CN113797327A

CN113797327A - Nucleic acid drug delivery carrier for carrying mRNA and preparation method and application thereof

Info

Publication number: CN113797327A
Application number: CN202111121386.8A
Authority: CN
Inventors: 杨振军; 潘宇飞; 邓晨昀; 周新洋
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2021-09-24
Filing date: 2021-09-24
Publication date: 2021-12-17

Abstract

The invention discloses a nucleic acid drug delivery carrier for carrying mRNA, a preparation method and application thereof. The carrier comprises neutral base liposome and cationic liposome, wherein the neutral base liposome is DNCA or DNTA, and the cationic liposome is CLD. The vector can deliver mRNA to cells and mice so as to express protein antigens, and can deliver influenza virus mRNA vaccines so as to activate humoral immunity and cellular immunity in the mice, induce generation of neutralizing antibodies, reduce lung injury of the mice infected by the virus and reduce death rate. Meanwhile, the Luciferase mRNA delivered by the mixed vector can express protein in mice. The invention lays a foundation for the wide clinical application of mRNA vaccines, and has wide application prospect in the field of gene therapy.

Description

Nucleic acid drug delivery carrier for carrying mRNA and preparation method and application thereof

Technical Field

The invention relates to a nucleic acid delivery vector, a preparation method and application thereof, in particular to a nucleic acid drug delivery vector for carrying mRNA, a preparation method and application thereof. The invention belongs to the technical field of biological medicines.

Background

Nucleic acid-based vaccines, including viral vectors, plasmid dna (pdna), and mRNA, are attracting increasing attention because they are capable of inducing a wide range of protective immune responses and can be produced by rapid and flexible manufacturing processes. Because the production process of the nucleic acid vaccine is independent of the encoding antigen, different vaccines based on the same nucleic acid platform can utilize the same production and purification method and production facility, and only adjustment on the verification method is needed, so that the cost and time for vaccine production are reduced. After vaccination, nucleic acid vaccines mimic viral infection, expressing vaccine antigens in situ, causing humoral and cytotoxic T cell responses. This advantage is crucial for the elimination of intracellular pathogens or infections where a powerful humoral and cellular immune response is required to achieve protection. In addition, nucleic acid vaccines have inherent adjuvant properties, as they can be recognized by specific Pattern Recognition Receptors (PRRs) and trigger innate immune responses, which are critical for the maturation of Dendritic Cells (DCs), thereby enhancing the induction of subsequent adaptive immune responses. However, pDNA is introduced into the nucleus of target cells with low efficiency, and viral vectors induce a specific immune response of the body to viral structural proteins, so that there is an increasing interest and research activity in mRNA vaccines.

Over the past two decades, technologies have been developed to develop prophylactic vaccines against infectious diseases based on mRNA. Technological advances in RNA biology, chemistry, stability and delivery systems have accelerated the development of fully synthetic mRNA vaccines. The effective, long-lasting and safe immune response observed in animal models, together with data from early clinical trials, make mRNA-based vaccination an attractive alternative to conventional vaccination approaches. In addition, the mRNA vaccine has the potential of simplifying vaccine discovery and development and promoting rapid response to emerging infectious diseases, and has the advantages of relatively simple and convenient mRNA preparation and synthesis, low cost and very wide prospect.

mRNA vaccines require a delivery system to develop their full potential, since naked RNA is not only susceptible to nuclease degradation,and the molecular weight is large, the negative charge is carried, and the membrane cannot pass through cell membranes passively. The cellular uptake rate of naked mRNA is estimated to be about one ten thousandth. Thus, the field of mRNA delivery has focused on the discovery and development of methods and materials that can transport RNA into cells. In 2018, FDA (US food and drug administration) approved the first siRNA drug

(Patisiran), which is a candidate therapy for hATTR (hereditary transthyretin amyloidosis), is delivered using liposomes composed of DLin-MC3-DMA, DSPC, PEG-DMG, and cholesterol. Thanks to the advances and efforts that have been made in the field of siRNA delivery, several major vehicles are currently under investigation, some encapsulating mRNA molecules into particles, others using positively charged polymers to bind RNA through charge interactions, and other attempts to create virus-like particles that can protect mRNA from both endo-and exo-enzyme interference, deliver mRNA efficiently into cells, and obtain sufficient levels of encoded protein. In all cases, the vector must cross the target cell membrane and, after uptake into the cell (usually by endocytosis), it must escape the endosome and release its mRNA into the cytoplasm where translation occurs.

