Enhancing Mucosal Immune Response of Newcastle Disease Virus

Nov 27, 2017 - Because mucosal sites are the entry ports of pathogens, immunization via mucosal routes can extremely enhance the immunity. To elevate ...
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Article Cite This: Mol. Pharmaceutics 2018, 15, 226−237

Enhancing Mucosal Immune Response of Newcastle Disease Virus DNA Vaccine Using N‑2-Hydroxypropyl Trimethylammonium Chloride Chitosan and N,O‑Carboxymethyl Chitosan Nanoparticles as Delivery Carrier Kai Zhao,*,†,# Jinyu Han,‡,# Yang Zhang,† Lin Wei,† Shuang Yu,† Xiaohua Wang,† Zheng Jin,*,‡ and Yunfeng Wang*,§ †

Key Laboratory of Microbiology, School of Life Science, Heilongjiang University, Harbin 150080, China Key Laboratory of Chemical Engineering Process and Technology for High-efficiency Conversion, College of Chemistry and Material Sciences, Heilongjiang University, Harbin 150080, China § Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, CAAS, Harbin 150001, China

Mol. Pharmaceutics 2018.15:226-237. Downloaded from pubs.acs.org by REGIS UNIV on 10/18/18. For personal use only.

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S Supporting Information *

ABSTRACT: Because mucosal sites are the entry ports of pathogens, immunization via mucosal routes can extremely enhance the immunity. To elevate the potential of N-2-hydroxypropyl trimethylammonium chloride chitosan (N-2-HACC) and N,Ocarboxymethyl chitosan (CMC) nanoparticles as a mucosal immune delivery carrier for DNA vaccines, we prepared the NDV F gene plasmid DNA with C3d6 molecular adjuvant (pVAX I-F(o)-C3d6) encapsulated in the N-2-HACC-CMC nanoparticles (N-2-HACC-CMC/pFDNA-C3d6 NPs). The N-2-HACC-CMC/pFDNA-C3d6 NPs had regular spherical morphology and low toxicity with a mean diameter of 309.7 ± 6.52 nm, zeta potential of 49.9 ± 4.93 mV, encapsulation efficiency of 92.27 ± 1.48%, and loading capacity of 50.75 ± 1.35%. The N-2-HACC-CMC had high stability and safety. The pVAX I-F(o)-C3d6 could be sustainably released from the N-2-HACC-CMC/pFDNA-C3d6 NPs after an initial burst release. Immunization intranasally of chickens with N-2-HACC-CMC/pFDNA-C3d6 NPs not only produced higher anti-NDV IgG and sIgA antibody than chickens in other groups did, but also significantly stimulated lymphocyte proliferation and triggered higher the IL-2, IL-4, and IFN-γ levels. These findings indicated that the N-2-HACC-CMC could be used as an efficient delivery carrier for the mucosal immunity of Newcastle disease virus DNA vaccine. The work laid a basis for the quaternized chitosan nanoparticles as efficient mucosal immunity delivery carrier for DNA vaccines and had immense application promise and potential for vaccines and drugs. KEYWORDS: Newcastle disease, F gene, chitosan derivative nanoparticles, intranasal delivery, mucosal immunity

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INTRODUCTION

that infect mucosal tissues or have a mucosal port of entry. Parenteral vaccination may protect in some instances, but usually a mucosal vaccination route is necessary. Mucosal vaccines can prevent the entrance of the pathogens into the

Most pathogens access the body through the mucosal membranes. Protective mucosal immune responses can be effectively triggered by a mucosal administration route such as oral, nasal, rectal, or vaginal routes. However, despite early success with the live attenuated oral Sabin polio vaccine over 50 years ago, only a few new mucosal vaccines in use today have been subsequently launched. Thus, there is an important requirement to develop vaccines against many of the pathogens © 2017 American Chemical Society

Received: Revised: Accepted: Published: 226

September 21, 2017 November 6, 2017 November 27, 2017 November 27, 2017 DOI: 10.1021/acs.molpharmaceut.7b00826 Mol. Pharmaceutics 2018, 15, 226−237

