Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via intra- and paracellular pathways

    Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via intra- and paracellular pathways Man Li, Mengnan Zhao, Yao Fu, You Li, Tao Gong, Zhirong Zhang, Xun Sun PII: DOI: Reference:

S0168-3659(16)30107-9 doi: 10.1016/j.jconrel.2016.02.043 COREL 8156

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

28 October 2015 30 January 2016 27 February 2016

Please cite this article as: Man Li, Mengnan Zhao, Yao Fu, You Li, Tao Gong, Zhirong Zhang, Xun Sun, Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via intra- and paracellular pathways, Journal of Controlled Release (2016), doi: 10.1016/j.jconrel.2016.02.043

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ACCEPTED MANUSCRIPT Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via intra- and paracellular pathways Man Li, Mengnan Zhao, Yao Fu, You Li, Tao Gong, Zhirong Zhang, Xun Sun*

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Key Laboratory of Drug Targeting, Ministry of Education, West China School of Pharmacy,

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Sichuan University, Chengdu, 610041, People’s Republic of China

*Corresponding author: Xun Sun

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Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17. Section 3. Southern Renmin Road, Chengdu 610041, People’s Republic of China.

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Tel: 86-28-8550 2037; Fax:86-28-85501615

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Email: [email protected] (X.Sun)

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ACCEPTED MANUSCRIPT Abstract Facing the threat of highly variable virus infection, versatile vaccination systems are urgently

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needed. Intranasal mRNA vaccination provides a flexible and convenient approach.

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However, the nasal epithelium remains a major biological barrier to deliver antigens to nasal

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associated lymphoid tissue (NALT). To address this issue, a potent polymer-based intranasal mRNA vaccination system for HIV-1 treatment was synthesized using cationic cyclodextrin-polyethylenimine 2k conjugate (CP 2k) complexed with anionic mRNA

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encoding HIV gp120. The delivery vehicle containing CP 2k and mRNA overcame the

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epithelial barrier by reversibly opening the tight junctions, enhanced the paracellular delivery of mRNA and consequently minimized absorption of toxins in the nasal cavity. Together with the excellent intracellular delivery and prolonged nasal residence time, strong system and

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mucosal anti-HIV immune responses as well as cytokine productions were achieved with a balanced Th1/Th2/Th17 type. Our study provided the first proof of evidence that cationic

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polymers can be used as safe and potent intranasal mRNA vaccine carriers to overcome the nasal epithelial barrier. The safe and versatile polymeric delivery system represents a

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promising vaccination platform for infectious diseases.

Graphic Abstract

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ACCEPTED MANUSCRIPT The self-assembled nanocomplex formulated with CP 2k could facilitate the delivery of mRNA vaccine through intracellular and paracellular pathways, and consequently induce

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both systemic and mucosal immune responses.

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Key Words:

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mRNA vaccination, CD-PEI, nasal epithelial barrier, intracellular delivery, paracellular delivery

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1. Introduction

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Vaccination has by far been the most effective approach to prevent the rapid spread of infectious diseases, such as human immunodeficiency virus infection and acquired immune

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deficiency syndrome (HIV/AIDS) [1-3]. Great efforts have been contributed to the design of

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HIV vaccines, and some showed promising results [4, 5]. Nonetheless, the development of potent vaccine still faces great challenges due to the high mutation rate and viral diversity of

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infectious diseases. In immunotherapy, a universally applicable vaccine may no longer provide sufficient protection due to the unique mutation frequently occurring in patients.

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Vaccination with mRNA offers a viable solution to this problem, which can be individually tailored and rapidly manufactured on demand [6]. Unlike plasmid DNA, mRNA can transfect both dividing and non-dividing cells without entering the nucleus, resulting in higher gene expression [7-10]. Techniques for effectively transfecting mRNA into cells have led to the development of various mRNA-based vaccination systems [11-13]. For infectious diseases with high mutation rates such as influenza, mRNA vaccine can be produced reliably in a scalable process thus allowing quick response to emergence of pandemic strains and consequently induce long-lived and protective immunity [14]. Therefore, mRNA vaccines are of great significance in the prevention of variable virus infection [15]. Currently, mRNA vaccines are injected intravenously [16], subcutaneously [17], or intradermally [18] in

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ACCEPTED MANUSCRIPT preclinical and clinical studies, which requires specially trained personnel. Also, the injection-associated pain and acute infections render the administration of mRNA less

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patient-friendly, which highlights the need to explore non-invasive routes of administration

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for mRNA vaccine delivery.

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The U.S. Food and Drug Administration approved the first intranasal vaccine against influenza (FluMist®) in 2003 [19]. As an alternative to systemic vaccine delivery, nasal immunization represents an effective and safe approach as compared to injection. The nasal

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cavity is the portal of entry for many pathogens, and nasal-associated lymphoid tissue

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(NALT) is rich in antigen-presenting cells (APCs), T and B lymphocytes [20], making NALT a suitable site for antigen internalization and subsequent mucosal and systemic immune

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responses [21]. Moreover, intranasal vaccination can induce immune response in distal

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mucosal tissues such as in the vagina, because most mucosal lymphocytes are functionally connected [22]. This may be particularly helpful when vaccinating against sexually

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transmitted pathogens such as HIV. Despite these advantages, intranasal mRNA vaccination faces following obstacles: the presence of enzymes in the nasal cavity may degrade mRNA;

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cilia movement may accelerate the clearance of mRNA antigen and shorten its nasal residence time, resulting in poor antigen uptake. Although the nasal cavity contains APCs, the nasal epithelium presents a major barrier to antigen delivery to these cells. Thus, overcoming the nasal epithelial barrier is the key to achieve enhanced immune responses. To ensure efficient delivery to APCs, multiple routes can be explored including (i) intracellular route via transfection of epithelial cells [23], (ii) transcytotic route of delivery via M cells to APCs such as dendritic cells and macrophages, and (iii) paracellular route of delivery via tight junctions to reach underlying APCs [24]. Particulate delivery systems are proven efficient carriers for vaccine delivery [25], and the most frequently used systems are lipoplex [26, 27] and liposomes [28-31]. While these

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ACCEPTED MANUSCRIPT lipid-based nano carriers can deliver mRNA efficiently, the epithelial barrier remains a challenge to intranasal delivery of mRNA vaccines. Thus, overcoming the epithelial barrier

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will likely lead to enhanced uptake of mRNA vaccine and stronger immune responses.