Currently, cationic liposome delivery is used in clinical and preclinical studies of RNA vaccines, relying on electrostatic interactions between the positive and negative charges of the mRNA to bind and entrap the mRNA. Many laboratories have successfully transfected cationic liposomes with mRNA in studies of various pathogens such as rabies virus, Zika virus, influenza virus, etc. However, cationic liposomes are highly toxic and are prone to binding by negatively charged serum proteins under physiological conditions, resulting in immunogenicity, hepatotoxicity and easy release of siRNA outside the target organ. These drawbacks limit the further use of cationic liposomes for drug delivery of entrapped mRNA.

The inventor previously designed and synthesized a basic acetamide glycerol ether molecule DNCA (CN108059619A), which has a head with basic properties and can combine and entrap single-stranded nucleic acid drugs and plasmids through hydrogen bonding and pi-pi stacking (CN 1084478807A). The 3 ', 3' double peptide-conjugated siRNA co-entrapped by DNCA and cationic lipid material CLD which is designed and synthesized by the inventor in advance and takes lysine as a head is successfully applied at the cellular level (Mol Pharm,2019,16, 4920). In the method, the entrapment method is optimized, an optimal DNCA/CLD transfection prescription is newly formulated and explored, the property of the mixed preparation applied in vivo is optimized, and an mRNA drug entrapment delivery system taking neutral lipid as a main body and being assisted by cationic lipid is successfully constructed. In vivo delivery of mRNA, an equimolar amount of DNTA may also be used instead of DNCA to achieve better transfection efficiency.

Disclosure of Invention

In order to improve the cell entrance efficiency and the effectiveness of intracellular release of RNA vaccines, the invention provides a high-efficiency low-toxicity nucleic acid drug carrier delivery strategy. The invention uses the mixture of neutral lipid and cationic lipid to encapsulate mRNA vaccine, so as to realize more effective, safe and nontoxic in vivo mRNA delivery, thereby improving the drug property of mRNA.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention relates to a nucleic acid drug delivery carrier for carrying mRNA, which is characterized by comprising neutral base liposome and cationic liposome, wherein the neutral base liposome is DNCA or DNTA, the cationic liposome is CLD, and the structural formulas of DNCA, DNTA and CLD are shown as follows:

wherein, the mass ratio of the neutral base liposome to the cationic liposome is preferably 1-5: 1-5.

Wherein, preferably, the carrier also comprises DSPE-PEG.

Further, the invention also provides a method for preparing the nucleic acid drug delivery carrier, which comprises the steps of dissolving and uniformly mixing the neutral base liposome and the cationic liposome by using ethanol, then freeze-drying, and re-dissolving by using a proper amount of ethanol when in use.

Preferably, the method further comprises the steps of adding DSPE-PEG into the ethanol solution dissolved with the neutral base liposome and the cationic liposome, and uniformly mixing by vortex.

Among them, it is preferable that the DSPE-PEG is used in an amount of 5% of the total molar amount of DNCA and CLD.

Furthermore, the invention also provides the application of the nucleic acid drug delivery carrier in preparing an mRNA carrying reagent.

Preferably, the mRNA is an mRNA vaccine or mRNA for the treatment of influenza and possibly other later conditions.

Among them, preferably, mRNA is dissolved in RNase-free water or physiological saline, and then forms a lipid complex with the nucleic acid drug delivery vehicle under ultrasonic conditions at room temperature.

Wherein, the mass ratio of the nucleic acid drug delivery carrier to the mRNA is preferably 2-6:1, and preferably 5: 1.

Compared with the prior art, the invention has the advantages that:

1. the nucleic acid drug delivery vector carrying influenza virus mRNA vaccine can express a large amount of HA protein in cells, successfully activate humoral cell immunity in mice, induce generation of neutralizing antibodies, and reduce death rate of influenza virus infected mice, and the mRNA vaccine HAs good safety and no obvious toxicity in cell level and animal level application, and lays a foundation for wide clinical application of the mRNA vaccine;

2. the neutral nucleoside lipid material has a base head, can be combined with mRNA through hydrogen bond action and pi-pi accumulation action, is more stable in vivo application compared with charge action between a cationic lipid material and mRNA, is not easy to adsorb charged particles or proteins in a circulation process, and avoids disintegration of a lipid complex outside a target organ and release of mRNA. And the dosage of the cationic lipid material in the preparation formula is reduced based on the combination of the non-electric effect, and the mass ratio of the cationic lipid material to mRNA is only 2-6:1, which is far lower than the dosage of the cationic lipid material in other reports.

