key: cord-0722958-rpadk1j7
authors: Ren, Jin; Cao, Yiming; Li, Lei; Wang, Xin; Lu, Haitao; Yang, Jing; Wang, Shengqi
title: Self-assembled polymeric micelle as a novel mRNA delivery carrier
date: 2021-09-02
journal: J Control Release
DOI: 10.1016/j.jconrel.2021.08.061
sha: ecfa64db2333fb9a95841160d60dc400e95b9074
doc_id: 722958
cord_uid: rpadk1j7

mRNA-based therapy has been evaluated in preclinical and clinical studies for the treatment of a wide variety of disease such as cancer immunotherapies and infectious disease vaccines. However, it remains challenging to development safe and efficient delivery system. Here, we have designed a novel self-assembled polymeric micelle based on vitamin E succinate modified polyethyleneimine copolymer (PVES) to delivery mRNA. In vitro, PVES could transfect mRNA into multiple cell lines such as HEK-293 T, HeLa and Vero and the transfection efficiencies were much higher than PEI 25 k. In addition, the cytotoxicity of PVES was much lower than PEI 25 k. Furthermore, mice administered intramuscularly with PVES/SARS-CoV-2 mRNA vaccine induced potent antibody response and show no obvious toxicity. These results demonstrated the potential of PVES as a safe and effective delivery carrier for mRNA.

Over the past decade, major technological innovation and research investment have enabled mRNA to become a promising therapeutic tool in the fields of vaccine development and protein replacement therapy [1, 2] . mRNA vaccines have several advantages over other vaccine approaches, such as a high safety, a flexibility to encode any protein as antigen, cost-effective and rapid production, which is of great importance in case of pandemic crisis [3, 4] . The recent coronavirus disease 2019 outbreak has demonstrated how quickly emerging infectious disease can spread and underlined the crucial need for a rapid response vaccine. As expected, mRNA vaccines (mRNA-1273 and BNT162b2) were the first kind of vaccines approved by FDA for emergency use. For mRNA-1273, it took only 25 days and 63 days respectively, from sequence selection to vaccine manufacture for the first clinical batch and the first human dosing. Moreover, there are four candidates have already advanced to clinical trials. The bright prospect of mRNA is attracting the attentions of scientists, investors, and even common people [5, 6] .

However, mRNA therapeutic is still facing the challenge of lacking safe and effective delivery system, because the large size and dense negative charge make naked mRNA difficult to pass through the cell membrane. In addition, mRNA is also an inherently unstable molecule that is susceptible to degradation [7] . Thus, the development of appropriate delivery systems is urgently required. Numerous effectiveness in supporting cell-mediated and humoral immune responses match or surpass other vaccines [10, 11] . However, more and more side effects causing by LNP were reported such as pain, redness, fever and flulike symptoms [12] . Recently, a study about inflammatory of LNP showed that LNPs used in many preclinical studies were highly inflammatory in mice. Intradermal injection of these LNPs led to rapid and robust inflammatory responses, characterized by massive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines. The same dose of LNP delivered intranasally led to similar inflammatory responses in lungs and a high mortality rate [13] . Thus, the development of more efficient and safe delivery systems is vital and highly necessary.

Although not as clinically advanced as lipid systems for mRNA delivery, polyethyleneimine (PEI) has been shown considerable potential in protein replacement, vaccine and other applications related to mRNA therapeutics [14] . The cationic charge and buffering capacity of PEI are beneficial for complexation with nucleic acids and endosomal/lysosomal release via "proton-sponge" effect [15] .

However, its broad therapeutic application has been limited by toxicity associated with high molecular weight. Thus, Low-molecular-weight PEIs were often modified and used for mRNA delivery to reduce toxicity [16] . For example, a polymer synthesized from stearic acid and branched PEI 2k was used to deliver HIV gag encoding mRNA. Following subcutaneous injection, immune responses were notably induced [17] . In another study, the cyclodextrin-PEI 2k conjugate was used for the intranasal administration of HIV gap120 mRNA, which resulted in a strong systemic and mucosal HIV-specific immune response [18] . temperature for 8 h. After the reaction was completed, solvent was removed by vacuum rotary evaporation. Product was dialyzed against distilled water for 24 h and lyophilized overnight.

