Redox-Responsive Biodegradable Polycation Poly(amido amine

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Redox-responsive biodegradable polycation poly(amido amine) used as intranasal vaccine delivery systems Zhong Guo, Sha Li, Meng Lv, Zonghua Liu, and Wei Xue ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00538 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 11, 2017

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ACS Biomaterials Science & Engineering

Redox-responsive biodegradable polycation poly(amido amine) used as intranasal vaccine delivery systems

Zhong Guo a #, Sha Li a #, Meng Lv a, Zonghua Liu a, *, Wei Xue a, b, *

a

Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of

Biomedical Engineering, Jinan University, Guangzhou, 510632, China b

Institute of Life and Health Engineering, Key Laboratory of Functional Protein Research of

Guangdong Higher Education Institutes, Jinan University, Guangzhou, 510632, China

#

The authors contributed equally to this work.

* Corresponding authors: [email protected] (Zonghua Liu) [email protected] (Wei Xue)

Tel and Fax: 86-20-85223062

ACS Paragon Plus Environment


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ABSTRACT Polycations such as polyethyleneimine and chitosan have been widely used as mucosal vaccine delivery

systems

due

to

their

permeation

enhancement

effect.

Preferably,

environmentally-responding biodegradable polycations would be better carrier materials for mucosal vaccine delivery. Disulfide bonds-based redox-sensitive polycations could respond to the higher intracellular glutathione concentration and degrade in the cytoplasm via the breakage of the disulfide bonds, which are particularly suitable for antigen delivery. In this work, we evaluated the potential of redox-sensitive, biodegradable polycation poly(amido amine) (PAA) as mucosal vaccine carriers. From the primary studies with ovalbumin used as a model protein antigen, it is found that PAA could complex with and encapsulate protein antigen via electrostatic attraction, enhance the cellular uptake of antigen by dendritic cell line DC2.4, prolong antigen residence in nasal cavity, and promote antigen permeation into nasal submucosal layer. Further, Balb/c mice were intranasally immunized with PAA-delivered recombinant hemagglutinin (HA) antigen protein of H7N9 influenza virus. The PAA/HA formulations induced significantly more potent systemic IgG response and mucosal IgA response, higher splenocyte proliferation activity, higher secretion levels of cytokines IFN-γ and IL-4 by splenocytes, more memory CD4+ and CD8+ T cells, and more DCs expressing MHC Ⅱ molecule. From the results, the redox-responsive polycation PAA as vaccine carriers helped elicit more potent cellular and humoral immune responses. Particularly, PAA induced much higher cellular immune response compared with previously reported carrier materials. The intelligent PAA could be developed as efficient mucosal vaccine delivery systems for clinical applications. KEYWORDS: poly(amido amine), redox-responsive, vaccine delivery systems, mucosal immunization


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1. INTRODUCTION In the field of immunotherapy, immune vaccination plays a key role in disease prevention and treatment, particularly for some intractable diseases such as cancers and HIV.1-2 In immune vaccination, development of vaccines has made a great progress and a variety of vaccines have been produced.3 However, these vaccines usually can not meet the concomitant requirement of both safety and efficacy.3 As a result, safer vaccines are preferred, but meanwhile their immunization efficacy discounted due to the sacrifice of their immunogenicity.2 Fortunately, the immune response effectiveness of the vaccines can be further improved by using proper vaccine delivery systems in immune vaccination.4 Natural and synthetic biomaterials are increasingly being used as vaccine carriers for delivering antigen and increasing their immunogenicity.5 Therefore, the development of efficient vaccine carriers has been receiving great attention for decades. In recent years, some polycations have been found to have promising potential as vaccine carriers for antigen delivery in various immune vaccinations. It was found that cationic polymers including polyethyleneimine, polylysine, cationic dextran and cationic gelatin could strongly promote Th1 response in vivo, characterized by specifically stimulating macrophages to secrete IL-12 via toll-like receptor-4.6 And higher cationic degree and larger molecular weight of the cationic polymers meant stronger stimulation ability. It was also reported that electrostatic attraction between positively charged vaccine carriers and anionic epithelial cell layer enhanced antigen delivery efficiency.7 Natural polycation chitosan and various chitosan derivatives with better physicochemical properties have been widely studied as potent vaccine carriers.8 Polyethyleneimine as the golden standard of gene delivery has found great potential as mucosal and systemic vaccine carriers to deliver protein or DNA vaccines.9-12 Spermine-conjugated dextran derivatives were also used to deliver protein and DNA vaccines.13-14 The carboxyl groups of poly(γ-glutamic acid) were particularly modified with amines to induce a stronger electrostatic attraction with anionic mucus and to improve antigen delivery efficiency.7 Collectively, the polycations have special advantages



