CN114848831A

CN114848831A - Coated nano preparation and preparation method and application of carrier thereof

Info

Publication number: CN114848831A
Application number: CN202210261679.4A
Authority: CN
Inventors: 宋相容; 魏霞蔚; 魏于全
Original assignee: Chengdu Westin Biomedical Technology Co ltd
Current assignee: Chengdu Westin Biomedical Technology Co ltd
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2022-08-05
Also published as: CN116763757A

Abstract

The invention belongs to the technical field of biology, and particularly relates to an encapsulated compound for reducing protein adsorption and/or resisting non-target cell adhesion, and a preparation method and application of an encapsulated nano preparation. The invention combines the anion drug excipient and the existing nano-carrier for coating, and explores a general design strategy for delivering the nano-drug compound, which has simple prescription, easy manufacture and clinical transformation. And the anion polymer is adopted to wrap the nano gene/drug compound to prepare a wrapped nano preparation, and then the delivery efficiency of the wrapped nano preparation in vivo and in vitro is explored. The invention provides a potential general design platform for developing high-efficiency low-toxicity anion nano-composites and also provides a simple and feasible method for clinically accelerating the transformation of the nano-composites.

Description

Coated nano preparation and preparation method and application of carrier thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an encapsulated compound for reducing protein adsorption and/or resisting non-target cell adhesion, and a preparation method and application of an encapsulated nano preparation.

Background

The relevant basic research of the nano preparation is wider, but the clinical transformation difficulty is very large, and one of the main reasons is that: after most of the nano preparations enter the body, the surface of the nano preparations can be adsorbed by protein or nonselective some cells, so that the nano preparations are aggregated or are difficult to reach a target position to play a role in high-efficiency treatment or prevention activity, and corresponding adverse reactions are induced. Based on a large amount of research accumulation, the invention creatively designs a type of anion-coated nano preparation, which can reduce the surface adsorption of protein, enhance the delivery and uptake of target cells, improve the activity of nano drugs and reduce the toxicity; and the preparation method of the coated nano preparation is simple and has good industrialization prospect. The invention provides a brand-new nano preparation platform and provides a new idea for the transformation research of nano preparations. The implementation of the preparation technology can promote the clinical transformation of the nano-drug and provide a better choice for the treatment and prevention of diseases.

First, the present invention successfully uses anion-coated nano-formulations for efficient delivery of mRNA. Due to its timeliness, effectiveness and simple manufacturing process, the mRNA drug is reputed to be the future of medicine, and has great application prospect in the fields of prevention and treatment of infectious diseases, tumors, cardiovascular diseases, metabolic diseases, genetic diseases and the like.

The key to the success of mRNA drug development is ensuring stable and efficient mRNA delivery to target tissues under physiological conditions. Thus, mRNA delivery vectors play a crucial role in stabilizing mRNA structure, controlling ribosome accessibility, and affecting translation mechanisms. The carrier materials widely studied at present are mainly cationic lipid materials and ionizable lipids, and the carriers mainly rely on the positive charge head group in the lipid materials when mRNA loading is realized, and the carriers have the following defects: (1) cationic liposomes cause acute cellular necrosis in a positive charge-dependent manner; (2) cationic nano-vaccines with high positive charge may destroy blood cells and cause hemolysis; (3) the serum protein can be adsorbed by the cationic nano preparation to form a precipitate, and then is removed by a reticuloendothelial system; (4) after endocytosis, most of the nano-drug compound is trapped in an endosome and a lysosome, and the stability of mRNA is influenced by the existence of a large amount of enzyme, so that the transfection efficiency is low; (5) the nano-drug complex with higher positive charge is not beneficial to being transported to immune organs, and can cause insufficient mRNA uptake by immune cells, so that the mRNA function is difficult to be exerted. To address the deficiencies of existing mRNA delivery vehicles, is there a simpler way to solve the above-mentioned problem? The answer to this field of the art is not given in the prior art at present! Successful delivery of mRNA remains a serious challenge.

In view of this, the inventors have attempted to solve the above-mentioned problems of mRNA drug delivery, especially mRNA delivery, with a special "coating" technique, exploring a generic design strategy for mRNA delivery that is simple to formulate, easy to manufacture, and clinically transformed. The strategy is also equally applicable to other nucleic acid drug delivery that rely on electrostatic interactions to effect loading, including but not limited to: DNA, plasmid, siRNA, microRNA, lncRNA, sarRNA or ASO, etc.

