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Enhanced Intranasal Delivery of mRNA Vaccine by Overcoming The Nasal Epithelial
Enhanced Intranasal Delivery of mRNA Vaccine by Overcoming The Nasal Epithelial
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: S0168-3659(16)30107-9
DOI: doi: 10.1016/j.jconrel.2016.02.043
Reference: COREL 8156
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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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
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*Corresponding author: Xun Sun
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Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17.
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Section 3. Southern Renmin Road, Chengdu 610041, People’s Republic of China.
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Abstract
Facing the threat of highly variable virus infection, versatile vaccination systems are urgently
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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
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mRNA vaccination system for HIV-1 treatment was synthesized using cationic
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cyclodextrin-polyethylenimine 2k conjugate (CP 2k) complexed with anionic mRNA
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
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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
mucosal anti-HIV immune responses as well as cytokine productions were achieved with a
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balanced Th1/Th2/Th17 type. Our study provided the first proof of evidence that cationic
polymers can be used as safe and potent intranasal mRNA vaccine carriers to overcome the
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nasal epithelial barrier. The safe and versatile polymeric delivery system represents a
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Graphic Abstract
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Key Words:
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mRNA vaccination, CD-PEI, nasal epithelial barrier, intracellular delivery, paracellular
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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
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infectious diseases, such as human immunodeficiency virus infection and acquired immune
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
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potent vaccine still faces great challenges due to the high mutation rate and viral diversity of
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
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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
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
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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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for mRNA vaccine delivery.
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The U.S. Food and Drug Administration approved the first intranasal vaccine against
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influenza (FluMist®) in 2003 [19]. As an alternative to systemic vaccine delivery, nasal
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immunization represents an effective and safe approach as compared to injection. The nasal
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
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a suitable site for antigen internalization and subsequent mucosal and systemic immune
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
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connected [22]. This may be particularly helpful when vaccinating against sexually
transmitted pathogens such as HIV. Despite these advantages, intranasal mRNA vaccination
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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
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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
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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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
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.
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Polyethyleneimine (PEI) with a molecular weight of 25 kDa was proven to open the epithelial
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tight junctions [32], and has been shown as an efficient mRNA carrier in in vitro transfection
[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
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mucus binding efficiency of PEI 25k, structural modification of low-molecular-weight PEI
biocompatible pharmaceutical excipient, has been selected to modify PEI 2k. Cyclodextrin
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was demonstrated to cause minimum damage to mucosal integrity and enhance drug
permeation via the nasal epithelium by transiently and reversibly opening tight junctions [34,
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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
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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
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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(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
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2. Materials and methods
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2.1 Materials and animals
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Branched polyethylenimine (PEI) with molecular weights of 2 or 25 kDa, β-cyclodextrin
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and chitosan (CS,
190-300 kDa, 75-85% deacetylated) were obtained from Sigma-Aldrich (St. Louis, MO,
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USA). Restriction enzymes were obtained from Fermentas (Thermo Fisher, USA). All other
BALB/c (female) mice 6-8 weeks old were obtained from Dashuo Biotechnology
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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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stand another 5 h. The resulting solution was dialyzed against distilled water for 3 days and
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
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(Shanghai, China); a Kozak consensus sequence was inserted, as were Hind III and Bam HI
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restriction sites at the ends. The sequence was then cloned into the pVAX1 vector (Life
Technologies, USA), which contains a T7 promoter and a poly(A) tail. A plasmid expressing
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the luciferase reporter gene was constructed by inserting the luciferase coding sequence into
the pVAX1 backbone. The recombinant plasmid was amplified and purified using an
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endo-free plasmid kit (Omega, USA) and linearized using Xba I. Synthetic mRNA was
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
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measuring the absorbance at 260 nm. After incubating the purified transcription products at
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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.
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PEI portion of CP 2k/phosphate in RNA). The mixture was vortexed for 10 sec, then
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
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RNase-free distilled water and equilibrated for 30 sec before measuring at a fixed angle.
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Measurements were made in triplicate.
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H-600, Hitachi, Japan). Samples were loaded on a copper grid and stained with
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phosphotungstic acid (1%) for 20 sec before observation.
The ability of CP 2k to bind RNA was checked using agarose gel electrophoresis. CP
2k/mRNA complexes were prepared as described above with 1 μg mRNA, mixed with 5 ×
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RNA loading buffer (Tiangen, Shanghai, China), and electrophoresed on a 4% agarose gel.