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as vaccine delivery systems over other materials due to their abundant positive charge. Compared with the above polycations, redox-sensitive biodegradable polycations would be more preferable carrier materials for vaccine delivery. Disulfide bonds-based polycations could respond to the higher intracellular glutathione concentration, degrade in the cytoplasm via the breakage of the disulfide bonds, and hence have unique advantages of redox-responsive biodegradability and better biocompatibility particularly suitable for vaccine delivery.15 In this work, disulfide-linked, bio-reducible hyperbranched poly(amido amine) (PAA) was evaluated as intranasal protein antigen delivery systems. Recently, PAA has gained much attention for gene delivery and demonstrated high transfection efficiency as well as good biosafety, resulting from the presence of bio-reducible disulfide linkages.16-18 As a result, PAA displayed higher transfection efficiency and better bio-safety compared with the gold standard of the gene carrier polyethyleneimine of 25 kDa.19 Moreover, the introduction of disulfide-linkages provided an intelligent release of RNA molecules into cytoplasm.20 On the basis of these considerations, PAA could also be a promising carrier material for antigen delivery. Particularly, PAA may has a special merit to release protein antigen directly to cytoplasm of antigen presenting cells (APCs) and hence promote MHC Ι presentation and cellular immunity. In this study, the effectiveness of PAA was evaluated as a carrier material for intranasal vaccine delivery.

2. MATERIALS AND METHODS 2.1. Materials Cholera toxin B subunit (CTB) and chicken egg ovalbumin (OVA) were purchased from Sigma-Aldrich (St. Louis, USA). Cy5.5 or Cy5-labeled OVA (OVA-Cy5.5 or OVA-Cy5) was obtained from Sungene Biotech (Tianjin, China). Polyinosinic : polycytidylic acid (poly(I : C)) was purchased from InvivoGen (San Diego, USA). Recombinant hemagglutinin (HA) protein of influenza virus (A/Anhui/1/2013, H7N9) was obtained from Sino Biological Inc. (Beijing, China).


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Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, IgG2a and IgA antibodies were purchased from Abcam (Cambridge, USA). Enzyme-linked immunosorbent assay (ELISA) kits (for IL-4 or IFN-γ) and ELISpotPLUS kits (for IL-4 or IFN-γ) were purchased from BioLegend (San Diego, USA). Fluorochrome-labeled CD4, CD8, CD44, CD62L, CD11c and MHC Ⅱ antibodies were purchased from eBioscience (San Diego, USA). Female Balb/c mice used in this study were purchased from the Southern Medical University (Guangzhou, China). All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, and were approved by the Experimental Animal Ethics Committee in Guangzhou. DC2.4 cell line was obtained from Biohysw (Shanghai, China). The DC2.4 cells or splenocytes collected from the immunized Balb/c mice were cultured in RPMI 1640 (Gibco) with 10% FBS (Gibco), 100 units mL-1 penicillin and 100 µg mL-1 streptomycin (Gibco) at 37°C in an incubator with 5% CO2 atmosphere. 2.2. Physicochemical characterization of PAA The molecular weight of PAA was measured by using gel permeation chromatography (GPC) (Malvern, UK). GPC measurements were performed with a VE 1122 solvent delivery system and a Model 270 DUAL detector and 3580 RI detector using pullulan as a standard. NaNO3 solution (0.02 M, containing 0.2‰ (w/v) sodium azide) was used as the eluent with a flow rate of 1 mL min-1. The hydrodynamic diameter and zeta potential of PAA were measured with a zeta potential analyzer (Malvern, UK). 2.3. Complex formation of PAA with OVA Fluorescein isothiocyanate (FITC)-labeled PAA (PAA-FITC) and OVA-Cy5 were used to evaluate the interaction between PAA and OVA. Briefly, mixed solutions of PAA-FITC (50, 100, 500 µg) and OVA-Cy5 (100 µg) in ultrapure water (500 µL) were incubated at room temperature for 1 h. After incubation, PAA, PAA-FITC, and PAA-FITC/OVA-Cy5 complexes were determined by using BECKMAN CytoFLEX (Beckman, USA). The measurement parameters were set: a threshold