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Molecular Pharmaceutics

properties. Chitosan as a drug delivery system has been extensively evaluated. Presently, chitosan and its derivatives have drawn much attention as both adjuvant and delivery carrier for vaccines.21 Chitosan can bind with negatively charged protein or plasmid DNA by electrostatic interaction and protect them from degradation; hence, it is widely used as a delivery carrier in the field of vaccines.22,23 However, chitosan can be dissolved in acidic solution, although not easily dissolved in the condition of physiological potential of hydrogen (pH).24 This poor solubility becomes the largest limitation of chitosan for medical application since it must be dissolved in aqueous solutions with a positive charge for transporting a target vaccine into cells.25 The abundant hydroxyl and amino groups on the chitosan skeleton make it easy for chemical modification. Chitosan derivatives with certain functional groups can be obtained by chemical modification of chitosan. Compared with chitosan, the performance of derivatives is often better than chitosan.26 Thus, nanoparticles based on water-soluble chitosan derivatives have become new drug carriers because of their stability and biocompatibility in biological solutions (pH 7.4). Nowadays, there are many studies on chitosan derivatives, such as acylation, alkylation, sulfation, hydroxylation, quaternization, and carboxymethylation.27−30 Recently, our group has synthesized O-2′-hydroxypropyl trimethylammonium chloride chitosan (O-2′-HACC),31 N-2-hydroxypropyl trimethylammonium chloride chitosan (N-2-HACC),32 N-2-hydroxypropyl dimethyl ethylammonium chloride chitosan (N-2-HFCC),33 and N,Ocarboxymethyl chitosan (CMC).32 O-2′-HACC, N-2-HACC, N-2-HFCC, and CMC have higher water solubility than chitosan,31−33 and N-2-HACC has higher water solubility and a more suitable nanoparticles size than O-2′-HACC and N-2HFCC.34 Additionally, the preparation process of N-2-HACC was easier, more convenient, and lower cost than that of O-2′HACC and N-2-HFCC, and there are no organic solvents in the preparation process, so antigen immunogenicity cannot be destroyed. Moreover, the synthesis of N-2-HACC was optimized, and the relationship between nanoparticle size and degree of N-2-HACC substitution has also been studied by our group.35 N,O-Carboxymethyl chitosan (CMC) is a carboxymethylated product of chitosan, with better solubility in water compared to N-2-HACC. However, CMC has lower encapsulation efficiency and drug loading than O-2′-HACC, N-2-HACC, and N-2-HFCC. Therefore, to further improve water solubility, encapsulation efficiency, and loading capacity, we developed two chitosan derivatives, positively charged N-2HACC and negatively charged CMC. In the present study, to prove whether the biodegradable polymers N-2-HACC and CMC nanoparticles can also be used as a delivery carrier to realize sustained release and induce desired mucosal immunity for DNA vaccines, we prepared the NDV F gene plasmid DNA with C3d6 molecular adjuvant (pVAX I-F(o)-C3d6) encapsulated in the N-2-HACC-CMC nanoparticles (N-2-HACCCMC/pFDNA-C3d6 NPs) by the polyelectrolyte complex method, and their characteristics for the intranasal delivery of NDV DNA vaccine were studied. In vitro and in vivo experiments were performed to systematically evaluate the adjuvant effect of N-2-HACC-CMC nanoparticles. This study has provided a foundation for the further development and immense application potential of mucosal vaccines and drugs encapsulated in biodegradable polymeric nanoparticles.

body, the inactivation of pathogens, and the dissemination of pathogens.1 Additionally, mucosal vaccines also have lots of advantages over injectable vaccines by being simpler to administer, having less risk of transmitting infections and potentially being easier to manufacture.2 Developing mucosal vaccines based on the biomaterial nanoparticles as adjuvant and delivery carrier can circumvent some shortcomings of conventional vaccines. The nanovaccines can enhance humoral, cellmediated, and mucosal immune response through the sustained release and protection against degradation of loaded antigen. Currently, DNA vaccines have displayed a huge potential for the development of new vaccines, and increasing attention has been focused on DNA vaccines because of their easy production, superior stability in ambient temperature, nonrequirement for cold chain, and their ability to generate antigen-specific immune responses.3,4 However, many studies have indicated that DNA vaccines usually administered via intramuscular injection can fail to reach the antigen-presenting cells (APCs) and therefore fail to induce immune responses because of the difficulty for them to pass through cell membranes.5,6 Also, weak immune responses and low levels of DNA vaccine expression will be induced, especially in large animals.7 At present, in order to improve the efficacy of DNA vaccines, several effective strategies are desirable, including optimization of plasmid DNA, delivery methods, selection of suitable target for effective antigen presentation, and the use of a powerful adjuvant to enhance immunogenicity.8−10 When it comes to gene delivery, the two major gene delivery methods are viral and nonviral (nanoparticles). Each delivery system has its own set of advantages and disadvantages. Although viral delivery of plasmid DNA is extremely efficient, nanoparticle (NP) technologies have also been proved to significantly improve effectiveness in the delivery of plasmid DNA compared to viral-based delivery systems, and the NPs approach is desirable.11 Various NPs have been developed, such as iron oxide NPs, gold NPs, cerium oxide NPs, carbon-based materials, and polymeric NPs.12−14 In all potential NP delivery systems, polymeric NPs when used as nanosized particulate carriers can either encapsulate or entrap the DNA vaccines, antigens, proteins, and drugs and deliver them to the desired site of action.2 NPs have lower cytotoxicity and can protect the antigens from being damaged under unfavorable conditions after systemic or mucosal administration, and the uptake of nanoparticles by APCs can increase and induce potent immune responses.15,16 Additionally, nanomaterials are widely used as vaccine delivery systems because they cause the vaccine antigen to be released slowly.17 Hence, the disadvantages of DNA vaccines can be avoided when biomaterial-based nanoparticles are used as delivery carrier of vaccine antigens.18 NPs have great advantages and are attracting much attention, especially in the field of vaccines and drugs delivery.19,20 In all of the natural polymers, dextran and chitosan are the two most popular polysaccharides for the formulation of NPs for the delivery of plasmid DNA. Dextran is neutral at physiological pH and therefore can not effectively bind DNA; thus, chitosan NPs are significantly more popular due to their cationic nature. Chitosan, also known as a linear amino polysaccharide, is rich in chitin material from the exoskeleton of crustaceans and insects, the endoskeletons of cephalopods, or the cuticles of arthropods and fungal cell walls.12 Chitosan, which consists of both 2-amino-2-deoxy-β-D-glucan units and 2acetamido-2-deoxy-β-D-glucan, has great potential in biomedicine because of its interesting biological and physicochemical 227