PVES/mRNA complexes were freshly prepared by mixing a fixed amount of mRNA stock solution and varying amounts of PVES in sterile distilled RNase-free water. The ratio of PVES/mRNA was calculated as the molar ration of nitrogen in PEI portion of PVES and phosphate in mRNA. After mild vortex, the mixture was incubated at room temperature for 30 min to allow particles form. As the control, PEI (1.8 k or 25 k)/mRNA complexes were similarly prepared.

Gel retardation assays were performed to confirm the ability of PVES to condense mRNA and provide protection from degradation. First, PVES/mRNA complexes with 1 µg mRNA were prepared as described above at various N/P ratios from 4 to 32 in a final volume of 5 µL. Each of these complexes was mixed with 1 µL 6  RNA loading buffer (Beyotime Biotechnology, Shanghai, China) and electrophoresed on a 1% (w/v) agarose gel for 45 min at 100 V. Then, mRNA retardation was visualized and photographed by ChemiDoc XRS imaging system (Tanon-5200 Multi, Shanghai, China). In the second study, RNase A (Beyotime Biotechnology, Shanghai, China) was incubated with naked eGFP mRNA (1.0 µg) or the equivalent amount of mRNA complexed with PVES at the N/P ratios of 16 and 32.

After 10 min, the mixture was also analyzed by electrophoresis.

Size distribution of PVES and PVES/mRNA complexes of N/P ratios from 4 to 48 were measured using dynamic laser light scattering (DLS) on a particle analyzer (Litesizer 500, Anton Paar, Austria). The zeta potential was analyzed with the same apparatus. The morphology of PVES and PVES/mRNA complexes were analyzed by transmission electron microscopy (TEM, Hitachi H-7650, Tokyo, Japan) using a negative stain technique. PVES (1.0 µg/µL) and PVES/mRNA complexes (N/P=32) were absorbed to a copper grid for 60 s and stained with phosphotungstic acid (1%) for 20 s before observation. 

The cytotoxicity of PVES/mRNA complexes was measured using a Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the instructions. HEK-293T, 7 were performed with PEI 25k/mRNA and PEI1.8k/mRNA at the same N/P ratio. After 24 h of incubation, the culture medium was removed and 100 µL fresh medium containing 10% CCK-8 was added to each well. The cells were incubated at 37 ℃ for 30 min. Absorbance was measured at a wavelength of 450 nm using a microplate reader (Sunrise, TECAN, Switzerland).

The cytotoxicity of PVES was further assessed on HEK-293T cells. Briefly, HEK-293T cells were seeded into a 96-well plate (5  10 3 cells/well). After 24 h of incubation, the culture medium was replaced with fresh medium and PVES at predetermined concentrations (0 to 80 µg/mL) were added to each well. In parallel, PEI 1.8k and PEI 25k were used with the same dosage for comparisons. After 48 h of incubation, the culture medium was removed and 100 µL fresh medium containing 10% CCK-8 was added to each well. The cells were incubated at 37 ℃ for 30 min.

Absorbance was measured at a wavelength of 450 nm using a microplate reader (Sunrise, TECAN, Switzerland).

HEK-293T cells were applied to test cellular uptake of PVES/mRNA complexes.

Briefly, Luciferase mRNA was labeled by Fluorescein Labeling Kit (Mirus, Madison, USA) and cells were seeded into 24-well plates (210 5 cells/well) and allowed to grow for 24 h. Then cells were treated with PVES/MFP488-mRNA complexes at N/P=32. After transfection for 30 min, 60 min, 120 min and 240 min, cells were washed with PBS, and harvested with 0.25% trypsin/EDTA. The cells were then resuspended in PBS and cellular uptake was analyzed by flow cytometry. 