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value of FSC-H was 5000, PAA-FITC was FITC-H, and OVA-Cy5 was APC-H. The data were analyzed by using FlowJo software (version 7.6.1). 2.4. Cellular uptake of OVA by DC2.4 cells DC2.4 cells were seeded on sterile coverslips (WHB, China) in 24-well plates with 5×104 cells/well, and cultured overnight. The old medium in each well was discarded and the cells were incubated with OVA-Cy5 solutions containing PAA or not for 6 h. The cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 1 h, and washed three times with PBS. The cells were observed by using a confocal laser scanning microscope (LSM 700, ZEISS, Germany). DC2.4 cells were seeded on 24-well plates with 1×105 cells/well, and cultured overnight. The old medium in each well was discarded and the cells were incubated with OVA-Cy5 solutions containing PAA or not for 6 h. The cells were washed three times with PBS. The cells were trypinised, centrifuged at 1100 rpm for 5 min, resuspended with PBS, and then analyzed with the BECKMAN CytoFLEX. 2.5. OVA antigen residence in nasal cavity OVA-Cy5.5 solutions (30 µL, containing PAA, CTB or Poly (I : C)) were intranasally dropped in female Balb/c mice (n=3) under light anesthesia with a 2.5% avertin solution (2, 2, 2-tribromoethanol-tert amyl alcohol, Sigma). Then, the mice were observed at 5 min, 20 min, 1 h, 2 h and 6 h by using an in vivo imaging system (PerkinElmer, USA). 2.6. OVA antigen permeation into nasal submucosal layer OVA-Cy5.5 solutions (30 µL, containing PAA or not) were intranasally dropped in female Balb/c mice under light anesthesia with the 2.5% avertin solution. After 1 or 6 h, the mice were euthanized, and the nasal cavities were surgically collected and fixed in 10% formalin for 24 h. After decalcification in EDTA-2Na solution, the tissues were paraffin-embedded and sectioned to slices of 4 mm thickness on polylysine-coated slides. The immunohistochemical staining of the


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tissues was performed at room temperature with streptavidin-perosidase method. Briefly, after deparaffinage, rehydration and antigen retrieval process, the sections were blocked by normal goat serum and incubated with primary rabbit anti-OVA antibody overnight at 4°C. The slides were washed three times with PBS and incubated with goat anti-rabbit IgG antibody for 1 h at 37°C. After counter-staining with hematoxylin, the sections were dehydrated, mounted, and then observed with a ZEISS Axio Observer A1 microscope (ZEISS, Germany) and POTEC DV200 (Chongqing, China). 2.7. Immunization study Female Balb/c mice (6-8 weeks, n=5) were intranasally vaccinated with 30 µL of HA solutions in PBS (10 µg HA per mouse) formulated with PBS, PAA (5, 10, or 50 µg), CTB (10 µg), or Poly (I : C) (10 µg). All the mice were anesthetized with 100 µL of 2.5% avertin solution before immunization. The immunization was performed three times at 2-weeks intervals. Two weeks after the third immunization, blood samples were collected from the immunized mice and incubated at room temperature for 1 h. The sera were obtained from the blood samples by centrifugation for 5 min at 1,000 rpm. The nasal and genital wash samples were collected by flushing with 100 µL of PBS through the nasal cavity and genital tract, respectively. After centrifugation for 5 min at 13,000 rpm, the supernatants of the wash samples were collected and stored at −20°C. 2.8. Determination of antibody titers The antibody titers of antigen-specific IgG in the sera or IgA in the mucosal wash samples were determined by ELISA. In brief, 96-well ELISA plates were pre-coated with 100 µL of HA protein (10 µg mL-1) dissolved in 0.1 M carbonate buffer overnight at 4°C. After washing once with PBS-Tween 20 (PBST), the plates were blocked with PBST containing 2% bovine serum albumin for 1 h at room temperature, and then washed four times with PBST. The serial 2-fold diluted samples (100 µL) in the blocking buffer were added to each well, incubated for 2 h at 37°C, and