Secondly, with the development of biotechnology, protein, polypeptide drugs and other small molecule drugs are increasingly widely applied in clinic. However, due to the common problems of low absorption efficiency and short half-life period of the drug, frequent injection administration for a long time is usually required, and great burden is brought to the psychology and the economy of patients. Therefore, the development of a new administration form is one of the most effective methods for solving this problem. The dosage form of drug administration is not just as simple as delivering the drug to the site of the lesion, in fact the drug delivery system mainly carries four core functions: drug targeting, drug controlled release, drug absorption promotion, and drug property enhancement. The research and development of excellent drug carrier materials have important significance for controlling the stable release of protein, polypeptide drugs and small molecule drugs, improving the curative effect of the drugs, lightening adverse reactions and improving the life quality of patients, for example, drugs which are difficult to directly penetrate into cells are modified on the surfaces of various carriers, so that the capacity of the drugs for penetrating through specific biological barriers (such as blood brain barriers and cell membranes) is improved, and the drug effect is improved. Meanwhile, the drug is helped to realize drug controlled release to a certain extent, and the side effect of the drug is reduced. Based on the above, the inventor uses a special coating technology to deliver protein, polypeptide drugs and small molecule drugs, and develops exploration on the contrary of improving the stability of the drugs, realizing targeted therapy and reducing adverse reactions of the drugs.

Disclosure of Invention

An object of the present invention is to provide an inclusion complex for reducing serum adsorption and/or resisting non-target cell adhesion, which enables stable and efficient delivery of nucleic acid drugs, protein drugs and small molecule drugs to a target tissue under physiological conditions.

In order to achieve the purpose, the invention adopts the following technical scheme:

an encapsulated composite for reducing serum adsorption and/or resisting adhesion of non-target cells, the encapsulated composite consisting of an outer encapsulating layer and an inner composite, the inner composite consisting of a nano-preparation easily adsorbed by serum and/or adhered by non-target cells; the external wrapping layer is anionic compounds such as sodium Hyaluronate (HA), Sodium Alginate (SA), sodium carboxymethylcellulose (CMC), sodium heparin (NA), Chondroitin Sulfate (CS), Dermatan Sulfate (DS), cutin sulfate (KS), Heparan Sulfate (HS), Sodium Dodecyl Sulfate (SDS) and the like and related derivatives thereof.

The known gene drug carrier generally has the defects of high toxicity, poor biocompatibility, low in-vivo efficiency and the like, cannot be widely applied to the market, and in addition, due to the common problems of low absorption efficiency and short half-life of protein, polypeptide drugs and small molecule drugs, the long-term frequent injection administration is generally needed, and the like, and the clinical requirement cannot be met. And the carrier formed by the anionic polymer coating has the advantages of strong transfection capability, strong lysosome escape capability and high biological safety, and the delivery problem of the medicine is well solved.

Further, the nano preparation can be used for loading nucleic acid drugs, protein polypeptide drugs or small molecule drugs and mixtures thereof.

Further, the nano-formulation is a cationic formulation, including but not limited to: liposome, nanogel, core-shell nanoparticles, HDL nanoparticles, lipid nanoparticles, solid lipid nanoparticles, polymer micelle lipid nanoparticles, polymer nanoparticles, micelles and emulsions.

Further, the cationic liposome contains any one or more of DOTAP, DOTMA, DOSPA, DTAB, DDAB and DC-CHOL lipid materials.

Further, the surface charge of the complex is 40 to-30 mV.

Further, the mass ratio of the wrapping layer to the nucleic acid drug, the protein polypeptide drug or the micromolecule drug is 1: 0.25-1: 4.

Further, the mRNA includes, but is not limited to, antigenic proteins encoding viruses, gene editing tool proteins, protein-complement therapy-associated proteins, cytokines. In particular, the protein can be used for encoding antigen proteins of EBV, HPV, HBV, SARS-Cov-2, antigen proteins of malaria, syncytial virus, dengue, Zika, rabies and influenza virus, antigen proteins related to tumor such as Mucin1, KRAS and the like, Cas9 encoding gene editing, and cell factor IL 12.

Further, the mRNA includes, but is not limited to LMP1-mRNA, LMP2-mRNA, EBNA1-mRNA (CN112237628A), EBV-mRNA for nasopharyngeal carcinoma treatment; E6/E7-mRNA (EP3011060B1), HPV-mRNA for the treatment of cervical and oropharyngeal cancer; RBD-mRNA (CN113527522A) and S-mRNA for preventing SARS-Cov-2.

Further, the complex does not require a membrane stabilizer to stabilize the lipid molecule layer of the liposome.