The SARS-CoV-2 RBD mRNA was prepared by in vitro transcription using the T7 standard mRNA production system (Cellscript, Madison, USA) from a linearized DNA template which encodes codon-optimized RBD region of SARS-CoV-2 (residues 319-541, accession number YP_009724390) and a cap capping system (Cellscript, Madison, USA) according to the manufacturer's instructions. The mRNA product was precipitated with phenol/chloroform and resuspended in RNase-free water. The concentration of mRNA was determined by Agilent 2100 bioanalyzer system (Agilent, Palo Alto, USA).

The expression of PVES/mRNA vaccine was verified by Western blot analysis.

Briefly, HEK-293T cells (110 6 cells/well) were seeded into a 6-well plate and incubated in a 5% CO 2 incubator at 37 ℃ for 24 h. The PVES/mRNA vaccine complexes at N/P ratio of 32 (5.0 µg mRNA/well) were transfected according to the previous transfection method. After 24 h, the cells were collected, and radio immunoprecipitation assay (RIPA) lysate and proteinase inhibitor were used to lyse the cells. The total protein of the cells was extracted, and a bicinchoninic acid assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration. The same amount of protein was taken for SDS-polyacrylamide gel electrophoresis separation, transferred on to the PVDF membrane, blocked with 5% skimmed milk, detected with SARS-CoV-2 RBD rabbit PAb (1:1000) (Sino Biological, Beijing, China) and secondary antibody (Sino Biological, Beijing, China). The blots were visualized with Clarity Western ECL Substrate (Applygen, Beijing, China) on

Chemiluminescence imaging system (Tanon-5200 Multi, Shanghai, China). 

Female BALB/c mice aged 6-8 weeks were randomized into 6 groups (5 animals per group) and immunized intramuscularly thrice with PVES/mRNA vaccine complexes (5 µg, 10 µg and 30 µg mRNA/mouse, N/P=32) at an interval of 14 days.

For the negative controls, the mice received the same volume of saline, 30 µg mRNA and PVES at the same time point. Blood was collected from the orbital vein at 10, 24 and 38 days post initial immunization. The collected blood was centrifuged at 4000 rpm to isolate serum (30 min, 4 ℃). The serum was stored in aliquots at -20 ℃ for subsequent detection of SARS-CoV-2 RBD specific IgG.

ELISA was used to measure SARS-CoV-2 RBD specific IgG antibody.

SARS-CoV-2 RBD specific IgG titer were determined by a commercial ELISA kit Plates were then washed five times with wash buffer and added with chromogen solution followed by 20 minutes of incubation at room temperature. The absorbance at 450 nm was read using a microplate reader (Sunrise, TECAN, Switzerland). The endpoint titers were defined according to the manufacturer's instruction.

An intracellular cytokine staining assay was performed to characterize antigen specific CD4 + and CD8 + immune responses. Briefly, spleens were removed from immunized mice at 4 weeks post immunization and splenocytes were isolated. Mouse splenocytes were added to a 12-well plate (1×10 6 cells/well) and then stimulated with the peptide pool (2 µg/mL of individual peptide) for 2 h. After that, Golgiplug (BD Biosciences) was added to a final concentration of 1 µL/mL and incubated for 4 h. different N/P ratios (4, 8, 16, 24, 32, 40 and 48) revealed that PVES could effectively condense mRNA to particle complexes at N/P≥8 (Fig. 1E ). Further increase of N/P ratios did not obviously change their particle sizes and zeta potentials.

The morphology of PVES and PVES/mRNA complexes (N/P=32) determined by TEM (Fig. 1C) illustrated that they were spherical in shape and the size were approximately 200 nm of PVES, 100 nm of PVES/mRNA complexes, which were consistent with the results of DLS.

Altogether, these results confirmed the ability to form stable nanoparticle of PVES and mRNA. 

