Polyethylenimine-Modified Fluorescent Carbon Dots As Vaccine

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Polyethyleneimine modified fluorescent carbon dots as vaccine delivery system for intranasal immunization Sha Li, Zhong Guo, Guandi Zeng, Yu Zhang, Wei Xue, and Zonghua Liu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00370 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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ACS Biomaterials Science & Engineering

Polyethyleneimine modified fluorescent carbon dots as vaccine delivery system for intranasal immunization Sha Li 1, Zhong Guo 1, Guandi Zeng 2, Yu Zhang 1, Wei Xue 1, Zonghua Liu 1, * 1

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

Biomedical Engineering, Jinan University, No. 601 West Huangpu Avenue, Guangzhou, 510632, China 2

Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes,

Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, No. 601 West Huangpu Avenue, Guangzhou 510632, China

* Corresponding author: Zonghua Liu ([email protected])

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ABSTRACT Fluorescent carbon dots (CDs) as a luminescent nano-material have obtained much attention in the biomedical field. To make good use of their luminescent property and nano-scaled size, CDs were developed as a vaccine delivery system for intranasal immunization in this work. To this aim, polyethyleneimine-modified CDs were prepared via a simple microwave method. Intranasal immunization was performed by using the CDs as an antigen carrier to deliver model protein antigen ovalbumin. The results showed that, the CDs as an intranasal vaccine delivery system enhanced the immunization efficacy by significantly increasing IgG titer, IgA induction in the local and distant mucous membrane sites, splenocyte proliferation, cytokine IFN-γ secretion by splenocytes, and memory T cells. From the results, the CDs could be used as vaccine delivery systems with the advantage of tracing the antigen transportation from administration site to the lymph organs.

KEYWORDS: fluorescent carbon dots, vaccine delivery systems, intranasal immunization, bio-imaging


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INTRODUCTION In the field of immunization prevention and therapy, the use of vaccine delivery systems can assist the delivery of antigens to achieve better immune response efficacy. The functions of vaccine delivery systems include protecting antigens from rapid degradation, rendering soluble antigens with particulate form, adjusting antigen delivery route, and releasing antigen in a controlled way, etc. Among them, tracking antigen delivery route is important to better understand the effect of antigen delivery route on the final immunization efficacy. To this aim, Korgel et al. prepared CuInSexS2−x/ZnS quantum dots as a bio-imaging agent to trace the transportation of poly(lactic-co-glycolic acid) microparticles for oral mucosal vaccine delivery in mice by encapsulating the quantum dots in the microparticles.1 This study put forward proof-of-principle in vivo tracing of vaccine delivery, but did not report the efficacy of the corresponding oral immunization. Fluorescent carbon dots (CDs) are a new generation of luminescent carbon nano-materials. Compared to conventional carbon nano-materials, the merits of CDs include tiny size, stable luminescence, strong anti-bleaching, easily-modified surface, good biocompatibility, and environmental friendliness. Replacing widely-used carbon quantum dots, CDs have gained much attention in biomedical fields for cellular imaging,2 bio-probing,3 and antibacterial materials.4 Moreover, nano-scaled CDs have also been used as drug or gene delivery systems.5-12 In these studies, the gene delivery function of CDs was well combined with their bio-imaging function, displaying a unique merit than ordinary gene carrier materials. By contrast, the utility of CDs as vaccine/antigen delivery systems has not been reported according to our literature search. We previously reported the blood compatibility evaluations of CDs and the conjugation of CDs



with hyperbranched polyglycerol to improve their bio-compatibility.13-14 In this work, fluorescent CDs were developed as vaccine delivery systems for intranasal immunization, in order to track vaccine delivery route and then better understand the effect of antigen transportation on the final immunization efficacy. To this aim, polyethyleneimine-modified CDs were prepared via a simple microwave method. Intranasal immunization was performed by using the CDs as an antigen carrier to deliver model protein antigen ovalbumin (OVA). This study could provide key information on the potential of bio-imaging agent fluorescent CDs as vaccine delivery systems.

