IgG2c/IgG1 superior to 0.75. Future studies should clarify why the balanced Th1/ Th2 immune response is not observed in all mice. Regarding the cellular immune response, results showed an increase in IFN-γ production of NP vaccinated mice suggesting that PCL/chitosan NPs induced a Th1-biased response, which appear to be adjuvant-dose dependent. IFN-γ is a cytokine secreted by Th1 CD4+ T cells involved in the virus neutralization and opsonization.45 In the literature, other polymeric NPs have already shown similar performance-poly(D,L-lactide) (PLA) NPs, when loaded with HBsAg lead to high levels of IFN-γ production and antibody isotypes, indicative of a Th1-type immune response.46 Likewise, hydrophobic NPs based on poly(D,L-lactide-co-glycolide) (PLGA) modified with chitosan or its derivatives also increased IFN-γ production on vaccinated mice.47,48

Another subset of CD4+ T cells (Th17 subset) produces IL-17 and IL-22, which have been implicated in protective cellular and memory responses, increasing immunity against a range of pathogens upon vaccination.49,50 PCL/chitosan NPs were efficiently able to induce IL-17 production which is coherent with a previous publication from our group also showed that nasal immunization with chitosan nanoparticles containing Bacillus anthracis protective antigen (PA), induced IL-17 and IL-22 production in mice spleen cells, after restimulation with PA.7 Although, authors have no knowledge of other published work revealing the induction of IL-17 cytokine in mice vaccinated with HBsAg associated with polymeric NPs, a recent report on a nanoscale water in oil emulsion adjuvant for HBsAg referred that this formulation administered through nasal route is able to induce the IFN-γ Th1 cytokine and the IL-17 Th17 cytokine, after spleen cell restimulation with the antigen.51 The role of Th17 responses in viral infections is still not as clear as it is for bacterial and fungal infections, but the authors suggested that the Th17 cell mediated immunity may be responsible for suppressing IgE responses,51 which we verified since PCL/ chitosan NPs did not induce IgE titers in immunized mice. Over the last years, several groups, including ours, have reported the effect of CpG-ODN in the redirection of Th-bias of immune response against several antigens, including HBsAg.18,52−54 In the work here presented, the evaluation of the response induced by the 807NPs+CpG formulation revealed increased Th1 specific antibody titers in all animals. Similar to this result, other observations from our group showed an increase in the Th1/Th2 antibodies ratio from 0.1 to 1.0, when 10 μg CpGODN were added to alginate-coated chitosan NPs loaded with HBsAg.18 Another interesting result regarding the introduction of CpG-ODN in the vaccine formulation, was the generation of antigen-specific IgE antibodies in all vaccinated animals. IgE is a Th2 antibody downregulated by Th1 type cytokines meaning the adjuvant effect of CpG-ODN was expected to avoid this cytokine production.55 In fact, this study was performed with CpG-ODN 1826, which is a mouse B-class CpG, and when compared for instance to A-class GpG is much more potent Th1 adjuvant.56 Nonetheless, a prophylactic vaccination study conducted by McGowen and co-workers41 also observed that when using Bacillus anthracis protective antigen adjuvanted with 10 μg of CpG-ODN by intradermal route, low IgE titers in 2 out of 4 mice were generated.41 In our study, we used a different immunization route and a 2-fold higher CpG-ODN dose which might explain detectable IgE titers in all mice immunized with 807NPs+CpG formulation. Moreover, we observed that the addition of CpG-ODN as coadjuvant inhibited the Th17 cellular-based immune response stimulated by PCL/chitosan NPs, which we previously suggested to be related to the inhibition of IgE production. Therefore, CpG-ODN might be preventing the Th17 pathway and therefore empowering the Th2 pathway and IgE production.57 In accordance to our work, Verwaerde and co-workers49 reported that the addition of CpG-ODN to a nanoparticulate carrier of heparin binding hemagglutinin antigen (HBHA) would increase IFN-γ but decrease IL-17 production by spleen cells of vaccinated mice, after HBHA stimulation. The authors suggested this inhibition could be related with the ability of CpG-ODN to polarize a Th1 immune response.

5. CONCLUSIONS PCL/chitosan NPs were shown to be a good adjuvant for the HBsAg antigen when administered by the subcutaneous route. 80