The second purpose of the invention is to provide an encapsulated nano preparation, compared with the traditional positive charge nano preparation, the nano preparation adopts an anionic polymer encapsulation strategy, and has higher transfection capability, stronger lysosome escape capability, stronger antiserum capability and lower toxicity.

the coated nano preparation consists of a nano preparation and a coating layer, wherein the coating layer is an anionic compound or a related anionic mixture; the nano preparation is prepared from a material with at least one positive cation center, the nano preparation material is a pharmaceutically acceptable carrier material for preparing micelles, emulsions, nanogels, core-shell nanoparticles, HDL nanoparticles, solid lipid nanoparticles, liposomes and/or polymeric micelles, and the nano preparation is prepared by a pharmaceutically acceptable method.

In vitro experiments prove that the encapsulated compound has stronger transfection capability, but the nano preparation with high positive charge can adsorb serum protein to form polymeric precipitate and is removed by a reticuloendothelial system, so that the nano preparation cannot smoothly reach a target organ. Although it has been reported that the addition of anionic phospholipids to cationic carriers can reduce the adsorption of serum albumin by vaccines by reducing the Zeta potential of liposomes. However, this strategy not only destroys the drug loading capacity of the carrier, but also causes poor stability of the formulation due to mixed precipitation of positive and negative ions. Therefore, compared with the traditional gene vector, the in vivo delivery of the nano material through the anionic polymer encapsulation strategy has more advantages.

Further, the coated nano-formulation comprises:

(a) the main medicine comprises nucleic acid medicine, protein medicine and micromolecular medicine;

(b) a cationic lipid consisting of 50-90 wt% of the total lipid present in the particle;

(c) a non-cationic lipid, which comprises a mixture of phospholipids and/or other carriers, accounting for 10-50 wt% of the total lipid;

(d) an anionic compound or a derivative thereof.

Further, the encapsulated compound or the encapsulated nano-preparation is applied to the preparation of vaccines.

Further, the diseases for which the vaccine is used for treatment include nasopharyngeal carcinoma, cervical carcinoma, head and neck squamous carcinoma, liver cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, viral infection and atherosclerosis.

The invention also aims to provide a preparation method of the encapsulated composite or the encapsulated nano preparation.

the preparation method of the encapsulated compound or the encapsulated nano preparation specifically comprises the following steps:

1) preparing the coated layer containing active ingredients by adopting a nano preparation pharmaceutical process, wherein the coated layer contains positive charges;

2) adding the anionic compound or the anionic mixture to be adsorbed by the coated layer to obtain the coated compound or the nano preparation.

Further, when the mRNA vaccine is prepared, the anion compound or the anion mixture is modified/coated on the surface of the nano preparation, so that the gene transfection efficiency of the nano preparation is improved.

Further, in the preparation of mRNA vaccine, the anion compound or the anion mixture is modified/coated on the surface of the nano preparation to change endocytosis path and/or enhance lysosome escaping capability.

Further, in the preparation of an mRNA vaccine, use of the anionic compound or the anionic mixture as an in vivo expression enhancer, a spleen targeting agent, an antigen-presenting ability enhancer, or a proliferation inducer for CTLs.

Furthermore, when the protein, the polypeptide and the small molecule drug are prepared, the anionic compound or the anionic mixture is used for improving the stability of the nano preparation and is used as a preparation stabilizer.

Further, when preparing protein, polypeptide and small molecule drugs, the anionic compound or the anionic mixture is used for improving the biocompatibility of the drugs and reducing the toxicity of the drugs.

Further, in the preparation of proteins, polypeptides and small molecule drugs, the use of the anionic compound or the anionic mixture for resisting non-target cell adhesion of the drug and promoting penetration of the drug through biological barriers.

The invention has the advantages that:

1) in the previous researches, the delivery of the nano vaccine is carried out by using a few coating strategies, and the first experiment proves that the existence of the lipid membrane stabilizer is not beneficial to the coating of the nano vaccine, so that a preliminary framework is provided for the future research and development of the nano vaccine based on the coating method.

2) According to the invention, the novel anionic mRNA nano-vaccine which is simplified in prescription, high in stability and easy to prepare is obtained by wrapping anions on the gene vaccine with high positive charge.

3) The invention firstly proves that the negative charge nano vaccine is useful for the immunotherapy of mouse tumor.

4) The negative charge nano vaccine based on the encapsulation strategy has the advantages which are not possessed by the traditional positive charge nano vaccine, including higher transfection capability, stronger antiserum capability, stronger lysosome escape capability and better biological safety.

5) The anion packaging strategy adopted by the invention successfully overcomes the defects of gene vectors in gene drug delivery.