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However, lipid-based carriers alone are unable to open the epithelial tight junctions to induce

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immune responses. To solve this problem, polymeric delivery systems offer a viable solution. Polyethyleneimine (PEI) with a molecular weight of 25 kDa was proven to open the epithelial tight junctions [32], and has been shown as an efficient mRNA carrier in in vitro transfection

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[33]. Nevertheless, PEI 25k-mRNA complexes showed poor colloidal stability and high

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cytotoxicity in mouse fibroblasts [33]. To overcome the epithelial barrier and enhance the mucus binding efficiency of PEI 25k, structural modification of low-molecular-weight PEI

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with bioadhesive sugar moieties represents a feasible approach. Cyclodextrin, as a

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biocompatible pharmaceutical excipient, has been selected to modify PEI 2k. Cyclodextrin was demonstrated to cause minimum damage to mucosal integrity and enhance drug

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permeation via the nasal epithelium by transiently and reversibly opening tight junctions [34, 35]. Moreover, linking cyclodextrin to PEI lowered the charge density of polyamine

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backbone, and consequently reduced the cytotoxicity of cationic PEI polymer while maintained a large number of protonatable groups, which led to high gene-delivery efficiency of DNA [36]. Based on these properties, we assume that cyclodextrin-PEI (CP) polymer may serve as an efficient carrier for nasal mRNA vaccine delivery. Here, we report the synthesis of CP polymer and the fabrication of a CP polymer-based intranasal delivery platform for mRNA vaccine. The HIV glycoprotein 120 (gp120) was selected as a model antigen. CP 2k polymer and mRNA formed nanoscale complexes via electrostatic interactions. We compared the ability of CD-PEI 2k (CP 2k) or PEI 25k to deliver mRNA encoding gp120 and to induce gp120-specific immune responses. These experiments were carried out in cultures of DC 2.4 cells, Madin-Darby canine kidney

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ACCEPTED MANUSCRIPT (MDCK) cells and Calu-3 cells to simulate the nasal epithelium. The in vivo interaction between this delivery vehicle and the epithelial tight junctions, as well as immune responses

Materials and methods

2.1 Materials and animals

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2.

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in mice intranasally immunized with mRNA complexes were investigated.

Branched polyethylenimine (PEI) with molecular weights of 2 or 25 kDa, β-cyclodextrin molecular

weight

1135),

1,1’-carbonyldiimidazole

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(CD,

(CDI),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and chitosan (CS,

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190-300 kDa, 75-85% deacetylated) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Restriction enzymes were obtained from Fermentas (Thermo Fisher, USA). All other

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reagents were of analytical purity.

China)

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in

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specific

pathogen-free,

light-cycled

and

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(Chengdu,

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BALB/c (female) mice 6-8 weeks old were obtained from Dashuo Biotechnology

temperature-controlled facility. All experiments were approved by the Institutional Animal

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Care and Ethics Committee of Sichuan University.

2.2 Synthesis of cyclodextrin-polyethylenimine conjugates CP 2k was prepared as reported with some modifications [37]. Briefly, 12 ml of N, N-dimethylformanide (DMF) containing β-cyclodextrin (0.84 g, 0.74 mmol) and CDI (1.60 g, 10.4 mmol) were stirred at room temperature for 1.5 h under nitrogen. The resulting CDI-CD was purified by precipitation with cold diethyl ether and filtered to remove soluble impurities. The product was dissolved in 10 ml of dimethylsulfoxide (DMSO) and stored at 4°C. Then CDI-CD in 10 ml DMSO and 0.6 ml triethylamine (Et3N) were added dropwise with stirring over 1.5 h to 18 ml of DMSO containing PEI 2k (9.0 g). Then the reaction was allowed to

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ACCEPTED MANUSCRIPT stand another 5 h. The resulting solution was dialyzed against distilled water for 3 days and

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lyophilized overnight.

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2.3 In vitro transcription of mRNA

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The DNA sequence of HIV gp120 (GenBank DQ667594.1) was synthesized by Sangon (Shanghai, China); a Kozak consensus sequence was inserted, as were Hind III and Bam HI restriction sites at the ends. The sequence was then cloned into the pVAX1 vector (Life

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Technologies, USA), which contains a T7 promoter and a poly(A) tail. A plasmid expressing the luciferase reporter gene was constructed by inserting the luciferase coding sequence into

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the pVAX1 backbone. The recombinant plasmid was amplified and purified using an endo-free plasmid kit (Omega, USA) and linearized using Xba I. Synthetic mRNA was

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prepared by in vitro transcription using the T7 high yield RNA synthesis kit and an

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m7G(5')ppp(5')G RNA cap structure analog (New England Biolabs, MA, USA) according to

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the manufacturer’s instructions. The mRNA product was precipitated with phenol/chloroform and re-suspended in RNase-free water. The concentration of mRNA was determined by

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measuring the absorbance at 260 nm. After incubating the purified transcription products at 70°C for 10 min, mRNA purity and size were analyzed by running an aliquot together with RiboRuler High Range RNA Ladder (Fermentas, Thermo Fisher, USA) on agarose gels and staining with SYBR green II dye. Labeling of mRNA with Cy3 or fluorescein was performed using the Label IT nucleic acid labeling kit (Mirus Bio, WI, USA) according to the manufacturer’s protocol.

2.4 Formulation and characterization of CP 2k/mRNA Cationic CP 2k was dissolved in sterile, distilled RNase-free water to a concentration of 1 mg/ml. The CP 2k/mRNA delivery vehicle was formed by adding pre-diluted CP 2k to

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ACCEPTED MANUSCRIPT pre-diluted mRNA at N/P ratios of 8, 16 or 24 (calculated as the molar ratio of nitrogen in PEI portion of CP 2k/phosphate in RNA). The mixture was vortexed for 10 sec, then

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incubated at room temperature for 20 min to allow particles to form. Size distribution and

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zeta potential of the delivery vehicle were measured by photon correlation spectroscopy using

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a Zetasizer Nano ZS90 (Malvern Instruments, UK). Samples were diluted to 1 ml using RNase-free distilled water and equilibrated for 30 sec before measuring at a fixed angle. Measurements were made in triplicate.