Drawings

FIG. 1 shows the result of agarose gel organization of encapsulation of HA mRNA by DNCA/CLD liposomes of different masses;

wherein: 1. marker 2000; DNCA/CLD; HA mRNA; DNCA/CLD HA mRNA 2: 1; DNCA/CLD HA mRNA ═ 3: 1; DNCA/CLD HA mRNA 4: 1; DNCA/CLD HA mRNA ═ 5: 1; DNCA/CLD HA mRNA ═ 6: 1;

FIG. 2 shows the morphology of DNCA/CLD/HA mRNA complexes under transmission electron microscopy;

FIG. 3 shows the particle size and potential of DNCA/CLD/HA mRNA complexes;

FIG. 4 is an inverted fluorescence microscope observation of the expression of green protein in HEK-293T cells of DNCA/CLD liposome-delivered EGFP mRNA;

FIG. 5 is a microplate type chemiluminescence apparatus for detecting the expression of Firefoy Luciferase delivered by DNCA/CLD liposome in HEK-293T cells;

FIG. 6 is a graph showing that an animal in vivo imaging system detects bioluminescence signals of DNCA/CLD liposome-delivered Luciferase mRNA after protein expression in BALB/c mice;

FIG. 7 is a Western Blot to verify the HA protein expression of DNCA/CLD liposome delivered influenza virus mRNA vaccine in HEK-293T cells;

note: m: marker; 1: 1. mu.g HA mRNA; 2: 2. mu.g HA mRNA; 3: 3. mu.g HA mRNA; 4, Control;

FIG. 8 is the in vivo neutralizing antibody levels following 3 rd immunization of BALB/c mice with influenza virus mRNA vaccine;

FIG. 9 shows the survival rate of influenza virus infected BALB/c mice within 14 days after immunization with influenza virus mRNA vaccine (80. mu.g);

FIG. 10 shows that in vivo animal imaging systems detect bioluminescence signals of DNCA/CLD, DNCA/CLD/PEG, DNTA/CLD/PEG liposome-delivered Luciferase mRNA after protein expression in BALB/c mice;

FIG. 11 is a graph of the effect of PEG incorporation on the particle size of DNCA/CLD/mRNA lipid complexes;

FIG. 12 is a graph of the effect of PEG incorporation on DNCA/CLD/mRNA lipid complex potential;

FIG. 13 is a screen showing the optimal ratio of bioluminescence signals after detection of DNCA/CLD by an animal in vivo imaging system and protein expression in BALB/c mice by delivering Luciferase mRNA.

Detailed Description

The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description of the specific embodiments proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1

This example mainly illustrates the optimal binding ratio of DNCA/CLD liposome to HA mRNA and the shape and particle size potential of the complex.

DNCA/CLD/HA mRNA preparation

HA mRNA is dissolved in RNase-free water, DNCA/CLD liposome dissolved in pure ethanol (1 mu l of DNCA/CLD is 1.76 mu g of DNCA +1.58 mu g of CLD, DNCA and CLD are dissolved and uniformly mixed by ethanol, then freeze-drying is carried out, when in use, proper amount of ethanol is used for redissolving) is added into HA mRNA solution, proper amount of RNase-free water is added, and ultrasonic treatment is carried out at room temperature for 10min, so as to obtain DNCA/CLD/HA mRNA compound.

Determination of optimal ratio of DNCA/CLD to HA mRNA

DNCA/CLD and HA mRNA were bound by the above method at different mass ratios (2: 1; 3: 1; 4: 1; 5: 1; 6:1), then the complex (1. mu.g HA mRNA) was added to 1% agarose gel, voltage was set at 100V for 30min, and finally the encapsulation of HA mRNA by DNCA/CLD was observed using a gel imager.

Determination of morphology particle size potential of DNCA/CLD/HA mRNA

The morphology of the DNCA/CLD/HA mRNA complex (5:1) was observed under a Transmission Electron Microscope (TEM), and the particle size and potential of the complex were measured by a particle sizer.