The gel was run at 120 V for 30 min and then visualized and photographed using the
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To assess the ability of CP 2k to protect the complexed mRNA from RNase degradation,
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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,
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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
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%
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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
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(mLuc), which was produced by in vitro transcription as described above. CP 2k/mLuc
25k/mLuc was prepared at N/P ratios of 8 or 16. Cells were seeded in 24-well plates at a
density of 1 × 105 cells/well and incubated for 24 h before transfection. Cells were incubated
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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
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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,
DC 2.4 cells were seeded on coverslips in 6-well culture plates for 24 h, then treated
with phosphate-buffered saline (PBS) containing 0.015% (w/v) heparin and stained with
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LysoTracker (Beyotime, China) for 1 h at 37°C. Then samples were washed with PBS and
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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
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intranasally to mice under anesthesia with intraperitoneal injection of 1% (w/v) sodium
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pentobarbital (5 μg Cy3-mRNA per animal). In parallel, some animals were given naked
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
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fluorescence in the nasal cavity was quantified over time using an excitation wavelength of
548 nm and an emission wavelength of 562 nm. Relative fluorescence was expressed as the
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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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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
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once-daily intranasal doses of CP 2k/mRNA or PEI 25k/mRNA (10 μg mRNA, optimal ratio
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N/P 16) or double-distilled water, followed by LPS (5 mg/kg) on days 1, 3, 5 and 7. Negative
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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analyzed on in vitro MDCK cell model. Briefly, 2 × 105 MDCK cells were seeded into the
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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
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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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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
junctions (n = 3).
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2.12 Animal immunization
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Female BALB/c mice aged 6-8weeks were randomized into 5 groups (5 animals per
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group), and immunized twice with mRNA encoding HIV gp120 at an interval of 2 weeks.
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,
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mice received no treatment or unrelated mRNA encoding luciferase complexed with CP 2k,
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
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immunization, all mice were sacrificed by neck dislocation, and the immune response was
analyzed.
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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.
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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was stopped with 2M H2SO4. Optical density was measured at 450 nm using a microplate
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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
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CD8+ T cells [40]. Splenocytes were isolated from naïve mice. Red blood cells were lysed
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using ACK buffer, and the cell suspension was divided equally into two aliquots. One was
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
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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
concentration of 20% (v/v). The two aliquots (1 × 107 cells/100 μl) were mixed equally, and
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the cell suspension was injected into the tail vein of untreated (control) or immunized mice.
After 18 h, splenocytes from recipient mice were prepared and analyzed by flow cytometry.
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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
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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
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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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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).
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Cells were centrifuged, then incubated in permeabilization buffer containing either
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polyethyleneimine (PE)-conjugated anti-mouse IFN-γ antibody or PE-conjugated anti-mouse
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
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CD8+/IFN-γ+ and CD4+/IL-4+were determined using Kaluza software (Beckman Coulter, CA,
USA).
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IFN-γ and IL-4 production in splenocytes culture supernatants and NALT supernatants
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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
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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
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
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Quantitative RT-PCR (qRT-PCR) was used to assay production of Th1 cytokines (IFN-γ
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
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TIANscript RT kit, and cytokine mRNA levels were analyzed using SsoFast TM EvaGreen
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Supermix on an iCycler iQTM 5 system (Bio-Rad, USA). β-actin served as the internal
control.
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2.18 Statistical analysis
Experiments were performed at least in triplicate unless otherwise noted. The data are
shown as the mean ± SD. Statistical difference was analyzed by Student’s t test and one-way
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ANOVA with Bonferroni post hoc test. All tests are accepted as statistically significant when
3. Results
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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
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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
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agarose gels in the absence of any free mRNA band (Figure 2B). The optimal ratio of CP 2k
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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
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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)
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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
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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
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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
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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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
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).
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After internalization, CP 2k/mRNA was expected to escape from the endo/lysosome to
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release mRNA cargo into the cytoplasm. To examine this process, we used confocal laser
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with CP 2k. At the same time, we localized endo/lysosomes using LysoTracker (red
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fluorescence). Co-localization of mRNA and endo/lysosomes was detected as yellow
fluorescence was observed in the cytoplasm than in the endo/lysosome (Figure 3C), which
indicates that CP 2k/mRNA escaped from the endo/lysosome after internalization thus
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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
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escape via the “proton sponge” mechanism [43] by increasing ionic concentration and
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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
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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
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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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
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
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nasal epithelial cells. Mean fluorescence due to Cy3 was 8.98 ± 0.31 in NALT and 10.34 ±
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1.34 in nasal epithelial cells, indicating significantly greater uptake for CP 2k/mRNA than for
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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
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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
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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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,
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
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no histological differences from untreated animals, while animals treated with PEI
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25k/mRNA exhibited obvious cell swelling (Figure 5B, arrows). As a positive control,
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
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intranasally administered CP 2k/mRNA does not facilitate nasal absorption of LPS, whereas
Next we sought to understand why PEI 25k, but not CP 2k, promoted LPS absorption.