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CP 2k/mRNA morphology was analyzed by transmission electron microscopy (TEM,

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H-600, Hitachi, Japan). Samples were loaded on a copper grid and stained with phosphotungstic acid (1%) for 20 sec before observation. The ability of CP 2k to bind RNA was checked using agarose gel electrophoresis. CP

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2k/mRNA complexes were prepared as described above with 1 μg mRNA, mixed with 5 × RNA loading buffer (Tiangen, Shanghai, China), and electrophoresed on a 4% agarose gel.

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The gel was run at 120 V for 30 min and then visualized and photographed using the ChemiDoc™ XRS system (Bio-Rad, Hercules, CA).

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To assess the ability of CP 2k to protect the complexed mRNA from RNase degradation, RNase (Sigma, USA) was incubated with naked gp120 mRNA (10 μg) or the equivalent amount of mRNA complexed with CP 2k at the optimal ratio N/P 16. At the indicated time points, EDTA was added to a final concentration of 10%, and the mixture was denatured at 65°C for 5 min. Then mRNA was released from the CP 2k/mRNA by adding 30 U of heparin, and the released mRNA was isolated by phenol/chloroform precipitation. Levels of gp120-mRNA were measured as Ct values in quantitative Real-Time PCR (qRT-PCR) using the SsoFastTM EvaGreen Supermix on an iCycler iQTM 5 system (Bio-Rad, USA).

2.5 Cell culture

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ACCEPTED MANUSCRIPT Madin-Darby canine kidney (MDCK) cells, Calu-3 human lung adenocarcinoma cells and DC 2.4 murine dendritic cells were obtained from the cell bank at the Chinese Academy

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of Science (Shanghai, China). Cells were maintained in Dulbecco’s Modified Eagle Medium

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High Glucose containing L-glutamine, or in RPMI 1640 medium (Hyclone, Life Technology,

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USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), and 1% penicillin/streptomycin.

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2.6 In vitro transfection

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DC 2.4, Calu-3 and MDCK cells were transfected with mRNA encoding luciferase (mLuc), which was produced by in vitro transcription as described above. CP 2k/mLuc

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containing 1 μg luciferase mRNA was prepared at N/P ratios of 8, 16 or 24 (calculated as the

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molar ratio of nitrogen in PEI portion of CP 2k to phosphate in DNA). In parallel, PEI 25k/mLuc was prepared at N/P ratios of 8 or 16. Cells were seeded in 24-well plates at a

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density of 1 × 105 cells/well and incubated for 24 h before transfection. Cells were incubated in serum-free medium for 4 h after transfection, the cell culture medium was replaced with

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0.5 ml of fresh complete culture medium, and the cells were incubated another 20 h at 37°C in a 5% CO2 incubator. Luciferase expression was measured using a luciferase assay system (Promega, WI, USA) following the manufacturer’s instructions. The total protein concentration in transfected cells was determined using the BCA protein assay kit (Pierce, Thermo Fisher, MA, USA).

2.7 Endosome escape assay DC 2.4 cells were seeded on coverslips in 6-well culture plates for 24 h, then treated with CP 2k/fluorescein-labeled mRNA (2 μg/well). At 2 or 6 h, cells were washed three times with phosphate-buffered saline (PBS) containing 0.015% (w/v) heparin and stained with

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ACCEPTED MANUSCRIPT LysoTracker (Beyotime, China) for 1 h at 37°C. Then samples were washed with PBS and

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observed using a confocal laser scanning microscope (FV1000, Olympus, USA).

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2.8 Nasal residence time

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CP 2k or PEI 25k were complexed with Cy3-labeled mRNA, and administered intranasally to mice under anesthesia with intraperitoneal injection of 1% (w/v) sodium pentobarbital (5 μg Cy3-mRNA per animal). In parallel, some animals were given naked

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Cy3-mRNA. All mice were scanned at 0, 0.5, 1, 2 and 3 h after administration using an

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IVIS® Spectrum imaging system (Caliper Life Sciences, Hopkinton, MA, USA). Absolute fluorescence in the nasal cavity was quantified over time using an excitation wavelength of

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548 nm and an emission wavelength of 562 nm. Relative fluorescence was expressed as the

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2.9 In vivo uptake

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percentage of the initial fluorescence in the nasal cavity.

CP 2k or PEI 25k containing 5 μg Cy3-labeled mRNA were administered intranasally to

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mice under anesthesia with intraperitoneal injection of 1% (w/v) sodium pentobarbital. As a control, some animals were given naked Cy3-mRNA. At 6 h after administration, mice were sacrificed by neck dislocation, and nasal epithelial cells were isolated as described [38]. Cells were digested by incubating for 2 h at 37°C with type IV collagen. Mouse palates were excised using a No. 22 scalpel blade, the palates were gripped behind the incisor teeth with fine forceps and gently pulled toward the molar teeth. The NALT was teased gently into the medium to release cells. After washing nasal epithelial cells and NALT cells with PBS, the cells were centrifuged, re-suspended in PBS, and analyzed by flow cytometry to measure uptake of Cy3-mRNA.

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ACCEPTED MANUSCRIPT 2.10 In vivo toxicity To investigate whether long-term intranasal administration of the polymer/mRNA

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complex can cause absorption of toxins present in the nasal cavity, toxicity was analyzed in

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vivo using a procedure similar to that described [39]. Female BALB/c mice weighing

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18.0-22.0 g were randomly divided into five groups (five mice/group). Mice received once-daily intranasal doses of CP 2k/mRNA or PEI 25k/mRNA (10 μg mRNA, optimal ratio N/P 16) or double-distilled water, followed by LPS (5 mg/kg) on days 1, 3, 5 and 7. Negative

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control animals received no treatment. Positive control animals received one intraperitoneal

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injection of LPS (5 mg/kg). Blood samples were collected via retro-orbital puncture on day 8.

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Livers were removed and sectioned for histology analysis.

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2.11 Influence on tight junctions in an in vitro epithelial cell model The effect of different polymer/mRNA complexes on tight junction opening were

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analyzed on in vitro MDCK cell model. Briefly, 2 × 105 MDCK cells were seeded into the apical chamber of 24-well transwell inserts (3 μm Transwell® inserts, Corning, USA) and

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incubated in a 5% CO2 incubator at 37°C with refreshing of apical and basolateral media every 2 days. The transepithelial electrical resistance (TEER) was monitored using Millipore ERS Ohm meter (Millipore, USA). The tight junction formation was identified when the TEER reached 200 Ω·cm2. Complete medium in both apical and basolateral chambers were replaced with serum free media for 30 min before experiment. The mRNA complexes were prepared with 1 μg luciferase encoding RNA at the optimal ratio with CP 2k or PEI 25k and added into the apical chamber with serum free media. Chitosan (CS, 0.02 mg/ml) was used as positive control. After 1h of incubation, both apical and basolateral chambers were washed with Hank’s balanced salt solution (HBSS) and cells were cultured with complete culture media for another 11 h. For quantitative analysis, cells were collected at 1, 6 and 12 h; total

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ACCEPTED MANUSCRIPT RNA was extracted and levels of ZO-1 mRNA were assayed by qRT-PCR. Besides, the TEER were monitored at prearranged time points to evaluate the recovery of epthelial tight

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junctions (n = 3).