As shown in FIGS. 1-3, the best binding of DNCA/CLD to HA mRNA was 5:1, and the formed complex was spherical nanoparticles with a particle size of 292.7 + -7.98 nm and a potential of 23.9 + -0.6 mV.

Example 2

This example mainly demonstrates that EGFP mRNA delivered by DNCA/CLD liposomes can express a large amount of green fluorescent protein in cells.

The material and the method are as follows:

HEK-293T cells, EGFP mRNA, DNCA/CLD liposomes (same as example 1), HEK-293T cells culture medium containing 10% fetal bovine serum DMEM (GIBCO) culture medium.

2. The expression of the EGFP mRNA delivered by the DNCA/CLD liposome in HEK-293T cells was observed by an inverted fluorescence microscope. DNCA/CLD/EGFP mRNA was prepared as in example 1.HEK-293T cells in DMEM (GIBCO) medium containing 10% fetal bovine serum at 37 deg.C with 5% CO₂Culturing in an incubator. The cells were observed to be well grown, and after culturing to a logarithmic growth phase, the cells were seeded into 12-well plates at 5X 10⁵One/well, 37 ℃ and 5% CO₂Culturing in an incubator, and performing transfection when the cell fusion degree reaches 80%. The experiment was divided into a cell control group, an EGFP mRNA group, and a DNCA/CLD/EGFP mRNA (5:1) group. The 12-well plate was removed, the medium was discarded, 1ml of Opti-MEM was added to each well, and 1. mu.g of EGFP mRNA was added thereto at 37 ℃ with 5% CO₂Culturing in incubator, changing the culture medium after 4 hr, changing into DMEM medium containing 10% fetal calf serum, and culturing at 37 deg.C under 5% CO₂Culturing in an incubator. Protein expression was observed under an inverted microscope after 24h or 48 h.

As a result:

EGFP mRNA groups

24h and 48h both had essentially no green fluorescent protein expression, and DNCA/CLD/

eGFP mRNA groups

24h and 48h both had significant green fluorescent protein expression (FIG. 4).

In conclusion, EGFP mRNA delivered by DNCA/CLD liposome can express a large amount of green fluorescent protein in cells.

Example 3

This example mainly illustrates that the fluc (Fireforcerase) mRNA delivered by DNCA/CLD liposome can express a large amount of Fireforcerase protein in cells.

Materials and methods

HEK-293T cells, FLUC mRNA, DNCA/CLD liposomes (same as example 1), HEK-293T cells culture medium is DMEM (GIBCO) culture medium containing 10% fetal bovine serum.

The Promega dual-luciferase reporter kit detects the expression of protein in HEK-293T cells of FLuc mRNA delivered by DNCA/CLD liposome. DNCA/CLD/FLUC mRNA was prepared as in example 1.HEK-293T cells in DMEM (GIBCO) medium containing 10% fetal bovine serum at 37 deg.C with 5% CO₂Culturing in an incubator. The cells were observed to be well grown, and after culturing to a logarithmic growth phase, the cells were seeded into 24-well plates at 1X 10⁵One/well, 37 ℃ and 5% CO₂Culturing in an incubator, and performing transfection when the cell fusion degree reaches 80%. The experiment was divided into a cell control group, a FLuc mRNA group, and a DNCA/CLD/FLuc mRNA (2:1, 3:1, 4:1, 5:1, 6:1, etc.) group. Taking out 24-well plate, adding 150ng Fluc mRNA, and reacting at 37 deg.C and 5% CO₂Culturing in an incubator, discarding the culture medium after 6h, lysing cells according to the specification of the dual-luciferase kit, transferring the lysate to a 96-hole white board, detecting by using a microplate chemiluminescence apparatus, and adding 50 mu l of lysate and 20 mu l of luciferase substrate into each hole.

As a result:

the FLuc mRNA group (M7) had essentially no Firefly luciferase expression, and each of the remaining DNCA/CLD/FLuc mRNA groups had Firefly luciferase expression at 6h, where the mass ratio of DNCA/CLD to mRNA in the M14 group was 5:1 (fig. 5).

In conclusion, the DNCA/CLD liposome delivered FLuc mRNA can express a large amount of Firefly luciferase in cells.