To evaluate the transfection efficiency of PVES/mRNA complexes, eGFP mRNA was used as the reporter gene. HEK-293T cells were transfected with PVES/mRNA complexes at different N/P ratios (Fig. 2) . The results indicated that the transfection efficiency greatly enhanced with the increase of N/P ratios and reached the plateau at N/P=32. Further increase of N/P ratios to 40 and 48 did not enhance eGFP expression obviously. At lower N/P ratios, the transfection efficiency of PVES/mRNA complexes was slightly lower than that of positive control PEI 25k/mRNA complexes. However, When N/P ratio was up to 32, the transfection efficiency of PVES/mRNA complexes was equivalent to PEI 25k/mRNA complexes. Thus, N/P=32 was used for subsequent evaluation experiments. As expected, almost no eGFP expression was observed for cells transfected with PEI 1.8k/mRNA complexes. We also compared the transfection efficiency of PVES with that of a classic nucleic acid transfection reagent lipofectamine 3000 to fully evaluate the potential of PVES as an mRNA delivery. The result indicated that the eGFP expression level of the cells transfected with PVES complexes was comparable to lipofectamine 3000. In order to further compare the cytotoxicity of PVES and PEI 25k on HEK-293T cells, different concentrations (0 to 80 µg/mL) were assessed. The result (Fig. 4B) showed that the cell viabilities of PVES were significantly higher than PEI 25k. The cell viabilities of PVES were still up to 80% at the concentration of 60 µg/mL and it was only 20% for PEI 25k at the same concentration. 

PVES/mRNA complexes (N/P=32) encoding firefly luciferase were inoculated into mice through intramuscular administration to evaluate the efficiency of PVES as a mRNA in vivo delivery vector. The images (Fig. 5) showed obvious fluorescence at the administration site 6 h after injection. Real-time monitoring showed that photo flux faded to undetectable levels 48 h after injection. 

HEK-293T cells were transfected with PVES/mRNA vaccine, and the expression of RBD was confirmed by Western blot analysis and indirect immunofluorescence.

The results (Fig. 5A) showed that RBD was successfully expressed. In indirect immunofluorescence assay (Fig. 5B) , the green fluorescence was observed in the cytoplasm. These results revealed that PVES can effectively encapsulate mRNA vaccine and transfect it into cells to express protein, which suggested it could be used for vaccine delivery.

J o u r n a l P r e -p r o o f 

To further verify whether PVES vector can be used for mRNA vaccine delivery and induce immune response in vivo, groups of mice (n = 5) were immunized with different doses of PVES/mRNA vaccine complexes (5 µg, 10 µg and 30 µg/mouse). (Fig. 7A) . Titers of RBD specific antibody were detected by ELISA assay to evaluate humoral immune response (Fig. 7B) . As a result, three doses of PVES/mRNA complexes elicited significant antibody titers at 10 days after the first immunization.

After boosting immunization, the antibody levels were rapid increased. The titers of RBD specific antibody immunized with the high dose were higher than those observed in mice immunized with lower doses. The mean endpoint titers after the third immunization of 30 µg group rose to >10 5 and were 1.3-and 4.2-fold higher than 10 µg and 5 µg groups. Therefore, we selected the 30 µg dose to immunize mice in the following studies.

To characterize the cellular immune responses induced by PVES/mRNA vaccine, intracellular cytokine staining (ICS) assays were performed (Fig. 7C, D) . The result showed that after re-stimulation with RBD peptide pools in vitro, the average percentage of IFN- expressing CD8 + T cells and IL-4 expressing CD4 + T cells in J o u r n a l P r e -p r o o f PVES/mRNA group were considerably higher than control groups, which implied that PVES/mRNA vaccine was able to induce the RBD-specific CD8 + T cell and CD4 + T cell response. 

Vitamin E (VE, -tocopherol) is not only a daily nutrient, but also an important pharmaceutical agent. VE has been included in FDA inactive ingredients list for intravenous, oral and topical use. In addition, VE has been used as an immune supplement in human, as an emulsion adjuvant component in several veterinary vaccines as well as an adjuvant used in an H1N1 pandemic vaccine (Pandemrix). [19, 20] . VE is also utilized as a drug delivery vehicle via the form of tocopherol polyethylene glycol succinate (TPGS) micelles [21, 22] . So far, most studies of VE in J o u r n a l P r e -p r o o f delivery field have focused on cancer therapy, there are few reports on nucleic acid.