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(7) Bento, D.; Staats, H. F.; Goncalves, T.; Borges, O. Development of a novel adjuvanted nasal vaccine: C48/80 associated with chitosan nanoparticles as a path to enhance mucosal immunity. Eur. J. Pharm. Biopharm. 2015, 93, 149−64. (8) Staats, H. F.; Fielhauer, J. R.; Thompson, A. L.; Tripp, A. A.; Sobel, A. E.; Maddaloni, M.; Abraham, S. N.; Pascual, D. W. Mucosal targeting of a BoNT/A subunit vaccine adjuvanted with a mast cell activator enhances induction of BoNT/A neutralizing antibodies in rabbits. PLoS One 2011, 6 (1), e16532. (9) Food and Drug Administration - Premarket Notification K123633. https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/ cfpmn/pmn.cfm?ID=K123633, (06/10/2017), 2013. (10) Food and Drug Administration - Premarket Notification K050845. https://www.accessdata.fda.gov/scrIpts/cdrh/cfdocs/ cfpmn/pmn.cfm?id=K050845, 2005, (accessed June 10, 2017). (11) Dash, T. K.; Konkimalla, V. B. Poly-epsilon-caprolactone based formulations for drug delivery and tissue engineering: A review. J. Controlled Release 2012, 158 (1), 15−33. (12) Jesus, S.; Fragal, E. H.; Rubira, A. F.; Muniz, E. C.; Valente, A. J. M.; Borges, O. The Inclusion of Chitosan in Poly-ε-caprolactone Nanoparticles: Impact on the Delivery System Characteristics and on the Adsorbed Ovalbumin Secondary Structure. AAPS PharmSciTech 2017, 1−13. (13) Lebre, F.; Hearnden, C. H.; Lavelle, E. C. Modulation of Immune Responses by Particulate Materials. Adv. Mater. 2016, 28 (27), 5525−41. (14) Ulery, B. D.; Petersen, L. K.; Phanse, Y.; Kong, C. S.; Broderick, S. R.; Kumar, D.; Ramer-Tait, A. E.; Carrillo-Conde, B.; Rajan, K.; Wannemuehler, M. J.; Bellaire, B. H.; Metzger, D. W.; Narasimhan, B. Rational design of pathogen-mimicking amphiphilic materials as nanoadjuvants. Sci. Rep. 2011, 1, 198. (15) Neumann, S.; Burkert, K.; Kemp, R.; Rades, T.; Rod Dunbar, P.; Hook, S. Activation of the NLRP3 inflammasome is not a feature of all particulate vaccine adjuvants. Immunol. Cell Biol. 2014, 92 (6), 535− 42. (16) Lin, Y. C.; Lou, P. J.; Young, T. H. Chitosan as an adjuvant-like substrate for dendritic cell culture to enhance antitumor effects. Biomaterials 2014, 35 (31), 8867−75. (17) Scherliess, R.; Buske, S.; Young, K.; Weber, B.; Rades, T.; Hook, S. In vivo evaluation of chitosan as an adjuvant in subcutaneous vaccine formulations. Vaccine 2013, 31 (42), 4812−9. (18) Borges, O.; Silva, M.; de Sousa, A.; Borchard, G.; Junginger, H. E.; Cordeiro-da-Silva, A. Alginate coated chitosan nanoparticles are an effective subcutaneous adjuvant for hepatitis B surface antigen. Int. Immunopharmacol. 2008, 8 (13−14), 1773−80. (19) Lebre, F.; Bento, D.; Ribeiro, J.; Colaco, M.; Borchard, G.; de Lima, M. C. P.; Borges, O. Association of chitosan and aluminium as a new adjuvant strategy for improved vaccination. Int. J. Pharm. 2017, 527 (1−2), 103−114. (20) Jesus, S.; Borchard, G.; Borges, O. Freeze Dried Chitosan/ Polye-Caprolactone and Poly-e-Caprolactone Nanoparticles: Evaluation of their Potential as DNA and Antigen Delivery Systems. J. Genet. Syndr. Gene Ther. 2013, 4, 164. (21) Kevin, W. Endotoxins: Pyrogens: LAL Testing, and Depyrogenation, Second ed.; Taylor & Francis, 2001; p 392. (22) Suchaoin, W.; Pereira de Sousa, I.; Netsomboon, K.; Lam, H. T.; Laffleur, F.; Bernkop-Schnurch, A. Development and in vitro evaluation of zeta potential changing self-emulsifying drug delivery systems for enhanced mucus permeation. Int. J. Pharm. 2016, 510 (1), 255−62. (23) Staats, H. F.; Kirwan, S. M.; Choi, H. W.; Shelburne, C. P.; Abraham, S. N.; Leung, G. Y.; Chen, D. Y. A Mast Cell Degranulation Screening Assay for the Identification of Novel Mast Cell Activating Agents. MedChemComm 2013, 4 (1), 88. (24) Fang, Y.; Zhang, T.; Lidell, L.; Xu, X.; Lycke, N.; Xiang, Z. The immune complex CTA1-DD/IgG adjuvant specifically targets connective tissue mast cells through FcgammaRIIIA and augments anti-HPV immunity after nasal immunization. Mucosal Immunol. 2013, 6 (6), 1168−1178.

The IgG titers obtained were comparable with the ones obtained with one of the commercial vaccines already in the market, Engerix-B, without inducing IgE antibodies. Moreover, the induction of IFN-γ and IL-17 production suggest a Th1/Th17 cellular based immune response, different from what was observed with the nonadjuvanted antigen or with the commercial vaccines, that are unable to induce a cellular immune response. The ability of this new HBsAg adjuvant to induce the secretion of the IFN-γ, opens new perspectives for HBV vaccination since the combination of strong humoral immune response with cell mediated immune response, represents the best way to achieve protection through vaccine immunization and may constitute a valuable alternative to alum-based vaccines. Particularly, this vaccine can be explored as an immunotherapy for chronic hepatitis B carriers or as a vaccine for infants born to HBV-infected mothers. Therefore, future studies would test these hypotheses.

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AUTHOR INFORMATION

Corresponding Author

*Tel: + 351 239 488 428; E-mail: [email protected]. ORCID

Olga Borges: 0000-0002-2215-5121 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is funded by FEDER funds through the Operational Programme Competitiveness Factors-COMPETE 2020 and national funds by FCT-Foundation for Science and Technology under the project PTDC/SAU-FAR/115044/2009, fellowship DFRH-SFRH/BD/81350/2011 and strategic project POCI-010145-FEDER-007440 (UID/NEU/04539/2013). TEM microscopy analyses were performed at IBILI under FCT founding contract REDE/1510/RME/2005. Authors want to acknowledge Dr. Butterfield for his generous gift of the human mast cell line HMC1.1; Dr. Paulo Santos for the provided human blood from IPST, IP; and Dr. Monica Zuzarte, Dr. Isabel Nunes, Dr. Luísa Cortes, and Dr. Ana Donato for technical expertise in TEM microscopy, flow cytometry, confocal microscopy, and spleen cell counting, respectively.

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REFERENCES

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Molecular Pharmaceutics

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DOI: 10.1021/acs.molpharmaceut.7b00730 Mol. Pharmaceutics 2018, 15, 72−82