EXPERIMENTAL Materials Chitosan, branched polyethyleneimine of 25kDa (BPEI25k), OVA, red blood cell lysis buffer, bovine serum albumin (BSA), Tween 20, the cholera toxin B subunit (CTB) and the polyinosinic-polycytidylic acid (poly(I:C)) were provided by Sigma-Aldrich (St. Louis, MO, USA). Preparation of Cationic Fluorescent CDs and CDs/OVA Nanoparticles Cationic fluorescent CDs were prepared via microwave-assisted pyrolysis of chitosan in the presence of BPEI25k, as illustrated in Figure 1. In brief, 125 µL of BPEI25k solution (200 mg/mL, dissolved in deionized water) and 5 mL of chitosan solution (10 mg/mL, dissolved in 1% v/v acetic acid solution) were mixed in a 50 mL beaker flask and stirred for 1 h for well mixing. The mixture was transferred into a microwave synthesis reactor and stirred under microwave radiation of 700 W at 180°C for 8 min to synthesize cationic fluorescent CDs. After cooling the reactor to room temperature, ultrapure water (10 mL) was added to the reactor, and the resulting solution was centrifuged at 5000 rpm for 10 min. The supernatant was loaded into a dialysis tube (MWCO 3500),


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and dialyzed against ultrapure water for three days. Then, the cationic CDs were collected by freeze drying. The cationic CDs (2 mg) or OVA (2 mg) were dissolved in 3 mL of phosphate buffered saline (PBS, pH7.4), respectively. CDs/OVA nanoparticles were prepared by mixing equal volume of the cationic CDs and OVA solutions. Characterization of the CDs and CDs/OVA Nanoparticles UV-visible absorption spectra of the CDs were recorded on a spectrophotometer (UV-2550, Shimadzu Corporation, Japan). Fluorescence emission spectra of the CDs were recorded by using a fluorescence spectrophotometer (Hitachi F-7000, Hitachi High-Technologies Corp., Japan). The chemical structures of the CDs were characterized by using transformed infrared spectrophotometer (FT-IR, BRUKE, VERTEX 70, Germany). The average hydrodynamic diameters and zeta potential of the CDs or CDs/OVA nanoparticles dispersed in ultrapure water were detected with a zetasizer analyzer (Malvern Instruments Ltd., UK). Antigen Internalization by DC2.4 Cells DC2.4 cells were cultured overnight in 24-well (1×105 cells/well) chamber slides. After that, the cells were washed and then incubated with PBS, OVA-Cy5, CDs/OVA-Cy5 or CDs suspensions (in RPMI 1640 medium). Then, the cells were washed and observed with a confocal laser scanning microscope (CLSM 700, Zeiss, Germany). Persistence of the CDs/OVA Nanoparticles in Nasal Cavity OVA was fluorescently labeled with Cy5.5 to track the OVA administered into test mice. Six-week-old female Balb/c mice (n=3 for each group) were intranasally administered by dropping 30 µL of OVA-Cy5.5, CDs/OVA-Cy5.5, CTB/OVA-Cy5.5 or poly(I:C)/OVA-Cy5.5 suspensions (10



µg OVA for each mouse) under anesthesia. Then, the Cy5.5 fluorescence signals were detected by using an in vivo imaging system (PerkinElmer, USA). Immunohistochemical Analysis Six-week-old female Balb/c mice were intranasally administered by dropping 30 µL of OVA or CDs/OVA suspensions (10 µg OVA for each mouse) under anesthesia. After 1 and 6 h, the mice were euthanized and the nasal cavities were separated for immunohistochemical assay. The nasal cavities were fixed with 10% formalin at 4°C for 24 h, and were decalcified immediately with ethylene diamine tetraacetic acid disodium salt solution for one month. The nasal cavity tissues were paraffin-embedded, sectioned into 4 mm slices and mounted on glass slides. After deparaffinage, rehydration and antigen retrieval process, the sections were acquired for immunohistochemical staining. Firstly, the sections were incubated with 10% goat serum (in PBS) for 0.5 h to prevent the non-specific binding of antibody. Subsequently, the sections were treated for 2 h with primary rabbit anti-OVA antibody (Life Span BioSciences, Inc., Seattle, USA), and then incubated for 1 h with goat anti-rabbit IgG antibody-horseradish peroxidase (HRP) (BOSTER, Wuhan, China). The sections were restained with hematoxylin and mounted. These slides were observed and recorded using an optical microscope (ZEISS Axio Observer A1, Germany). Animal Immunization Female Balb/c mice (6-8 weeks old, five mice/group, purchased from Southern Medical University, Guangzhou, China) were intranasally immunized with 30 µL suspensions of free OVA, CDs/OVA, CTB/OVA or poly(I:C)/OVA in saline (10 µg OVA per mouse). All the mice were vaccinated thrice with 10 days intervals. Seven days post the third immunization, the mice blood was collected, placed at room temperature for 1 h, and then subject to centrifugation for 5 min at