Only in 2016, Liu et al developed a series of vitamin E modified PEI 1.8k for DNA delivery and the results showed that VE labeling greatly enhance the cellular uptake of GFP DNA plasmid and could successfully deliver pDNA to the liver and lung of living mice [23] .

PEI is a water-soluble cationic polymer and VE is a hydrophobic molecule, the covalent conjunction of VE to PEI forms amphiphilic copolymers, which could self-assemble to produce stable micelles [24, 25] . It was proved by the homogeneous spheroidal nanoparticles observed by TEM, and the lower polydispersity index (PDI ˂ 0.2) in hydrodynamic diameter. The particle size of PVES was about 180 nm, while it was about 144 nm after condensing negatively charged mRNA. The lower size indicated that PVES/mRNA complexes could form more stable and compact nanoparticles by electrostatic interaction, which is important to allow cellular uptake by endocytosis and avoid rapid clearance by the reticuloendothelial system (RES) in system delivery applications [26] . Thus, we speculate that PVES delivering mRNA vaccine has dose dependent effect on RBD specific antibody titers, indicating that it has the potential to be an efficient J o u r n a l P r e -p r o o f vaccines vehicle. Additionally, PVES/mRNA vaccine is administrated with the most common used intramuscular injection for human use.

Cell-mediated immune responses play a critical role in combating viral infections.

They are comprised of T-cell responses, which fundamentally differ from antibody (humoral) responses in the way they bring about infection control [31] . SARS-CoV-2 mRNA vaccines have been reported that they could induce strong cell immune responses, especially type 1 T cellular responses [32, 5] . Here, RBD specific CD4 + and CD8 + T cell responses were evaluated by intracellular cytokine staining assay.

PVES/mRNA vaccine elicited antigen-specific CD8 + T cells expressing type 1 (Th1) immune response cytokine (IFN-) and CD4 + T cells expressing type Ⅱ cytokine (IL-4). Therefore, PVES/mRNA vaccine could induce a substantial T cell response against SARS-CoV-2 RBD antigen aside from humoral immune responses. In addition, the level of IFN-/CD8 + T cells in immunized group was approximately 10-fold higher than control group whereas IL-4/CD4 + T cells was only 2-fold, which revealed it induced a Th1-biased cellular immune response.

The safety of vaccine has been a public concern. Recently, there are two reports about the thrombosis and thrombocytopenia after ChAdOx1 COVID-19 vaccination [33, 34] . In the case of mRNA vaccine, some researchers suspect the immune system response to the delivery vehicle is causing the side effects [12] . Thus, we evaluated the safety of PVES vector and PVES/mRNA vaccine and the results showed that following immunization, no local inflammation response at the injection site or other adverse effects were observed during the observation periods. After all the experiments were completed, the histopathology of liver, spleen and kidney showed no obvious pathological alteration of groups of PVES, mRNA and PVES/mRNA compared to the control group.

However, after immunization with PVES or PVES/mRNA vaccine, some cytokines levels such as IL-6 and TNF- were slightly higher than control group at 24 h. They might return to normal levels because the levels at 24 h were much lower than 6 h. Moreover, we determined the cytokines levels at 48 h post immunization and the J o u r n a l P r e -p r o o f levels were lower than 24 h. Because of almost all the cytokine levels were less than the minimum detection limit and undetectable, we did not present here. Thus, we believe PVES is a potential mRNA vaccine delivery system without serious toxicity.

Taken together, PVES may be a promising mRNA delivery vector. Further study will be carried out such as evaluation of the adjuvant properties and improving targeting.

In summary, we developed a mRNA vaccine delivery system based on modified 

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The study was supported by the National Natural Science Foundation of China