DOI: 10.1021/acs.molpharmaceut.7b00826 Mol. Pharmaceutics 2018, 15, 226−237

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MATERIALS AND METHODS Synthesis of N-2-Hydroxypropyl Trimethylammonium Chloride Chitosan and N,O-Carboxymethyl Chitosan. The two water-soluble chitosan derivatives with positive charge, N-2-hydroxypropyltrimethylammonium chloride chitosan (N-2-HACC) and N,O-carboxymethyl chitosan (CMC), are synthesized by our laboratory as the nanomaterials for the delivery of vaccine antigen. Syntheses of N-2-HACC and CMC were carried out as described previously.32 Preparation of the N-2-HACC-CMC Nanoparticles Encapsulating Plasmid pVAX I-F(o)-C3d6. The eukaryotic expression plasmid pVAX-optiF that carries and drives the expression of F gene of NDV was provided by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China). The eukaryotic expression plasmid pVAX-optiF with C 3d6 molecular adjuvant (pVAX I-F(o)C3d6) was constructed by our Laboratory in School of Life Science of Heilongjiang University, China. The pVAX I-F(o)C3d6 was encapsulated in N-2-HACC-CMC nanoparticles (N2-HACC-CMC/pFDNA-C3d6 NPs) by the polyelectrolyte complex method as follows. Two milliliters of the 100 mg/mL pVAX I-F(o)-C3d6 solution was slowly added to 5 mL of 1.0 mg/mL N-2-HACC solution under stirring at 300 r/min for 3 min, followed by stirring again but at 1200 r/min for 1 min. Next, 2 mL of the 0.85 mg/mL CMC solution was slowly added to the mixture, and stirring continued at 1200 r/min for 40 min. Finally, after stirring at 4 °C for 20 min at 12000 r/min, the supernatant was removed, and the precipitate was resuspended in ddH2O at 4 °C. The process was repeated three times, and the final precipitate dissolved in 2 mL of ddH2O to obtain N-2-HACC-CMC/pFDNA-C3d6 NPs. Characterization of the N-2-HACC-CMC/pFDNA-C3d6 NPs. The N-2-HACC-CMC/pFDNA-C3d6 NPs were observed under transmission electron microscopy (JEM-200EX, Hitachi Ltd., Japan) to assess their surface characteristics and morphology. The zeta potential and particle size of N-2HACC-CMC/pFDNA-C3d6 NPs were measured by a Zeta Sizer ZS90 (Malvern Instruments Ltd., Southborough, MA, USA). Loading capacity (LC) and encapsulation efficiency (EE) were measured as described previously.31 DNase I Protection of the N-2-HACC-CMC/pFDNAC3d6 NPs. To test stability of the N-2-HACC-CMC/pFDNAC3d6 NPs, 100 μL of N-2-HACC-CMC/pFDNA-C3d6 NPs suspension were incubated with 25 μL of DNase (TaKaRa, Dalian, China) at 37 °C for 30, 60, 120, and 180 min, respectively. The final concentration of the DNase is 1.0 U/mL. Next, 100 μL of termination solutions (400 mmol/L NaCl, 100 mmol/L EDTA, pH 8.0) was added into the reaction system at 65 °C for 10 min to terminate the reaction. The plasmid pVAX I-F(o)-C3d6 as the negative control was incubated at 37 °C for 30 min. The integrity of plasmid pVAX I-F(o)-C3d6 was analyzed by using 0.8% agarose gel electrophoresis at 100 V for 30 min. In Vitro Release of the N-2-HACC-CMC/pFDNA-C3d6 NPs. To determine the release of pVAX I-F(o)-C3d6 from the nanoparticles, the N-2-HACC-CMC/pFDNA-C3d6 NPs suspension was centrifuged at 4 °C at 16000 r/min for 30 min, and the precipitate was resuspended in 2 mL of PBS buffer (pH 7.4) and stirred at 100 r/min at 4 °C. Samples (100 μL) were withdrawn at different time intervals, and the same volume of fresh PBS was added. The sample was centrifuged at 4 °C at 12,000 r/min for 20 min. The concentration of the released

pVAX I-F(o)-C3d6 in the supernatant was determined by UV spectrophotometry. All the experiments were repeated five times. Safety of the N-2-HACC-CMC/pFDNA-C3d6 NPs. In Vitro Cytotoxicity Assay. To test safety of the N-2-HACCCMC NPs as adjuvant and plasmid DNA delivery carrier for mucosal immunity, in vitro cytotoxicity was evaluated by the Cell Counting Kit-8 (CCK-8) reagent (Dojindo Ltd., Kumamoto, Japan), and the survival rate of chicken embryonic fibroblast (CEF) cells was determined by measuring OD450 according to ref 31. In Vivo Safety Assay. To test in vivo biological safety, 30 4week-old SPF chickens obtained from Animal Center of Harbin Veterinary Research Institute Laboratory were randomly assigned into three groups and inoculated with ten times the dose. Chickens in Group 1 were administered i.m. with N-2HACC-CMC/pFDNA-C3d6 NPs suspension; chickens in Group 2 were administered i.n. with N-2-HACC-CMC/ pFDNA-C3d6 NPs suspension; and chickens in Group 3 were administered i.m. with 0.2 mL of PBS (pH 7.4). The three groups were monitored continuously for 14 days, and any abnormal changes including feeding, drinking, mental state, and body weight were recorded. In Vitro Expression of the N-2-HACC-CMC/pFDNAC3d6 NPs. An in vitro transfection assay was performed according to the instructions from the Lipofectamine 2000 reagent kit (Invitrogen, USA), and the expression of pVAX IF(o)-C3d6 in the transfected cells was detected by using an indirect immunofluorescent test. This transfection experiment was divided into four groups. Group 1 was N-2-HACC-CMC/ pFDNA-C3d6 NPs containing 4 μg of plasmid pVAX I-F(o)C3d6, which was transfected into 293T cells; Group 2 was 4 μg of plasmid pVAX I-F(o)-C3d6; Group 3 was blank N-2HACC-CMC nanoparticles included as a negative control; and Group 4 was cell control. The NDV positive serum and fluorescein isothiocyanate-labeled goat-antichicken IgG from Sigma was diluted at 1:100 and 1:5, respectively. Epifluorescence images were obtained using an Axio observer Z1 microscope (Zeiss). Immunization of the N-2-HACC-CMC/pFDNA-C3d6 NPs. One hundred 1-day-old healthy SPF chickens obtained from Animal Center of Harbin Veterinary Research Institute Laboratory were randomly assigned into five groups with 20 chickens in each group. Chickens in Group 1 were immunized i.m. with PBS buffer as a negative control; chickens in Group 2 were immunized i.m. with blank N-2-HACC-CMC-NPs; chickens in Group 3 were immunized i.m. with 0.1 mL of pVAX I-F(o)-C3d6 (200 μg); chickens in Groups 4 and 5 were immunized with 0.1 mL of N-2-HACC-CMC/pFDNA-C3d6 NPs containing 200 μg of pVAX I-F(o)-C3d6 i.m. and i.n., respectively. At 2 weeks post-first-immunization, booster immunization was performed with the same dosages and routes as the first immunization. The experimental protocol was approved by the Animal Ethics Committee as stipulated in the guide to the care and use of experimental animals of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The chickens were euthanized by intravenous injection of pentobarbital. Detection of Serum IgG Antibody. Blood samples were collected via the wing veins from three chickens in each of the five groups post-immunization per week. The serum samples were obtained by centrifugation at 4 °C at 3000 r/min for 10 228