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2.12 Animal immunization

Female BALB/c mice aged 6-8weeks were randomized into 5 groups (5 animals per group), and immunized twice with mRNA encoding HIV gp120 at an interval of 2 weeks.

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Mice were anesthetized with 1% (w/v) sodium pentobarbital and given intranasal doses of CP

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2k/mRNA, PEI 25k/mRNA or naked mRNA (10 μg mRNA/mouse). As negative controls, mice received no treatment or unrelated mRNA encoding luciferase complexed with CP 2k,

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which excludes effects of RNA-mediated immune stimulation. Mice were held upright for

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some time to ensure maximal dosing and to prevent swallowing. At 10 days after the second

analyzed.

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immunization, all mice were sacrificed by neck dislocation, and the immune response was

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2.13 Serum and mucosal antibody response ELISA was used to measure serum levels of antigen-specific antibodies (IgG total, IgG1 and IgG2a), as well as levels of secreted IgA antibodies in nasal and vaginal mucosal washes. Polystyrene 96-well plates were coated overnight at 4°C with 100 μl/well of 1 μg/ml HIV gp120 Con_B (Immune Technology, Suzhou, China). Plates were washed with PBS containing 0.1% Tween-20 (PBST), then incubated with 100 μl of serial dilutions of serum or mucosal washes; dilutions for assay of IgG, IgG1 and IgG2 arranged from 1:8 to 1:512. Plates were washed, incubated with 100 μl of horseradish peroxidase-conjugated anti-mouse IgG antibody (diluted 1:10000 for IgG or 1:5000 for IgG1 and IgG2a) or IgA antibody (diluted 1:5000). TMB substrate (BD PharMingen, CA, USA) was added and the reaction

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ACCEPTED MANUSCRIPT was stopped with 2M H2SO4. Optical density was measured at 450 nm using a microplate

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reader (VarioSkan, Thermo Fisher Scientific, MA, USA).

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2.14 Cytotoxicity T lymphocyte (CTL) assay

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An in vivo CTL killing assay was used to measure the cytotoxicity of antigen-specific CD8+ T cells [40]. Splenocytes were isolated from naïve mice. Red blood cells were lysed using ACK buffer, and the cell suspension was divided equally into two aliquots. One was

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incubated for 2 h at 37°C with 2 μg/ml HIV gp120 peptide and the other with medium only.

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Then the peptide-pulsed cells were stained for 10 min at 37°C with 2 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, CA, USA), while the non-pulsed cells were stained with 0.2 μM CFSE. CFSE labeling was then quenched by addition of FBS to a final

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concentration of 20% (v/v). The two aliquots (1 × 107 cells/100 μl) were mixed equally, and the cell suspension was injected into the tail vein of untreated (control) or immunized mice.

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After 18 h, splenocytes from recipient mice were prepared and analyzed by flow cytometry. The percentage of specific lysis was calculated as follows:

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  ratio of CFSE low / CFSE high cells re cov ered from naive mice   Specific Lysis  %   100  1    low high   ratio of CFSE / CFSE cells re cov ered from immunized mice  

2.15 Intracellular cytokine staining Ten days after second immunization, mice were sacrificed by neck dislocation. Splenocytes were harvested by homogenizing through a 70-μm cell strainer (BD Falcon) in 10% RPMI 1640 medium, and red blood cells were lysed using ACK lysis buffer (0.15 M NH4Cl, 10.0 mM KHCO3, 0.1 mM EDTA, pH 7.4). Splenocytes were washed, cultured in RPMI 1640 medium supplemented with 10% FBS, 2-mercaptoethanol and 2 μg/ml HIV gp120

peptide

sodium

from

the

consensus

B

V3

region

(TRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAH; Kaijie, Chengdu, China). After 1-h 13

ACCEPTED MANUSCRIPT incubation, brefeldin A (eBioscience, CA, USA) was added to each sample to a final concentration of 1 μg/ml and incubated another 5 h at 37°C. Cells were washed, then

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incubated at 4°C with flourescein isothiocyanate (FITC)-conjugated anti-mouse CD8a

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antibody or FITC-conjugated anti-mouse CD4 antibody (eBioscience). Cells were washed

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again, fixed, and permeabilized with Fixation and Permeabilization Buffers (eBioscience). Cells were centrifuged, then incubated in permeabilization buffer containing either polyethyleneimine (PE)-conjugated anti-mouse IFN-γ antibody or PE-conjugated anti-mouse

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IL-4antibody. Cells were washed, diluted in PBS containing 1% BSA, and analyzed by

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two-color flow cytometry (Beckman Coulter, CA, USA). Percentages of cells positive for CD8+/IFN-γ+ and CD4+/IL-4+were determined using Kaluza software (Beckman Coulter, CA,

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USA).

2.16 Cytokine production by ELISA

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IFN-γ and IL-4 production in splenocytes culture supernatants and NALT supernatants were measured by ELISA (BD PharMingen, CA, USA) according to the manufacturer’s

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protocol. The splenocytes isolated from immunized mice were cultured in RPMI 1640 medium with supplementary 10% FBS in 96-well plates (1 × 105 cells/well) with the presence of 2 μl/ml HIV gp120 peptide and incubated for 72 hours. Then the supernatants were collected by centrifugation. Each sample was examined in duplicate. To analyze the cytokine production in NALT culture supernatant, the palate of immunized mice were isolated. After washing with RPMI 1640 medium supplemented with 10% FBS, the palate was incubated with 200 μl RPMI 1640 medium in 96 well plate. After 48 hours’ incubation, the supernatant was collected to determine the content of IFN-γ and IL-4 produced by NALT cells.