Example 4

This example mainly demonstrates that mRNA delivered by DNCA/CLD liposomes can express proteins in BALB/c mice.

The material and the method are as follows:

1. female BALB/c mice, SPF grade, 12-14g, supplied by Beijing Wintolite laboratory animal technologies, Inc.; luciferase mRNA provided by vitamin technology ltd.

Preparing a reagent:

DNCA/CLD/Luciferase mRNA complexes (prepared per 10. mu.g of Luciferase mRNA): the concentration of Luciferase mRNA was 1. mu.g/. mu.l, 40. mu.l of Luciferase mRNA was added to 500. mu.l of physiological saline, 3.4. mu.l of DNCA/CLD liposome (same as example 1) was added at a concentration of 60. mu.g/. mu.l, and finally 256.6. mu.l of physiological saline was added and sonicated for 10 min.

2. Grouping experiments: blank, DNCA/CLD/Luciferase mRNA. 200 μ l of mRNA was injected intramuscularly and the blank group was injected with the corresponding dose of saline. 30mg/ml D-luciferin (Perkin Eimer) was injected in the abdominal cavity after 24h and 48h, anesthetized with gas for 3min after 5min, and finally bioluminescent signals in the mice were detected by a small animal in vivo imaging system.

As a result:

bioluminescent signals were detected in mice injected with DNCA/CLD/Luciferase mRNA for both 24h and 48h as found by the small animal in vivo imaging system (FIG. 6). The DNCA/CLD liposome delivered mRNA was shown to express protein in BALB/c mice.

Experimental example 5

This example illustrates the use of DNCA/CLD liposomes for the delivery of influenza virus mRNA vaccines.

The material and the method are as follows:

1. female BALB/c mice, SPF grade, 12-14g, supplied by Beijing Wintolite laboratory animal technologies, Inc.; the H1N1 type FM1 strain influenza virus and influenza virus mRNA vaccine is provided by six departments of the second institute of military medical sciences, Beijing.

Preparing a reagent:

the pentobarbital sodium solution is prepared by weighing 60mg of pentobarbital sodium and preparing 6mL of 10mg/mL pentobarbital sodium solution by using physiological saline.

DNCA/CLD/HA mRNA complexes (80. mu.g each HA mRNA prepared as in example 1): the HA mRNA concentration is 4 mug/mul, and 120 mul HA mRNA is added with 750 normal saline, then 40 mul DNCA/CLD liposome with 60 mug/mul is added, finally 290 mul normal saline is added, and the ultrasonic treatment is carried out for 10 min.

Western Blot method to determine expression of antigenic proteins in cells by DNCA/CLD delivered influenza virus mRNA vaccines. (1) HEK-293T cells in DMEM (GIBCO) medium containing 10% fetal bovine serum at 37 deg.C with 5% CO₂Culturing in an incubator. The cells were observed to be well grown, and after culturing to a logarithmic growth phase, the cells were seeded into 6-well plates at 1X 10⁶One/well, 37 ℃ and 5%CO₂Culturing in an incubator, and performing transfection when the cell fusion degree reaches 80%. The experiment was divided into a cell control group and a DNCA/CLD/HA mRNA group (HA mRNA content of 1, 2 and 3. mu.g). Taking out 12-well plate, discarding culture medium, adding 2ml opti-MEM into each well, adding 1, 2, 3 μ g HA mRNA, 37 deg.C, 5% CO₂Culturing in incubator for 4 hr, changing the culture medium, changing to 2ml DMEM (GIBCO) culture medium containing 10% fetal calf serum, and culturing at 37 deg.C with 5% CO₂The incubator is used for 24 h. (2) Total cell protein was extracted from each well and Western Blot was performed. Carrying out 10% SDS-polyacrylamide gel electrophoresis (80v electrophoresis for 30min, and then 120v electrophoresis for 60 min); after electrophoresis is finished, taking gel, and reserving the gel at a required position according to the position of a protein marker; and (3) shearing a PVDF membrane and filter paper with corresponding sizes according to the gel, activating the PVDF membrane in methanol for 1min, and then placing the PVDF membrane and the filter paper in a membrane transferring solution for 10 min. Respectively placing the filter paper, the PVDF membrane, the gel and the filter paper on a membrane rotating instrument from bottom to top in sequence. Setting current according to the size of the filter paper for 1.5-2 h; after the transfer is finished, putting the membrane into 50mL of 5% skimmed milk powder, shaking, incubating at 40r/min, incubating for 40min, and sealing overnight; after the sealing is finished, the membrane is transferred into HA primary antibody (Yiqiao Shenzhou, 1:1000), and is incubated at room temperature for 2 hours at 40 r/min; washing the membrane with 1 × blocking solution for 3 times at a speed of 5min, 5min and 20min, 80r/min respectively; transferring the washed membrane into a secondary antibody (Proteitech, 1:2000), and shaking for 1h at room temperature; washing the membrane with 1 × blocking solution for 3 times at a speed of 5min, 5min and 20min, 80r/min respectively; putting the PVDF film into an imager and developing; ② quantifying the internal reference, washing the PVDF membrane for 15min by using 1 multiplied sealing liquid at 80 r/min; putting the washed membrane into membrane regeneration liquid for 30min, and washing the membrane for 15min by using 1 × sealing liquid at a speed of 80r/min after the membrane regeneration; the membrane was transferred to beta-action primary antibody (Proteintetech, 1: 50000) and incubated overnight; washing the membrane with 1 × blocking solution for 3 times at a speed of 5min, 5min and 20min, 80r/min respectively; transferring the washed membrane into a secondary antibody (Proteitech, 1:2000), and shaking for 1h at room temperature; washing the membrane with 1 × blocking solution for 3 times at a speed of 5min, 5min and 20min, 80r/min respectively; the PVDF film is placed in an imager and developed.