Drawings

FIG. 1 shows the particle size and zeta potential of formulas 5 to 8 in example 1, i.e., the particle size and zeta potential of SA @ DOTAP/CHOL-mRNA in different mass ratios;

FIG. 2 shows the particle size and zeta potential of formulas 1 to 4 in example 1, namely the particle size and zeta potential of SA @ DOTAP-mRNA with different mass ratios;

FIG. 3 is a graph of GFP expression in DC2.4 cells from formulas 5 to 8 of example 1, i.e., SA @ DOTAP/CHOL-GFP, in the absence of serum and 10% serum;

in FIG. 4, FIGS. 4-A and 4-B are the results of transfection of prescriptions 5 to 8 in example 1, i.e., the quantitative analysis of SA @ DOTAP/CHOL-GFP in DC2.4 cells under serum-free and 10% serum-free conditions, respectively, (mean. + -. SD, n ═ 3);

FIG. 5 is a graph showing the results of transfection of prescriptions 1 to 4 in example 1, i.e., the expression of GFP in DC2.4 cells by SA @ DOTAP-mRNA, in the absence of serum and 10% serum;

in FIG. 6, FIGS. 6-A and 6-B are the results of transfection of formula 1 to formula 4 in example 1, i.e., the quantitative analysis of SA @ DOTAP-mRNA in DC2.4 cells (G, H), (mean. + -. SD, n ═ 3), in the absence of serum and 10% serum, respectively;

FIG. 7 is an image of the expression of formula 1, formula 5 and formula 4 in DC2.4 cells in example 1 without serum and 10% serum;

in FIG. 8, FIGS. 8-A and 8-B are the quantitative analysis of prescription 1, prescription 5 and prescription 4 at DC2.4 in example 1, under serum-free and 10% serum conditions, respectively;

FIG. 9 is a graph showing the particle size distributions of formula 4 and formula 8 of example 1, namely SA @ DOTAP/CHOL-GFP and SA @ DOTAP-GFP;

FIG. 10 is a representation of the morphological structures of formula 4 and formula 8, SA @ DOTAP/CHOL-GFP and SA @ DOTAP-GFP, from example 1;

FIG. 11 is a graph of the particle size, potential and PDI of formula 1, formula 4, formula 5, DOTAP/CHOL-GFP, DOTAP-GFP and SA @ DOTAP-GFP in example 1 as a function of time;

FIG. 12 shows gel retardation studies on the mRNA loading stability of formula 1, formula 4 and formula 5 in example 1;

FIG. 13 shows the particle size and zeta potential of HA @ DOTAP-mRNA of formulas 1 to 4, i.e., different mass ratios, in example 2;

FIG. 14 is an image of GFP expression in DC2.4 cells at 10% serum conditions for HA @ DOTAP-mRNA of formulas 1 to 4, i.e., different mass ratios, of example 2;

in fig. 15, quantitative analysis of prescription 1 to prescription 4P in example 2 in DC2.4 cells under 10% serum conditions, (mean ± SD, n ═ 3);

FIG. 16 in example 3, recipes 1-8 are GFP expression images of encapsulated complexes prepared by encapsulation with different anions in DC2.4 cells;

FIG. 17 shows the particle size and zeta potential of micellar nanoparticles SA @ DOTAP/DOPE-mRNA of different mass ratios from formulation 1 to formulation 7 in example 4;

FIG. 18 is an image of GFP expression in DC2.4 cells under 10% serum conditions for micellar nanoparticles (SA @ DOTAP/DOPE-mRNA) of formulas 1 to 7, i.e., different mass ratios, of example 4;

FIG. 19 shows the particle size, PDI (A) and Zeta potential (B) of the shell-core nanoparticles CS in example 5 at different PLGA/lipid mass ratios;

FIG. 20 shows the particle size and potential of SA @ CS-mRNA under different mass ratios of mRNA to SA, from formulation 1 to formulation 4 in example 5;

FIG. 21 is a graph showing the expression of GFP in DC2.4 cells at 10% serum conditions for SA @ CS-mRNA at different mass ratios from prescription 1 to prescription 4 in example 5;

FIG. 22 is a graph of GFP expression in DC2.4 cells for formulations 26-27 of example 1, i.e., liposomal nanoparticle DOTMA-mRNA before SA modification and SA @ DOTMA-mRNA after SA modification at 10% serum conditions;

FIG. 23 shows the GFP expression images of liposome nanoparticle DOSPA-mRNA before SA modification and SA @ DOSPA-mRNA after SA modification in DC2.4 cells under 10% serum conditions, in the formulations of example 1 at 28-29;

FIG. 24 shows the GFP expression images of the liposome nanoparticle DC-CHOL-mRNA before SA modification and the SA @ DC-CHOL-mRNA after SA modification in DC2.4 cells under 10% serum conditions, in example 1, the formulations are 30-31