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washed four times with PBST. The plates were incubated with 100 µL of a 1:5000 dilution of HRP-conjugated anti-mouse IgG (IgG total, IgG1 or IgG2a) and IgA (1:2000 dilution) antibodies. After incubation at room temperature for 1 h, the plates were washed four times with PBST and incubated with 100 µL of peroxidase substrate tetramethylbenzidine (BD, PharMingen). After 15 min, the reaction was stopped with 100 µL 2 N sulfuric acid and the plates were read within 30 min at 450 nm using a microplate reader (Thermo Fisher scientific, USA). 2.9. Splenocyte proliferation assay Splenocytes were collected from the vaccinated mice with the grinding method at 14 days after the third immunization. The splenocytes (2×105 cells/well) were seeded in triplicate on 96-well plates and stimulated with HA antigen (10 µg mL-1) at 37°C. After 72 h, 20 µL of CCK-8 solution (Dojindo, Japan) was added to each well, and the plates were incubated for an additional 4 h. The absorbance at 450 nm was measured using the microplate reader. The results were expressed as the proliferation index (PI), calculated based on the following formula: PI = OD450 nm for stimulated cultures/OD450 nm for non-stimulated cultures. 2.10. Determination of cytokine secreting levels The splenocytes (4×106 cells/well) were seeded on 12-well plates and stimulated with 10 µg mL-1 HA protein. After 48 h, the cell supernatants were collected by centrifugation and stored at -80°C. IL-4 and IFN-γ levels in the supernatants were measured by ELISA kits according to the manufacturer’s protocols. 2.11. Determination of cytokine secreting cells by ELISpot assay In brief, 96-well plates were incubated with RPMI 1640 containing 10% FBS for 30 min at room temperature. The splenocytes (2.5×105 cells/well) were seeded on the plates and stimulated with 10 µg mL-1 HA antigen. After 18 h for IFN-γ or 36 h for IL-4, IFN-γ or IL-4 secreting splenocytes were measured by ELISpot kits and counted with an AID iSpot Spectrum Reader. 2.12. Determination of memory T cell responses


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The splenocytes (1.5×106 cells/well) were seeded on 24-well plates and stimulated with 10 µg mL-1 HA antigen for 60 h. Then, the cells were centrifuged at 1,500 rpm for 5 min, and washed with PBS. Further, the cells were stained with the fluorochrome-conjugated anti-mouse antibodies: FITC-anti-CD4, PerCP-Cy5.5-anti-CD8α, PE-anti-CD44 and APC-anti-CD62L for 30 min. The cells were collected after centrifugation, resuspended with PBS, examined by the BECKMAN CytoFLEX. 2.13. Expression of MHC Ⅱ on DC cells in spleen The splenocytes (1×106 cells) were washed with PBS, and centrifuged at 1,500 rpm for 5 min. Then, the cells were stained with the fluorochrome-labeled anti-mouse antibodies FITC-anti-CD11c and PE-anti-MHC Ⅱ for 30 min. The cells were collected after centrifugation, resuspended with PBS, examined by the BECKMAN CytoFLEX. 2.14. Statistical analysis All results are expressed as mean ± SD. Statistical significance was determined by two-tailed Student’s t test for two-group analysis or one-way ANOVA for multiple group comparisons. Significance is indicated by p values of

Redox-Responsive Biodegradable Polycation Poly(amido amine

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