DOI: 10.1021/acs.molpharmaceut.7b00826 Mol. Pharmaceutics 2018, 15, 226−237

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Figure 1. TEM morphology (A), particle size distribution (B), and zeta potential (C) of the N-2-HACC-CMC/pFDNA-C3d6 NPs.

Figure 2. Stability and in vitro release analysis of the plasmid pVAX I-F(o)-C3d6 after encapsulation in the N-2-HACC-CMC nanoparticles. (A) DNase I protection of the pVAX I-F(o)-C3d6. M, DL 15000 Marker; Lanes 1−4, N-2-HACC-CMC/pFDNA-C3d6 NPs treated by DNase I for 30, 60, 120, and 180 min, respectively; Lane 5, pVAX I-F(o)-C3d6 treated by DNase I for 30 min; Lane 6, plasmid pVAX I-F(o)-C3d6. (B) In vitro release profiles of the pFDNA-N-2-HACC/CMC NPs in the PBS solution (pH = 7.4). Data are presented as the mean ± standard deviation (n = 5).

IL-2 ELISA (enzyme-linked immunosorbent assay) Kit (Thermo Fisher Scientific Inc., MA, USA) in accordance with the manufacturer’s instructions, respectively. All the operations were performed according to the procedures described for the cytokine ELISA kits. Protection against NDV Strain F48E9. The protection conferred by N-2-HACC-CMC/pFDNA-C 3d6 NPs on chickens against NDV infection was investigated using a virulent strain of NDV (NDV strain F48E9) with genotype IX (Harbin Pharmaceutical Group Biovaccine Co. Ltd.). When the levels of NDV-specific antibody in serum of every immune group increased to 6.0 log2 post-first-immunization, seven chickens were selected randomly from each of the five groups and were infected i.m. with 100 μL of strain F48E9 at a viral titer of 104.5 EID50/0.1 mL for challenge studies. Meanwhile, feed, water, mental state, clinical symptoms, and mortality of chickens were continuously observed and recorded for 35 d. Serum samples were collected from three chickens at 1, 2, 3, 4, and 5 weeks after challenge, the contents of IgG, IL-2, IFN-γ, and IL-4 were determined. The negative control chickens and infected chickens were euthanized, and the glandular

min. The titers of anti-NDV IgG antibody were detected by hemagglutination inhibition (HI) test (n = 3). Detection of IgA Antibody. To detect the mucosal immune response, serum, tracheal fluid, bile, and Harderian glands were collected from three chickens at 1 to 10 week postimmunization. Mucosal extracts were obtained by centrifugation, and the supernatant was collected. The titers of IgA antibody were detected by using the NDV IgA ELISA Kit (Rapidbio Co. Ltd., Beijing, China) (n = 3). Lymphocyte Proliferation Response. The cellular-mediated immune responses of immunized chickens were assessed at 1, 2, 3, 4, 5, 6, 7, 8 9, and 10 weeks post-immunization, and the MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide) colorimetric assay was performed to evaluate lymphocyte proliferation as described previously.31 Splenic lymphocytes were prepared from all the experimental chickens using the standard protocol. Detection of IL-2, IL-4, and IFN-γ. For cytokines assays, serum samples were collected from three chickens at 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 weeks post-immunization. The concentration of IFN-γ, IL-2, and IL-4 in spleen cell culture supernatants were detected by using a chicken IFN-γ, IL-4, and 229

DOI: 10.1021/acs.molpharmaceut.7b00826 Mol. Pharmaceutics 2018, 15, 226−237

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Figure 3. Histopathological analyses of glandular stomach and duodenum. (A,C,E) Tissues of the glandular stomach from the SPF chickens immunized with the PBS i.m., N-2-HACC-CMC/pFDNA-C3d6 NPs i.m., and N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. (B,D,F) Tissues of the duodenum from the SPF chickens immunized with the PBS i.m., N-2-HACC-CMC/pFDNA-C3d6 NPs i.m., and N-2-HACC-CMC/pFDNA-C3d6 NPs i.n.