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We examined the effects of CP 2k/mRNA and PEI 25k/mRNA on epithelial tight junction
integrity in an in vitro epithelial MDCK cell model. We focused on tight junction protein
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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
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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
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
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Figure 5. In vivo toxicity and paracellular delivery of polymer/mRNA . (A) Serum levels of
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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
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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)
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To test whether the higher transfection efficiency, longer nasal residence time and
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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
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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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
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.
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These results suggest that condensation processcan endow the mRNA with the ability to elicit
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sIgA secretion in distal mucosa, which suggests its potential use in vaccines against sexually
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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.
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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
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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.
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Splenocytes were stained with carboxyfluorescein succinimidyl ester (CFSE) at 2 μM
(CFSEhigh) or 0.2 μM (CFSElow). The percentage was calculated from the ratio of the number
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of cells in the CFSEhigh population after pulsing with gp120 peptide to the number of cells in
the non-pulsed CFSElow population. CTL activity was 22.8 ± 1.31% after two intranasal
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immunization with PEI 25k/mRNA (p < 0.01, Figure 7A). CTL activity in these groups was
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significantly higher than in the group vaccinated with naked mRNA (p < 0.05). Similar
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results were obtained when intracellular cytokine staining (ICS) was used to measure the
levels of gp120-specific CD8+ and CD4+ T cell responses. CP 2k/mRNA induced similar
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levels of CD8+ and CD4+ T cell responses, which were significantly higher than those
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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
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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
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(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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immunogenicity
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To assess the bias of induced immune responses, we analyzed the cytokine productions
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of polymer-based mRNA vaccine. Th1 cytokine IFN-γ and Th2 cytokine IL-4 in the
splenocytes culture supernatant and NALT culture supernatant were determined by ELISA.
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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
response (Figure 8A-8E). Similar results were observed when levels of IFN-γ and IL-4 in
(Figure 8A and 8B). IL-4 is involved in the maturation of B cells, and the secretion of IFN-γ
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plays a critical role in the elimination of HIV infected cells. Thus, the augmentation of IL-4
and IFN-γ, as well as other cytokines, provided strong evidence regarding the potency of this
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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
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intranasal administration of our mRNA vaccines. This induction was reflected in the
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up-regulation of co-stimulatory molecules (Supporting Information Figure S5), as well as the
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,
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leading to secretion of type I interferon [47]. This poses a problem for mRNA vaccines
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
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interferon production than naked mRNA (Figure 8E). This suggests that antigen expression
of the mRNA may be higher when CP 2k polymer is used. In addition, either CP 2k/mRNA
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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
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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
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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).
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4. Discussion
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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
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intranasally can efficiently deliver vaccine to distal mucosal sites, which is important for
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vaccines against mucosally transmitted diseases. These delivery vehicle do not appear to
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
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nasal epithelial barrier [48]. Combining the high mucosal affinity of cyclodextrin[49] with
the good adjuvanticity of the cationic PEI polymer, stronger immune reponses were elicited.
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[50].
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Our results suggest that CP polymer efficiently deliver their mRNA cargo by two
mechanisms. One is intracellular delivery into nasal epithelial cells: first these cells take up
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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
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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
Next, CP polymer were found to efficiently deliver their mRNA cargo into 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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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
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
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probably helps explain why it is much less toxic than PEI 25k.
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The mucoadhesive properties of polymers are critical to the efficiency of the carrier
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
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25k (Figure 4A). Moreover, good colloidal stability and mucus-binding ability are vital for
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,
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polymeric vaccine delivery systems, safety and efficacy are two major factors to be
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PEI 2k with β-cyclodextrin, exhibited minimum cytotoxicity and kept the intactness of
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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
5. Conclusion
Here, a simple and versatile intranasal mRNA vaccine delivery system was developed
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which carries the high mucosal affinity of cyclodextrin and the good adjuvanticity of the
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
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causing absorption of toxins present in the nasal cavity. By reversibly opening tight junctions
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in the nasal epithelium, CP 2k not only facilitated paracellular antigen delivery but also
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
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Th1/Th2/Th17 profile. Using CP 2k or PEI 25k, condensation of mRNA into particles also
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
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immunity against gp120. Thus, this cationic CP polymer represents an excellent platform
Acknowledgement
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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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