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1000 rpm to obtain sera. The sera were kept at -20°C for antibody (IgG, IgG1, and IgG2a) analysis. The mice were sacrificed under anesthesia. The spleens and nasal/genital wash samples were collected. The nasal/genital wash samples were centrifuged for 5 min at 13,000 rpm. The resulting supernatants were kept at -20°C for antibody IgA analysis. The splenocytes were obtained by grinding the spleens for further measurements. Assessment of Serum IgG/Isotypes and Mucosal IgA Antibody Titers The titers of OVA-specific serum antibodies (total IgG, IgG1 and IgG2a isotypes) or mucosal antibody IgA were measured by using enzyme-linked immuneosorbent assay (ELISA). Briefly, 96-well ELISA plates were sensitized with 0.1 mL OVA solution (10 µg/mL, in 0.1 M carbonate buffer) at 4°C overnight. After washing with PBS-Tween 20 (PBS-T, containing 0.05% Tween 20), the plates were treated with 2% BSA solution for 1 h at 37°C, and then washed with the PBS-T. The plates were coated for 2 h at 37°C with the serial dilutions of the tested samples in the blocking buffer, washed again, and incubated for 1 h at 37°C with HRP-conjugated anti-mouse IgG, IgG1, IgG2a (2000-fold dilution) or IgA (1000-fold dilution) antibody (Abcam, Cambridge, USA). Finally, the detection of antigen-antibody complexes was performed by adding 100 µL TMB (BD, San Diego, USA) solution to each well. After reacting for 15 min, 0.1 mL of H2SO4 solution (2 M) were used to prevent the enzymatic reaction, and the optical absorbance at 450 nm was read by using a Thermo Fisher Scientific microplate reader (USA). Assessment of Splenocyte Proliferation The splenocytes (2×106 cells/mL, 100 µL/well) were seeded on a 96-well plate and re-stimulated with OVA antigen (10 µg/mL) for 3 days. After the re-stimulation, the cell counting kit 8 reagent (Dojindo, Japan) was added to the plate for 1-4 h of incubation. Then, the absorbance



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of each well at 450 nm was detected using the microplate reader. The splenocytes non-restimulated with the antigen of OVA were considered as a negative control. Finally, the splenocyte proliferation index (PI) was calculated according to the formula: PI=OD450 (restimulated wells)/OD450 (non-restimulated wells). Measurement of the Levels of the Cytokines Secreted by the Splenocytes The splenocytes (2×106 cells/mL, 2 mL/well) were seeded on a 12-well plate and restimulated with OVA antigen (10 µg/mL) for 60 h. After that, the supernatants were separated by centrifugation. IFN-γ and IL-4 levels were determined with ELISA kits (Biolegend, San Diego, CA, USA). ELISpot Analysis of the Cytokine-Secreting Splenocytes The splenocytes (2.5×106 cells/mL, 100 µL/well) were cultured in a 96-well plate that was pre-treated for 0.5 h with RPMI 1640 medium (containing 10% FBS). Then, 100 µL of OVA solution (10 µg/mL) was put to each well to restimulate the splenocytes for 18 h (IFN-γ) or 36 h (IL-4) at 37°C. The splenocytes non-restimulated with OVA were treated as a negative control. After the incubation, IFN-γ- or IL-4-secreting splenocytes were measured with ELISpot kits (Mabtech A B, Nacka Strand, Sweden) and recorded with an iSpot Spectrum Reader (AID, Germany). Memory T Cells The splenocytes (1×106 cells/mL) were seeded on a 24-well plate and restimulated with OVA antigen (10 µg/mL) for 60 h. After that, the cells were harvested and stained for 0.5 h with fluorochrome-labeled

anti-mouse

antibodies

FITC-anti-CD4,

PerCP-Cy5.5-anti-CD8α,

PE-anti-CD44 and APC-anti-CD62L (eBioscience, San Diego, CA, USA). Then, the cells were washed and detected by using a flow cytometry (Beckman CytoFLEX, Germany). Data Analysis


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The obtained data were expressed as the mean ± standard error of the mean (SEM). The data were analyzed by using GraphPad Prism 5 software (San Diego, CA, USA) by two-sided student’s t-test (95% confidence level). A probability value (p)

Polyethylenimine-Modified Fluorescent Carbon Dots As Vaccine

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