degradation by DNase I, the plasmid pVAX I-F(o)-C3d6 and N-2-HACC-CMC/pFDNA-C3d6 NPs were treated with DNase I, and then the digested products were analyzed with agarose gel electrophoresis. As shown in Figure 2A, the naked plasmid pVAX I-F(o)-C3d6 was degraded by incubation with DNase I within 30 min (Lane 5, Figure 2A), whereas the pVAX I-F(o)-C3d6 encapsulated in the N-2-HACC-CMC nanoparticles was protected from degradation by DNase I and was not degraded until it was treated for 3 h (Lanes 1−4, Figure 2A). The results indicated that the plasmid DNA encapsulated in the N-2-HACC-CMC nanoparticles could be protected from degradation by DNase I. As shown in Figure 2B, the amount of pVAX I-F(o)-C3d6 released from the N-2-HACC-CMC/pFDNA-C3d6 NPs was increased to 68.87 ± 1.43% from 0 to 72 h (n = 3), which reveals that the burst release mainly takes place during the first 72 h, followed by a continuous and slow release, and reached 74.80 ± 2.00% at 120 h and 81.67 ± 0.92% at 240 h (n = 3). In vitro release results suggested that the N-2-HACC-CMC nanoparticles could be used as a delivery carrier of plasmid DNA for sustained release. Biological Safety of the N-2-HACC-CMC/pFDNA-C3d6 NPs. The survival rate of chicken embryo fibroblast in the N-2HACC-CMC/pFDNA-C3d6 NPs was 90.05 ± 1.42% (n = 3), and compared with those of the control cells, there was no significant changes in cell morphology (P > 0.05). In vivo

duodenum, stomach, and myocardium were collected for histological staining assay. Statistical Analysis. The immunization experiments were repeated three times under the same conditions. Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA) statistical test with Tukey’s post test was performed using Origin 7.5 software (OriginLab Corporation, USA) to determine the significance of the differences between various groups. P < 0.05 was considered as significant.

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RESULTS Characterization of N-2-HACC-CMC/pFDNA-C3d6 NPs. As shown in Figure 1, the N-2-HACC-CMC/pFDNAC3d6 NPs had regular shape, smooth surface, and good dispersion and did not have subsidence or adhesion damage (Figure 1A). The mean diameter of nanoparticles was 309.7 ± 6.52 nm (Figure 1B), and zeta potential was 49.9 ± 4.93 mV (Figure 1C). The EE was 92.27 ± 1.48%, and LC was 50.75 ± 1.35% (n = 5). These results showed that the polyelectrolyte complex method was feasible to prepare the N-2-HACCCMC/pFDNA-C3d6 NPs, and the particle size was suitable for the delivery of plasmid DNA. DNase I Protection and in Vitro Release of the N-2HACC-CMC/pFDNA-C3d6 NPs. To determine whether the N-2-HACC-CMC nanoparticles can prevent DNA from 230

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Figure 4. In vitro expression of the N-2-HACC-CMC/pFDNA-C3d6 NPs in 293T cells assayed by indirect immunofluorescence (×40): (A) N-2HACC-CMC/pFDNA-C3d6 NPs group; (B) pVAX I-F(o)-C3d6 group; (C) N-2-HACC-CMC NPs group; (D) 293T cell group as the negative control.

Figure 5. Serum IgG antibody titers (A) and IgA antibody titers (B) following administration of N-2-HACC-CMC/pFDNA-C3d6 NPs i.n., N-2HACC-CMC/pFDNA-C3d6 NPs i.m., pVAX I-F(o)-C3d6 i.m., N-2-HACC-CMC NPs i.m., and PBS i.m. Values represent mean ± SD (n = 3). **P < 0.01, significantly different when compared topVAX I-F(o)-C3d6.

cytotoxicity analysis demonstrated that the drinking, feeding, weight, and other behavior of chickens immunized with the N2-HACC-CMC/pFDNA-C3d6 NPs i.n. or i.m. were normal compared with those of the control group, and the morbidity and mortality of chickens were 0% in both groups, indicating that immunization of the chickens with a high dose of N-2HACC-CMC/pFDNA-C3d6 NPs is safe. Histopathological analyses of glandular stomach and duodenum were shown in Figure 3A−F. These results indicated that the N-2-HACCCMC NPs as delivery carriers had little cytotoxicity but had higher safety level by the intranasal administration route. In Vitro Expression of the N-2-HACC-CMC/pFDNAC3d6 NPs. As shown in Figure 4, intensive fluorescence was observed in 293T cells transfected with the N-2-HACC-CMC/ pFDNA-C3d6 NPs (Figure 4A) and pVAX I-F(o)-C3d6

(Figure 4B), and the level of expression was greater for nanoparticle-mediated delivery than the pVAX I-F(o)-C3d6, probably due to the protection from DNase degradation. In contrast, no fluorescence was detected in the 293T cells transfected with N-2-HACC-CMC nanoparticles (Figure 4C) and the negative cells control group (Figure 4D). These results indicate that the pVAX I-F(o)-C3d6 can be effectively encapsulated by the N-2-HACC-CMC nanoparticles and expressed in vitro. Immune Efficacy in SPF Chickens. Serum HI Antibody. As shown in Figure 5A, N-2-HACC-CMC/pFDNA-C 3d6 NPs i.n., N-2-HACC-CMC/pFDNA-C3d6 NPs i.m. ,and pVAX IF(o)-C3d6 i.m. induced significant antibody responses in chickens when they were immunized, and the antibody titers of chickens were quickly increased and peaked at the sixth week 231

DOI: 10.1021/acs.molpharmaceut.7b00826 Mol. Pharmaceutics 2018, 15, 226−237

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Figure 6. IgA antibody titers in tracheal fluid (A), bile (B), Harderian gland (C), and lymphocyte proliferation response (D) of SPF chickens immunized with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.n., N-2-HACC-CMC/pFDNA-C3d6 NPs i.m., pVAX I-F(o)-C3d6 i.m., N-2-HACCCMC NPs i.m., and PBS i.m. IgA antibody content in these samples were detected with ELISA. Values represent mean ± SD (n = 3). **P < 0.01, significantly different when compared to pVAX I-F(o)-C3d6.