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ACCEPTED MANUSCRIPT 2.17 Cytokine productions by qRT-PCR Quantitative RT-PCR (qRT-PCR) was used to assay production of Th1 cytokines (IFN-γ

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and IL-2), Th2 cytokines (IL-4 and IL-10), Th17 cytokine (IL-17) and type I IFN cytokines

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(IFN-α4 and IFN-β1) by splenocytes from immunized mice. Total RNA was extracted from

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spleens using an RNA isolation kit (TianGen, China), it was reverse-transcribed using the TIANscript RT kit, and cytokine mRNA levels were analyzed using SsoFast TM EvaGreen Supermix on an iCycler iQTM 5 system (Bio-Rad, USA). β-actin served as the internal

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control.

2.18 Statistical analysis

Experiments were performed at least in triplicate unless otherwise noted. The data are

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shown as the mean ± SD. Statistical difference was analyzed by Student’s t test and one-way ANOVA with Bonferroni post hoc test. All tests are accepted as statistically significant when

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3. Results

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the p value is less than 0.05.

3.1 Synthesis and characterization of CP 2k polymer β-cyclodextrin-PEI 2k (CP 2k) was synthesized (Figure 1A) with a molar ratio of cyclodextrin:PEI of 0.36:1, and the structure was verified by 1HNMR. Signals from ethylene protons in PEI (–CH2CH2NH–) appeared at 2.4–3.0 ppm. Signals assigned to the hydrogens C1 and C2-C6 of β-cyclodextrin were observed at 5.1 and 3.0-4.0 ppm, respectively (Supporting Information Figure S1). The molecular weight of CP 2k was 80 kDa based on gel permeation chromatography.

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Figure 1. Synthesis of CP 2k and formation of CP 2k/mRNA. (A) Synthesis of CP 2k. (B) Formation of CP 2k/mRNA. CP 2k and mRNA were dissolved in sterile, distilled RNase-free water. Particles were formed by electrostatic interaction.

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3.2 Formulation and characterization of the CP 2k/mRNA

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HIV gp120 mRNA was transcribed and identified (Figure 2A). CP 2k and HIV gp120 mRNA were complexed by electrostatic interaction (Figure 1B). CP 2k completely

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condensed mRNA even at an N/P of 8, where N/P refers to the molar ratio of nitrogen in PEI to phosphate in DNA. This complete condensation was observed as slowed mobility on

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agarose gels in the absence of any free mRNA band (Figure 2B). The optimal ratio of CP 2k to mRNA was found to be N/P 16, based on an in vitro transfection assay. The average size of CP 2k/mRNA was 117.3 ± 3.44 nm at N/P 16; and they showed spherical morphology with a ζ-potential of 26.4 ± 2.8 mV (Figure 2C and Table 1). The ability of CP 2k to protect the mRNA from RNases was measured using qRT-PCR. Even after 4 h incubation with RNase, a low Ct value was detected for CP 2k/mRNA , indicating a relatively high gp120 mRNA level (Figure 2D). These results suggest that the delivery vehicle could protect mRNA from degradation in the nasal cavity.

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Figure 2.Characterization of the CP 2k/mRNA . Particles were formed by adding mRNA solution to a CP 2k solution at N/P ratios 8, 16 and 24 (calculated as the molar ratio of nitrogen in PEI portion of CP 2k/phosphate in DNA) with gentle mixing. (A) Characterization of the 1600-nt (nucleotides) mRNA by gel electrophoresis. (B) Gel retardation assay to detect condensation of mRNA into CP 2k at different N/P ratios. (C) Size distribution and morphology of CP 2k/mRNA formed at an N/P ratio of 16. The optimal ratio was determined by in vitro transcription, and morphology was examined by transmission electron microscopy. Scale bar, 200 nm. (D) Ability of CP 2k to protect mRNA from nucleases. Heparin was used to release mRNA from complexes, and mRNA levels were determined using qRT-PCR targeting the gp120 coding region (n = 3). Table 1. Size and ζ potential of CP 2k/mRNA complex at different N/P ratios (n=3) Size (nm)

PDI

ζ potential (mV)

CP2k:mRNA (N/P=8)

137.2 ± 5.43

0.203 ± 0.031

13.1 ± 2.3

CP2k:mRNA (N/P=16)

117.3 ± 3.44

0.161 ± 0.028

26.4 ± 2.8

CP2k:mRNA (N/P=24)

112.4 ± 3.56

0.189 ± 0.025

27 ± 3.1

3.3 Enhanced mRNA transfection by CP 2k/mRNA delivery vehicle To assess the transfection efficiency of the CP 2k/mRNA complex, mRNA encoding luciferase (mLuc) was used, and complexes were transfected into DC 2.4 murine dendritic cells; MDCK epithelial cells, which form tight junctions [41]; and Calu-3 bronchial epithelial cells [42]. The CP 2k/mLuc complex efficiently transfected all three cell lines, and the

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ACCEPTED MANUSCRIPT highest luciferase expression was achieved at N/P 16. Under these conditions, luciferase expression was significantly higher than that observed with PEI 25k/mLuc in all three cell

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lines (p < 0.01; Figure 3A and 3B; Supporting Information Figure S2). In addition, CP

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2k/mLuc (N/P 16) was less cytotoxic than PEI 25k/mLuc to DC 2.4 and MDCK cells, based

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on MTT assays (Supporting Information Figure S3).

After internalization, CP 2k/mRNA was expected to escape from the endo/lysosome to release mRNA cargo into the cytoplasm. To examine this process, we used confocal laser

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scanning microscopy to track fluorescein-labeled mRNA (green fluorescence) complexed

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with CP 2k. At the same time, we localized endo/lysosomes using LysoTracker (red fluorescence). Co-localization of mRNA and endo/lysosomes was detected as yellow

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fluorescence. After 2 h incubation following transfection into DC 2.4 cells, most

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fluorescein-mRNA co-localized with endo/lysosomes. After 6 h incubation, more green fluorescence was observed in the cytoplasm than in the endo/lysosome (Figure 3C), which

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indicates that CP 2k/mRNA escaped from the endo/lysosome after internalization thus facilitating the expression of mRNA in the cytoplasm. The successful endosomal escape of

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our delivery vehicle may reflect the potent buffering capacity of PEI, which allows mRNA to escape via the “proton sponge” mechanism [43] by increasing ionic concentration and eventully rupturing the endosome membrane.