3. The influenza virus animal model verifies the immune protective efficacy of influenza virus mRNA vaccine. BALB/c mice were randomly divided into 3 groups of 5 mice each, including a blank group, a model group, and a DNCA/CLD/HA mRNA group. The mRNA vaccine was injected into each of 200. mu.l each by intramuscular injection, and the blank group and the model group were injected with the corresponding dose of physiological saline. Immunizations were 3 times, 2 weeks/time. On day 10 after 3 immunizations, tail vein blood was taken, serum was separated, and neutralizing antibodies were detected by hemagglutination inhibition assay. On day 14, a lethal dose of H1N1 type FM1 strain influenza virus is used for nasal challenge, and the specific method is as follows: weighing the weight of the mouse, anesthetizing with 0.15mL/20g of pentobarbital sodium, and carrying out nasal administration with 20 muL/influenza virus, weighing and observing the mouse within 14 days after administration, and recording the death condition.

As a result:

western Blot experiments demonstrated that influenza virus mRNA vaccines delivered by DNCA/CLD liposomes expressed HA protein in cells (FIG. 7). Serum inhibition experiments prove that neutralizing antibodies can be generated in mice after the mice are immunized (figure 8), and a lethal dose of influenza virus is adopted for virus challenge experiments, so that the survival rate of the mice in an unimmunized group after being challenged for 14 days is 20 percent, while the survival rate of the mice in an immunized group is 100 percent (figure 9), and the influenza virus mRNA vaccine delivered by DNCA/CLD liposome can play a role in preventing and protecting the mice.

Example 6

This example illustrates that mRNA delivered by DNCA/CLD/PEG or DNTA/CLD/PEG liposomes can express proteins in BALB/c mice.

The material and the method are as follows:

1. male BALB/c mice, SPF grade, about 25g, supplied by Beijing Wittingerihua laboratory animal technology, Inc.; luciferase mRNA supplied by zhahiralida biotechnology limited.

Preparing a reagent:

DNCA/CLD/Luciferase mRNA complexes (prepared per 3. mu.g of Luciferase mRNA): the concentration of Luciferase mRNA is 1.5 mu g/mu l, 2 mu l of Luciferase mRNA is added into 30 mu l of GenOpti, then 2 mu l of DNCA/CLD liposome (same as example 1) with the concentration of 8.26 mu g/mu l is added, vortex mixing is carried out, and finally 66 mu l of GenOpti is added, and room temperature ultrasound is carried out for 10-15 min.