Figure 7. IL-2 (A), IL-4 (B), and IFN-γ (C) levels in the supernatant of splenocytes harvested from the SPF chickens immunized with the N-2HACC-CMC/pFDNA-C3d6 NPs i.n., N-2-HACC-CMC/pFDNA-C 3d6 NPs i.m., pVAX I-F(o)-C3d6 i.m., N-2-HACC-CMC NPs i.m., and PBS i.m. IFN-γ, IL-2, and IL-4 levels in the supernatant were analyzed in a chicken IFN-γ, IL-2, and IL-4 enzyme-linked immunosorbent assay. Values represent mean ± SD (n = 3). **P < 0.01, significantly different when compared to pVAX I-F(o)-C3d6.

post-immunization. After the sixth week, chickens immunized with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. or N-2HACC-CMC/pFDNA-Cd6 NPs i.m. produced higher titers of anti-NDV than those immunized with the pVAX I-F(o)-C 3d6 i.m. until the tenth week (P < 0.05). The differences in antibody titers between N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. and N-2-HACC-CMC/pFDNA-C3d6 NPs i.m. was not

significant before the ninth week (P > 0.05); however, there was significant difference at the tenth week (P < 0.01), indicating that the N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. are able to elicit the immune responses of chickens and maintain higher antibody levels for a long time via an intranasally administered route. 232

DOI: 10.1021/acs.molpharmaceut.7b00826 Mol. Pharmaceutics 2018, 15, 226−237

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Figure 8. Serum IgG antibody titers (A) and IL-2 (B), IL-4 (C), IFN-γ (D) levels in the supernatant of splenocytes harvested from the immunized SPF chickens after challenge with the highly virulent NDV strain F48E9. IFN-γ, IL-2, and IL-4 levels in the supernatant were analyzed in a chicken IFN-γ, IL-2, and IL-4 enzyme-linked immunosorbent assay. Values represent mean ± SD (n = 3). **P < 0.01, significantly different when compared topVAX I-F(o)-C3d6.

sIgA Antibody Titers in Mucosa Extracts. The titers of antiNDV sIgA antibody in chickens immunized with N-2-HACCCMC/pFDNA-C3d6 NPs i.n. were significantly higher (P < 0.01) in serum (Figure 5B), tracheal fluid (Figure 6A), bile (Figure 6B), and Harderian gland (Figure 6C), and the periods of IgA antibody secretion were also longer than those of chickens in other groups (P < 0.01). These results showed that intranasal delivery of the N-2-HACC-CMC/pFDNA-C3d6 NPs induced higher IgA titers than intramuscular delivery. Lymphocyte Proliferation. As shown in Figure 6D, the lymphocyte proliferation was significantly higher in chickens treated with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. group than in those treated with the N-2-HACC-CMC/ pFDNA-C3d6 NPs i.m., pVAX I-F(o)-C3d6 i.m., and control groups (P < 0.01). However, the chickens immunized with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.m. and pVAX I-F(o)C3d6 i.m. had no significant difference at 7−10 weeks postimmunization (P > 0.05). These findings indicated that immunization intranasally with the N-2-HACC-CMC/ pFDNA-C3d6 NPs led to greater and longer lymphocyte proliferation response, albeit not pronounced in the case of the i.m. route.

Assay of Cytokine Responses. As shown in Figure 7, the N2-HACC-CMC/pFDNA-C3d6 NPs delivered i.n. or i.m. triggered significantly more IL-2, IFN-γ, and IL-4 than immunization with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.m. (P < 0.01), and the levels of IL-2 (Figure 7A), IL-4 (Figure 7B), and IFN-γ (Figure 7C) were significantly higher in chicken immunized with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. than those of chickens in the N-2-HACC-CMC/pFDNA-C3d6 NPs i.m. group (P < 0.01). These results suggested that the N2-HACC-CMC/pFDNA-C3d6 i.n. could elicit cellular immune responses by inducing more cytokines. Protective Effect. The highly virulent NDV strain F48E9 had killed all chickens treated with PBS or N-2-HACC-CMC NPs in the 2 to 5 days after challenge; four chickens in the pVAX I-F(o)-C3d6 i.m. group died at the sixth day; two chickens in the N-2-HACC-CMC/pFDNA-C3d6 NPs i.m. group died at the seventh day; and the chickens in the N-2HACC-CMC/pFDNA-C3d6 NPs i.n. group had no death (Table S1). The protective efficacy was 100%, 71%, and 43% for the chickens immunized with the N-2-HACC-CMC/ pFDNA-C3d6 NPs i.n., N-2-HACC-CMC/pFDNA-C3d6 NPs i.m., and pVAX I-F(o)-C3d6 i.m., respectively (Table 233