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Figure 3. CP 2k facilitates in vitro gene expression and endosomal escape of mRNA vaccine. At 24 h after transfection, expression of the luciferase reporter gene was analyzed in (A) DC 2.4 cells, (B) MDCK cells (n = 3, **p < 0.01). (C) Release of CP 2k from endosomes in DC 2.4 cells was analyzed using confocal laser scanning microscopy. DC 2.4 cells were incubated for the indicated times with fluorescein-mRNA (green) complexed with CP 2k (CP2k/m) and then stained with LysoTracker (red). Co-localization of fluorescein-mRNA and endosome/lysosomes appears as yellow fluorescence. Scale bar, 10 μm. 3.4 Prolonged nasal residence time and efficient in vivo delivery of mRNA by CP 2k Given the promising intracellular results, we attempted to explore the in vivo behavior of the delivery vehicle. Prolonged nasal retention time may increase the probability of in vivo uptake. CP 2k containing Cy3-labeled mRNA encoding gp120 were administered intranasally to BALB/c mice. In vivo imaging was used to determine whether condensing mRNA with CP could prolong retention time in the nasal cavity (Figure 4A). Nasal residence time was only 2

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ACCEPTED MANUSCRIPT h for naked mRNA, whereas it was 3 h for CP 2k/mRNA, at which time residual fluorescence was still 23.8 ± 4.52% of the initial level. In contrast, residual fluorescence for PEI

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25k/mRNA was 17.7 ± 6.24% at 2 h and it was barely detectable at 3 h. These results suggest

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that the cationic CP 2k polymer shows stronger mucosal adhesion than PEI 25k, leading to

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longer nasal retention time of the mRNA, which further increases uptake by NALT and by nasal epithelial cells. Mean fluorescence due to Cy3 was 8.98 ± 0.31 in NALT and 10.34 ± 1.34 in nasal epithelial cells, indicating significantly greater uptake for CP 2k/mRNA than for

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either PEI 25k/mRNA or naked mRNA (p < 0.05, Figure 4B).

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Figure 4. CP 2k facilitates in vivo delivery of mRNA vaccine and prolongs nasal residence time. (A) Relative fluorescence intensity of Cy3-mRNA complexed with CP 2k (CP 2k/mRNA) or PEI 25k (PEI 25k/mRNA) in the nasal cavity was determined as the percentage of initial fluorescence (n = 4). (B) In vivo uptake efficiency of Cy3-mRNA complexed with CP 2k (CP2k/m) or PEI 25k (PEI25k/m) was determined by flow cytometry at 6 h after intranasal administration (n = 4, *p < 0.05). 3.5 Efficient delivery of mRNA vaccine through the epithelial barrier with higher safety One potential disadvantage of using polymers to deliver mRNA is that they may facilitate bioabsorption of toxins that may be present in the nasal cavity. To examine this problem in CP 2k, we co-administered CP 2k/mRNA, PEI 25k/mRNA (encoding HIV gp120) or double-distilled water and lipopolysaccharide (LPS) into mice every other day over 7 days. LPS absorption into the nasal cavity should lead to hepatotoxicity. Serum levels of alanine

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ACCEPTED MANUSCRIPT transaminase (ALT) were higher in animals given PEI 25k/mRNA + LPS than in those given CP 2k/mRNA + LPS or double distilled water + LPS (p < 0.05, Figure 5A). In contrast,

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serum levels of ALT and aspartate transaminase (AST) were similar between animals treated

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with CP 2k/mRNA + LPS or with double-distilled water + LPS. Liver sections were analyzed

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for histological changes. Groups treated with CP 2k/mRNA or double-distilled water showed no histological differences from untreated animals, while animals treated with PEI 25k/mRNA exhibited obvious cell swelling (Figure 5B, arrows). As a positive control,

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animals were injected intraperitoneally with LPS, and they showed severe liver damage,

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inflammatory infiltration and necrosis (Figure 5B, arrows). These results suggest that intranasally administered CP 2k/mRNA does not facilitate nasal absorption of LPS, whereas

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PEI 25k/mRNA permits it enough to lead to moderate liver damage.

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Next we sought to understand why PEI 25k, but not CP 2k, promoted LPS absorption. We examined the effects of CP 2k/mRNA and PEI 25k/mRNA on epithelial tight junction

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integrity in an in vitro epithelial MDCK cell model. We focused on tight junction protein ZO-1, which plays a vital role in maintaining the defense function of epithelium [44].

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Treating MDCK cells with either type of delivery vehicle significantly lowered ZO-1 protein expression 6 h later, based on qRT-PCR and immunofluorescence (Figure 5C). Surprisingly, by 12 h after treatment with CP 2k/mRNA, the level of ZO-1 protein recovered and normal tight junction morphology was restored. The ZO-1 protein remained disassembled even at 12 h after treatment with PEI 25k/mRNA. Measurements of TEER during 12 h of incubation were consistent with these observations (Supporting Information Figure S4). These results suggest that while PEI 25k irreversibly alters tight junction integrity, CP 2k reversibly opens tight junctions, thereby promoting intranasal delivery through paracellular pathways while still maintaining the integrity of the epithelial barrier.

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Figure 5. In vivo toxicity and paracellular delivery of polymer/mRNA . (A) Serum levels of ALT and AST were determined in mice intranasally given CP 2k/gp120-mRNA (CP2k/m), PEI 25k/gp120-mRNA (PEI25k/m) or double-distilled H2O together with LPS (5 mg/kg) every other day for 7 days (mean ± SD, n = 5, *p < 0.05). (B) Liver sections of mice treated with polymer/mRNA delivery vehicles and LPS. Arrows indicate pathological changes such as cell swelling or necrosis (n = 5, 200 ×). (C) ZO-1 protein expression in an in vitro epithelial cell model was analyzed by qRT-PCR after treatment with CP 2k/mRNA (CP2k/m) or PEI 25k/mRNA (PEI25k/m). Results were expressed as normalized fold expression (n = 3). 3.6 Enhanced humoral and cellular immune responses induced by CP 2k/mRNA

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To test whether the higher transfection efficiency, longer nasal residence time and superior epithelial permeation profile of CP 2k/mRNA translated into stronger immune responses, mice were intranasally vaccinated twice 2 weeks apart. Titers of HIV gp120-specific IgG and two IgG subtypes IgG1 and IgG2a were assayed in BALB/c mice 10 days after the second immunization in order to evaluate the humoral immune response. CP 2k/mRNA elicited significantly more IgG production than either PEI 25k/mRNA or naked mRNA (p < 0.01, Figure 6A). Naked mRNA and luciferase mRNA complexed with CP 2k (CP 2k/mLuc) induced barely detectable levels of gp120-specific antibodies. CP 2k/mRNA also induced significantly higher titers of IgG1 and IgG2a than did naked mRNA (p < 0.05, Figure 6B and 6C). Since mucosal vaccines administered at one site have been shown to elicit