DNCA/CLD/PEG/Luciferase mRNA or DNTA/CLD/PEG/Luciferase mRNA complexes (prepared per 3. mu.g Luciferase mRNA): the concentration of Luciferase mRNA is 1.5 mu g/mu l, 2 mu l of Luciferase mRNA is added into 30 mu l of GenOpti, then 2 mu l of DNCA/CLD liposome (same as example 1) with the concentration of 8.26 mu g/mu l is added, vortex mixing is carried out, 2 mu l of DSPE-PEG with the concentration of 1.5 mu g/mu l is added, vortex mixing is carried out, finally 64 mu l of GenOpti is added, and room temperature ultrasound is carried out for 10-15 min.

2. Grouping experiments: blank set, DNCA/CLD/Luciferase mRNA set, DNCA/CLD/PEG/Luciferase mRNA set. 100 μ l of DNCA/CLD/Luciferase mRNA or DNCA/CLD/PEG/Luciferase mRNA was injected by intramuscular injection, and PBS was injected in the blank group at the corresponding dose. After 6h, 100. mu.l of Luciferase Assay Reagent II (Promega) was injected intramuscularly, anesthetized with isoflurane gas for 3min, and finally the bioluminescent signal in the mice was detected by a small animal in vivo imaging system.

As a result:

it was found by a small animal in vivo imaging system that significant bioluminescence signals were detectable in mice injected with DNCA/CLD/PEG/Luciferase mRNA groups at 6h (FIG. 10), and the bioluminescence signals were very weak in the DNCA/CLD/Luciferase mRNA groups. It was shown that mRNA delivered by DNCA/CLD/PEG liposomes expressed proteins in BALB/c mice and that PEG had important roles in formulation ingredients, including reducing the particle size of the formulation and masking the excessively negative potential at the liposome surface (fig. 11, fig. 12). Adjusting the DNCA (DNTA) to CLD ratios, including 1:5, 3.5:3 and 5:1(DNCA or DNTA and CLD total mass is unchanged), the ratio of 5:1 is the most effective. (FIG. 13)

The information shown and described in detail herein is sufficient to achieve the above-mentioned objects of the present invention, and therefore the preferred embodiments of the present invention represent the subject matter of the present invention, which is broadly encompassed by the present invention. The scope of the present invention fully encompasses other embodiments that may become obvious to those skilled in the art, and that the scope of the present invention is therefore not limited by anything other than the appended claims, in which the singular form of an element used herein does not mean "one and only" one "unless explicitly so stated, but rather" one or more ". All structural, compositional, and functional equivalents to the elements of the above-described preferred embodiments and additional embodiments that are known to those of ordinary skill in the art are therefore incorporated herein by reference and are intended to be encompassed by the present claims.

Moreover, no apparatus or method is required to address each and every problem sought to be solved by the present invention, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. It will be apparent, however, to one skilled in the art that various changes and modifications in form, reagents and synthetic details may be made without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. A nucleic acid drug delivery carrier for carrying mRNA, which is characterized by comprising neutral base liposome and cationic liposome, wherein the neutral base liposome is DNCA or DNTA, the cationic liposome is CLD, and the structural formulas of DNCA, DNTA and CLD are as follows:

2. the nucleic acid drug delivery vehicle of claim 1, wherein the mass ratio of neutral base liposomes to cationic liposomes is 1-5: 1-5.

3. The nucleic acid drug delivery vehicle of claim 1, further comprising DSPE-PEG.

4. A method for preparing the nucleic acid drug delivery carrier of claim 1 or 2, characterized in that the neutral base liposome and the cationic liposome are dissolved and mixed by ethanol, then freeze-dried, and re-dissolved by a proper amount of ethanol when in use.

5. The method of claim 4, further comprising the step of adding DSPE-PEG to the ethanol solution of neutral base liposomes and cationic liposomes, and vortexing and mixing.

6. The method of claim 5, wherein the amount of DSPE-PEG used is 5% of the total molar amount of DNCA and CLD.

7. Use of the nucleic acid drug delivery vector of any one of claims 1-3 in the preparation of an mRNA carrier reagent.

8. The use according to claim 7, wherein the mRNA is an mRNA vaccine or mRNA for the treatment of influenza and possibly other later conditions.

9. The use of claim 7 or 8, wherein mRNA is solubilized with rnase-free water or physiological saline and then forms a lipid complex with the nucleic acid drug delivery vehicle under room temperature sonication.

10. The use of claim 8, wherein the nucleic acid drug delivery vector is present in a mass ratio of 2:1 to 6:1, preferably 5:1, to the mRNA.