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electroporation, infusion, sonication, and gene gun, has been used to improve their efficiency. All these improvement techniques have not only resulted in inducing mucosal immunity but also allowed the use of a much lower dose of plasmid DNA through an increase in transfection efficiency in vivo. This achievement, notwithstanding the development of a new immune delivery system, is necessary to further improve DNA vaccination. While viral delivery systems have better transfection efficiencies than NPs-based systems,42 NPs are easy to synthesize, and their molecular structures can be easily manipulated due to accessible functional groups. Furthermore, they generally have a low production cost compared to viral delivery systems, can accommodate large vector sizes, and possess a favorable safety profile.11 NPs that have been formulated for gene delivery fall into one of several categories, including metal NPs, lipid NPs, and polymer NPs. In all potential NP delivery systems, chitosan nanoparticles possess promising abilities to promote delivery of adsorbed DNA to APCs and protect encapsulated nucleic acidbased antigens. However, the natural polymer chitosan suffers from low solubility at the physiological pH value.43 Introducing amine groups into chitosan by modification of functional groups has been shown to improve plasmid DNA delivery to target tissues, as indicated by increased gene expression compared to unmodified chitosan NPs.44 Consequently, functional forms of chitosan have attracted great interest to enhance stability, permeability, conductivity, and controlled/ extended antigen release profiles at mucosal sites.45,46 Herein, this study serves to demonstrate that the modified chitosan nanoparticles can serve not only as efficient plasmid DNA delivery systems but also act as potent adjuvants for stimulating humoral, cellular, and mucosal immune response. Our group has synthesized the water-soluble biodegradable polymer quaternized chitosan, N-2-HACC, and CMC.32 To evaluate N-2-HACC and CMC as an adjuvant and delivery carrier for the pVAX I-F(o)-C 3d6, we prepared the N-2HACC-CMC/pFDNA-C3d6 NPs to develop a nasal vaccine against NDV by the polyelectrolyte electrolysis complex method. The ability of nanoparticles to transfer across the mucosal surface to uptake and transport antigen, and to stimulate the body to produce an effective immune response is dependent on the particle size, zeta potential and surface characteristics.47 A few studies have shown that by reducing the particle size to nano-range, adhesion property will be increased significantly.48 The average diameter of the N-2-HACC-CMC/ pFDNA-C3d6 NPs was 309.7 ± 6.52 nm (Figure 1B) and zeta potential was 49.9 ± 4.93 mV (Figure 1C), which was ideal for entry into the cells and enhanced ability. To obtain efficient antigen delivery from nasal route, the size must be less than 10 μm, which was the case for the produced nanoparticles that could efficiently interact with the immune cells and had a great potential for adhesion.49 In addition, it is important to elevate the effectiveness of delivery and to express corresponding antigens.49 Because the CMC have a negative zeta potential, which is opposite to that of N-2-HACC, we encapsulated the pVAX I-F(o)-C3d6 containing N-2-HACC with CMC, which improves both cell permeability and antigen stability, and N-2HACC-CMC also shows good adsorption performance for the pVAX I-F(o)-C3d6, enabling it to be efficiently encapsulated into nanoparticles. The encapsulation efficiency and loading capacity was 92.27 ± 1.48% and 50.75 ± 1.35%, respectively. The N-2-HACC-CMC/pFDNA-C3d6 NPs also can supply a relatively high level of protection of DNA against degradation

S1). Drinking, feeding, weight, and other aspects of behavior were normal, and there was no pathological or histopathological changes found in chickens treated with N-2-HACCCMC/pFDNA-C3d6 NPs i.n. Additionally, all the dead chickens displayed the typical pathological changes of ND, including mucosal hemorrhages in proventriculus papillae,duodenum, the whole intestines, and fatty heart. The anti-NDV titers in the chickens immunized with the N2-HACC-CMC/pFDNA-C3d6 NPs i.n., N-2-HACC-CMC/ pFDNA-C3d6 NPs i.m., and pVAX I-F(o)-C3d6 i.m. significantly increased and reached the peak at the third week. The chickens immunized with the nanoparticles i.n. produced higher IgG titers than those immunized with the N-2HACC-CMC/pFDNA-C3d6 NPs i.m. and pVAX I-F(o)-C3d6 i.m. after the fourth week (P < 0.01), in a slow release and sustainable fashion (Figure 8A). Also, IL-2 (Figure 8B), IL-4 (Figure 8C), and IFN-γ (Figure 8D) levels were significantly higher in the chickens immunized with the N-2-HACC-CMC/ pFDNA-C3d6 NPs i.n. than those immunized with the N-2HACC-CMC/pFDNA-C3d6 NPs i.m. and pVAX I-F(o)-C3d6 i.m. (P < 0.01).

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DISCUSSION C3d fragment is one of the cleavage fragments encoding chicken complement C3, and its chain cannot be cleaved by protease and remains the smallest fragment covalently linked to antigen, which has certain biological significance. Therefore, C3d is viewed as a molecular adjuvant that improves vaccine immune responses. The fusion 4 or 6 copies of C3d fragment could significantly improve the immune responses of NDV DNA vaccine, and 6 or more fusion copies were of great significance to enhance NDV F gene DNA vaccine.36 However, the conjugation between different C3d copies and antigen will also cause a negative regulation to immune response. One copy of C3d inhibited the specific antibody, while 2 copies of C3d showed a more pronounced inhibitory effect and a decrease in IL-4 and IFN-γ secretion, indicating that 1−2 copies of C3d molecules could elicit a negative regulatory effect on antigenspecific humoral and cellular immunities.37 So, we constructed the transfer vectors with a different number of C3d molecular adjuvants (n = 1, 2, 4, 6), and the vector was cloned into the optimal eukaryotic expression plasmid (pVAXI-optiF) that expressed the F gene of NDV. The results indicated that the pVAX I-F(o)-C3d6 had stronger fluorescence and induced better immune responses than the other plasmids. DNA vaccines are potentially powerful tools for immunization against a variety of pathogens. DNA immunization can induce both humoral and cellular immune responses38−40 and can be administered many times without eliciting any antivector immunity. Other advantages of DNA vaccine include its ability to polarize T-cells. Compared with protein-based vaccines, DNA vaccine formulations are relatively more stable and with longer shelf life, which in turn promotes their lower preparation cost, storage, and shipping. However, the immunogenicity of DNA vaccines has been restricted by some problems related to their delivery, including degradation of the DNA by lysosomes or DNases and poor cellular uptake of DNA, and it is difficult for DNA vaccines to pass through cell membranes, resulting in only a few reaching APCs to elicit immune responses.41 Additionally, nasal vaccination provides all the prerequisites for a successful needle-free vaccine delivery and also for better vaccine immune efficacy. A variety of strategies, which includes 234