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ACCEPTED MANUSCRIPT an immune response in distal mucosal tissues via movement of effector cells between mucosal tissues [27], we also assessed the immune response in both the nasal and vaginal

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compartments (Figure 6D). The immune response in mucosa was measured by assaying

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levels of secreted sIgA. CP 2k/mRNA induced greater sIgA secretion than PEI 25k/mRNA in

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vaginal washes (p < 0.01), while naked mRNA led to a relatively low level of sIgA secretion. These results suggest that condensation processcan endow the mRNA with the ability to elicit sIgA secretion in distal mucosa, which suggests its potential use in vaccines against sexually

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transmitted pathogens such as HIV. Animals treated with a CP 2k containing luciferase

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mRNA (CP 2k/mLuc) showed barely detectable sIgA levels in either the nasal or vaginal

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washes. This suggests that the observed sIgA induction was gp120-specific.

Figure 6. Humoral immune response induced by mRNA vaccination analyzed by ELISA. BALB/c mice were intranasally immunized twice with 10 μg gp120-mRNA in naked form or complexed with CP 2k or PEI 25k. Mice treated with unrelated CP 2k/mLuc were set as negative control. At 10 days after the second immunization, samples of serum and of lavage from nasal and vaginal cavities were obtained. (A) Serum titers of anti-gp120-specific IgG. Serial two-fold dilutions of serum were analyzed. (B) Serum titers of IgG1 and IgG2a subtypes. (C) Ratio of IgG1/IgG2a titers in different animal groups. (D) Levels of secreted 23

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IgA in nasal wash and vaginal wash. To obtain nasal washes, mice were washed with 1 ml of PBS containing 1% BSA. The vaginal cavity of mice was washed twice with 150 μl of PBS containing 1% BSA. Nasal washes were measured in the ELISA without further dilution, while vaginal washes were diluted two-fold before ELISA (mean ± SD, n = 5, *p < 0.05, **p < 0.01, n.s, non-significant)

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Besides the humoral immune response, the induced cellular immune response was

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evaluated in mice by measuring gp120-specific cytotoxic T lymphocyte activity (CTL), as well as CD8+ and CD4+ T cell responses. CTL activity was expressed as the percentage of

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gp120-specific cytotoxic lysis in vaccinated mice relative to that in untreated mice. Splenocytes were stained with carboxyfluorescein succinimidyl ester (CFSE) at 2 μM

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(CFSEhigh) or 0.2 μM (CFSElow). The percentage was calculated from the ratio of the number of cells in the CFSEhigh population after pulsing with gp120 peptide to the number of cells in

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the non-pulsed CFSElow population. CTL activity was 22.8 ± 1.31% after two intranasal

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immunizations with CP 2k/mRNA, significantly greater than 14.4 ± 1.46% after

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immunization with PEI 25k/mRNA (p < 0.01, Figure 7A). CTL activity in these groups was significantly higher than in the group vaccinated with naked mRNA (p < 0.05). Similar results were obtained when intracellular cytokine staining (ICS) was used to measure the

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levels of gp120-specific CD8+ and CD4+ T cell responses. CP 2k/mRNA induced similar levels of CD8+ and CD4+ T cell responses, which were significantly higher than those induced by PEI 25k/mRNA (p < 0.05, Figure 7B).

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Figure 7. Cellular immune response induced by CP 2k/gp120-mRNA, PEI 25k/gp120-mRNA, naked mRNA, unrelated CP 2k/mLuc. Untreated mice were set as blank control. At 10 days after the second immunization, splenocytes were obtained. Cytotoxic T lymphocyte activity (CTL) and Intracellular cytokine staining (ICS) were analyzed by flow cytometry. (A) The percentage of CD+8 T cell lysis that was gp120-specific was calculated (mean ± SD, n = 5, *p < 0.05, **p < 0.01). (B) CD+8 T cells producing IFN-γ and CD4+ T cells producing IL-4 were analyzed by ICS. Mean percentages of double-positive cells were calculated as the percentage of cytokine-positive cells to total cells (n = 5, *p < 0.05).

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3.7 CP 2k/mRNA induced a Th1/Th2/Th17 immune response and reduced mRNA

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immunogenicity

To assess the bias of induced immune responses, we analyzed the cytokine productions of polymer-based mRNA vaccine. Th1 cytokine IFN-γ and Th2 cytokine IL-4 in the

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splenocytes culture supernatant and NALT culture supernatant were determined by ELISA. Moreover, qRT-PCR was used to assay levels of Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-4 and IL-10) and Th17 cytokine (IL-17) in splenocytes of immunized mice. Traditional DNA vaccine was reported to induce a balanced Th1/Th2 immunity [4]. With our mRNA vaccination regimen, CP 2k/mRNA led to higher production of Th1 and Th2 cytokines than either PEI 25k/mRNA or naked mRNA. CP 2k/mRNA also induced quite high levels of Th17 cytokine, indicating that CP 2k/mRNA evoked a Th1/Th2/Th17 immune response (Figure 8A-8E). Similar results were observed when levels of IFN-γ and IL-4 in splenocytes culture supernatant were measured by enzyme-linked immunosorbent assay (Figure 8A and 8B). IL-4 is involved in the maturation of B cells, and the secretion of IFN-γ

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mRNA vaccine delivery system.

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Naked mRNA is known to activate TLR3/7 pathways [45, 46], and this adjuvanticity

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explains why naked mRNA encoding HIV gp120 induced cytokine production at the site of intranasal administration of our mRNA vaccines. This induction was reflected in the up-regulation of co-stimulatory molecules (Supporting Information Figure S5), as well as the

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high cytokine levels in NALT culture supernatant (Figure 8C and 8D). Naked mRNA has

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also been shown to induce innate immunity by activating the TLR3 signaling pathway, leading to secretion of type I interferon [47]. This poses a problem for mRNA vaccines

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because TLR3 activation can lower transgene expression, hampering the induction of an

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antigen-specific immune response [17]. In our experiments, CP 2k/mRNA led to lower type I interferon production than naked mRNA (Figure 8E). This suggests that antigen expression

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of the mRNA may be higher when CP 2k polymer is used. In addition, either CP 2k/mRNA or PEI 25k/mRNA led to 1- to 2-fold higher production of type I interferon than what was

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measured in untreated control mice. This suggests that the mRNA still triggers a slight innate immune response after condensation into polyplexes, which is desirable given that the innate response provides initial defenses in pathogen invasion. These results indicate that CP polymer-based polyplexes may achieve a balance between antigen-specific immune response and innate immunity.