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Molecular Pharmaceutics (Figure 2A), which is crucial for antigen delivery via mucosal surfaces. Therefore, it met the basic requirements for nanoparticles applied to the organism and had great application potential in mucosal immune delivery systems. It was reported previously that the swelling of the nanoparticles and pH values of the released medium can influence the release of the plasmid DNA encapsulated in the nanoparticles.50 Hence, in vitro release assay was performed by incubating N-2-HACC-CMC/ pFDNA-C 3d6 NPs in PBS solution (pH 7.4), and the cumulatively released amounts were monitored at different times, and the results showed that the pVAX I-F(o)-C3d6 could be released slowly (Figure 2B). In vitro cytotoxicity also showed that the N-2-HACC-CMC/pFDNA-C3d6 NPs had low cytotoxicity and high safety by either i.n. or i.m. and that no pathological changes were observed in the immunized chickens. These findings indicated that the N-2-HACC-CMC NPs can serve as a control-released delivery carrier and have little cytotoxicity but higher safety level, prolonging the expression duration of plasmid DNA in the body. ND is transmitted mainly through respiratory and digestive tracts, and the nasal mucosa is the first portal for antigen to enter and induce mucosal immune response.51 Furthermore, DNA vaccines are usually administered by intramuscular injection and can fail to reach the APCs, and therefore, immune response fails to be induced because it is difficult to cross the cell membranes.5,6,50 Thus, to detect the protective efficacy and antigenicity of the N-2-HACC-CMC/pFDNAC3d6 NPs after nasal administration, we performed two strategic approaches. The chickens were immunized with the both N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. and i.m. Analyses of anti-ADV IgG and IgA antibody responses revealed that intranasal immunization induced stronger immune responses and obtained a longer sustained release than immunization with the N-2-HACC-CMC/pFDNA-C3d6 NPs i.m. and the pVAX I-F(o)-C3d6 i.m. (Figures 5 and 6), and the sIgA antibody titers were much higher than the previously reported 800−1000 ng/mL (P < 0.05).52 This could be attributed to muco-adhesion potential of chitosan nanospheres and their more prolonged presence in contact with nasal mucosa.53 Intranasal immunization is very effective in inducing systemic and mucosal immune responses. Our results indicate that the nasal administration of N-2-HACC-CMC/pFDNAC3d6 NPs is effective in eliciting the immune response against ND. It is possible that the absorption-enhancing effect may have been caused by opening the intercellular tight junctions to work on the intercellular negative area and change the F-actin in the cytoskeleton from the original filamentous to a sphere, which makes the nanoparticles go through the mucosal epithelial cell barrier or be absorbed directly by M cells to enhance the immune effect.54 Cytokines play critical roles in the development of cellular immunity and prevention of viral infections.33 T helper cells are the key regulatory cells that regulate humoral and cellular immunity. The functional active regions of T helper cells are divided into two cell subpopulations, Th1 and Th2. Th1-type cytokines (IFN-γ, IL-2, and IL-12) help DNA vaccine to induce and enhance Th1 immune response, and Th2-type cytokines (IL-4) enhance Th2 immune response. IL-2 mainly enhances cellular immunity, IL-4 mainly regulates humoral immunity, and IFN-γ mainly regulates immune response by helping T helper cells differentiate into Th1. Hence, the production levels of IFN-γ, IL-2, and IL-4 were detected, and the enhanced lymphocyte proliferation and cellular responses have been

verified in the N-2-HACC-CMC/pFDNA-C3d6 NPs i.n. group (Figures 6D and 7), which indicated a better induction of Th1 and Th2 type responses. Thus, it was reasonably deduced that the quaternized chitosan played a key role in supporting cellular uptake by cell-N-2-HACC interactions and enhanced the antigen presentation. Currently, the chitosan-based nanomaterials as a potential adjuvant and carrier for animal DNA vaccines are still at an early stage. Thus, it is very necessary to research and develop efficient, stable, and safe vaccine adjuvants and delivery carriers that can induce humoral, cellular, and mucosal immune responses to prevent and control certain infectious diseases.2 More studies on biodegradable polymeric nanoparticles should be carried out for the delivery of antigens via mucosal route, specifically clinical trials. With the development of nanotechnology, all of these issues will eventually be resolved in future, and this study builds a basis for the application of quaternized chitosan nanoparticles as safe and efficient adjuvant and delivery carrier for the mucosal delivery of DNA vaccine; and showed great promise and application potential in the administration of vaccines and drugs by the intranasal route.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00826. After the immunized SPF chickens were challenged with the highly virulent NDV strain F48E9; mortality, morbidity and protective efficacy of the chickens are supplied (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Kai Zhao: 0000-0001-6139-1912 Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Key Research and Development Program of China (2017YFD0500706), National Natural Science Foundation of China (31570929 and 31771000), Natural Science Foundation of Heilongjiang Province of China (C2017058), Key Project of Research and Development of Shandong Province of China (2016GSF121020), Key Scientific and Technological Planning Project of Harbin (2016AB3BN036), Technological innovation talent of special funds for outstanding subject leaders in Harbin (2017RAXXJ001), Project of Graduate Innovative Scientific Research Foundation of Heilongjiang University (YJSCX2017158HLJU), and Special Project of Graduate Entrepreneurship of Heilongjiang University (20170160907). 235

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