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Figure 8. The cytokines produced by gp120-mRNA vaccine. Th1 cytokines (IFN-γ, IL-12) and Th2 cytokines (IL-4, IL-10) were determined by ELISA and qRT-PCR. Ten days after second immunization, the splenocytes of immunized mice were isolated and seeded into 96-well plates at a density of 1 × 105 cells/well. The IFN-γ (A) and IL-4 (B) in splenocytes culture supernatant were analyzed (n = 5, *p < 0.05, **p < 0.01). The NALT of mice were isolated and cultured with 200 μl RPMI 1640 medium for 48 h. The IFN-γ (C) and IL-4 (D) in NALT culture supernatant were analyzed (n = 5, *p < 0.05). (E) Quantitative RT-PCR measurement of cytokine production in splenocytes from vaccinated mice. Levels of transcripts encoding Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-4 and IL-10) and Th17 cytokine (IL-17) were determined using specific primers. In addition, levels of transcripts encoding the type I interferons IFN-α4 and IFN-β1 were analyzed. Results were expressed as normalized fold expression relative to the level in non-vaccinated animals (n = 5). 27

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4. Discussion

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Here we explored the application of CP polymer in the field of intranasal mRNA

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vaccination in an effort to overcome the nasal epithelial barrier and induce enhanced systemic

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and mucosal immunity. We provide in vivo evidence that CP polymer/mRNA administered intranasally can efficiently deliver vaccine to distal mucosal sites, which is important for vaccines against mucosally transmitted diseases. These delivery vehicle do not appear to

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suffer the short nasal residence time or clearance by cilia that can significantly reduce

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delivery of mRNA vaccines, and consequently enchanced the vaccine delivery across the nasal epithelial barrier [48]. Combining the high mucosal affinity of cyclodextrin[49] with

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the good adjuvanticity of the cationic PEI polymer, stronger immune reponses were elicited.

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[50].

Our results suggest that CP polymer efficiently deliver their mRNA cargo by two

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mechanisms. One is intracellular delivery into nasal epithelial cells: first these cells take up the mRNA and translate the encoded antigen; then the transfected cells are recognized and

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lysed by natural killer cells, releasing the antigen; and finally the antigen is taken up by dendritic cells and cross-presented to T and B cells [23]. This intracellular mechanism was elucidated by Frank Wegmann and colleagues, who showed that antigen complexed with PEI 25k could be taken up by DC cells and epithelial cells [50]. We also found that both nasal epithelium and NALT took up larger amounts of mRNA delivered with CP 2k than with PEI 25k (Figure 4B). Next, CP polymer were found to efficiently deliver their mRNA cargo into the underlying NALT by a paracellular pathway in which the CP 2k polymer disassembles the ZO-1 protein (Figure 5C), and reversibly opened tight junctions and lowering TEER (Supporting Information Figure S4). This may allow efficient delivery of the mRNA to the

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ACCEPTED MANUSCRIPT underlying dendritic cells, which then express the antigen and consequently induce immune responses. In contrast, we also provided evidence that PEI 25k irreversibly opened tight

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junctions, and their integrity remained compromised at 12 h after treatment with PEI

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25k/mRNA. As a result, co-administration of this delivery vehicle and LPS led to obvious

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liver damage (Figure 5A and 5B). The observation that CP 2k reversibly opens tight junctions probably helps explain why it is much less toxic than PEI 25k.

The mucoadhesive properties of polymers are critical to the efficiency of the carrier

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system for an intranasal vaccine. In our experiments, the presence of cyclodextrin rendered

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CP polymer highly mucoadhesive which likely results in longer nasal residence time than PEI 25k (Figure 4A). Moreover, good colloidal stability and mucus-binding ability are vital for

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intranasal drug delivery systems [51]. The longer nasal residence time of our mRNA vaccine

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may also reduce the risk that antigen is delivered to the lung or the central nervous system, reducing clearance and increasing antigen uptake. In terms of clinical application of

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polymeric vaccine delivery systems, safety and efficacy are two major factors to be considered. In our study, CP 2k polymer, synthesized by conjugating low-molecular weight

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PEI 2k with β-cyclodextrin, exhibited minimum cytotoxicity and kept the intactness of epithelium after intranasal vaccination, showing good biocompatibility. Currently, PEI-based DNA delivery system has entered clinical trial for treatment of bladder cancer. Therefore, CP 2k may hold the promise for clinical application. Taken together, our results indicate that CP 2k circumvented the nasal epithelial barrier by facilitating intra- and paracellular delivery of mRNA and prolonging the nasal residence time, and consequently elicited stronger mucosal and systemic T cell and B cell responses.

5. Conclusion Here, a simple and versatile intranasal mRNA vaccine delivery system was developed

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cationic PEI polymer. Our study provided the first in vivo evidence that CP polymer

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administered intranasally efficiently delivered an mRNA vaccine across the nasal epithelial

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barrier and induced potent immune responses against HIV-1 gp120 model antigen without causing absorption of toxins present in the nasal cavity. By reversibly opening tight junctions in the nasal epithelium, CP 2k not only facilitated paracellular antigen delivery but also

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reduced absorption of toxins present in the nasal cavity. Intranasal inoculation of mice with

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CP 2k/mRNA led to strong mucosal and systemic immune responses in a balanced Th1/Th2/Th17 profile. Using CP 2k or PEI 25k, condensation of mRNA into particles also

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reduced the ability of mRNA to stimulate an innate immune response, likely translating into

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higher expression of the antigen encoded in the mRNA and therefore higher antigen-specific immunity against gp120. Thus, this cationic CP polymer represents an excellent platform

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material for intranasal mRNA vaccination.

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Acknowledgement

We acknowledge the financial support of the National

Natural Science Foundation of China

(No.81173011, 81422044) and the National Science & Technology Major Project of China (No. 2012ZX09304004).

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