FIG. 25 is a laser scanning microscope (LSCM) image of DC2.4 cells treated for 5h with DOTAP/CHOL-CY5, DOTAP-CY5, and SA @ DOTAP-CY 5;

FIG. 26 is a flow assay of mean fluorescence intensity after 2h treatment of DC2.4 cells with PBS, DOTAP/CHOL-CY5, DOTAP-CY5, and SA @ DOTAP-CY 5;

FIG. 27 is a graph showing cellular uptake assays for DOTAP/CHOL-CY5, DOTAP-CY5, and SA @ DOTAP-CY5 after treatment with different cellular uptake inhibitors;

FIG. 28 is a CLSM image showing lysosomal escape of DOTAP/CHOL-CY5, DOTAP-CY5, and SA @ DOTAP-CY5 in DC2.4 cells;

FIG. 29 is a graph of DOTAP/CHOL-GFP, DOTAP-GFP, SA @ DOTAP-GFP in 50% serum, particle size over time;

FIG. 30 shows the transfection efficiency of DOTAP/CHOL-GFP, DOTAP-GFP, SA @ DOTAP-GFP in 10% and 30% fetal calf serum;

FIG. 31 is a flow cytometry analysis of purity of BMDC cells after 7 days of culture;

FIG. 32 is a graph of antigen presentation in BMDC cells of DOTAP/CHOL-OVA, DOTAP-OVA and SA @ DOTAP-OVA, and FIG. 32-B is a graph of stimulation of BMDC cell maturation by DOTAP/CHOL-OVA, DOTAP-OVA and SA @ DOTAP-OVA;

FIG. 33 is the expression of luciferase in mice following intravenous injection of DOTAP/CHOL-OVA, DOTAP-LUC and SA @ DOTAP-LUC;

FIG. 34 is a graph of the quantification of luciferase expression in lung (FIG. 34-A) and spleen (FIG. 34-B) DOTAP/CHOL-LUC DOTAP-LUC and SA @ DOTAP-LUC (. + -. SD, n ═ 3);

FIG. 35 is a flow cytometer analysis of spleen after OVA-specific CTL proliferation three passes through vein-injected DOTAP/CHOL-OVA DOTAP-OVA and SA @ DOTAP-OVA (. + -. SD, n. sup.3);

FIG. 36 is a graph of lymph node weight in mice in the DOTAP/CHOL-OVA, DOTAP-OVA and SA @ DOTAP-OVA groups after treatment was completed;

FIG. 37 is a schematic view of an immunotherapy protocol;

FIG. 38 is a graph of tumor growth in groups of mice;

figure 39 is the tumor volume increase in each group of mice (mean ± SD, n ═ 6);

figure 40-a is the tumor weight (mean ± SD, n ═ 6) after treatment for each group of mice, and figure 40-B is the tumor inhibition rate for DOTAP/CHOL-OVA, DOTAP-OVA and SA @ DOTAP-OVA;

fig. 41 is FACS analysis OVA-specific CTL proliferation in tumors (mean ± SD, n ═ 3);

FIG. 42 is a toxicity test of liposome/mRNA complexes on DC2.4 cells;

FIG. 43 is a liver function assay for mice treated differently;

FIG. 44 is a renal function assay in mice treated differently;

FIG. 45 is H & E staining of cardiac, liver, spleen, lung, and kidney physiological tissues on day10 after primary immunization.

Note: in fig. 1 to 45, p <0.05, and p < 0.01.

Detailed Description

EXAMPLE 1 preparation of liposomes/complexes

Table 1 recipe composition

Cationic liposomes containing CHOL and not containing CHOL without main drug were prepared by thin film hydration method (see Table 1 for the specific formula). The method comprises the following specific steps:

1) adding ethanol solution of cation carrier and auxiliary carrier lipid into round-bottom flask, and drying under vacuum condition to prepare lipid membrane.

2) RNase-free water was added, hydrated at 60 ℃ and the lipid membrane solution was collected.

3) And (3) placing the lipid membrane solution in an ice bath, and carrying out 100W ultrasonic treatment for 3 minutes to obtain the liposome without drug loading.

4) The drug nano delivery systems with different N/P or mass ratios are prepared by adopting a co-incubation method of liposome without drug loading and main drugs, and the specific preparation method comprises the following steps: taking the liposome without drug loading as A according to the proportion in the formula table; adding the main drug into RNase-free water, and mixing uniformly to obtain B; mixing B and A, and incubating at room temperature for 10min to obtain different liposome composites.

5) The preparation method of the encapsulated composite comprises the following steps: adding sodium alginate solution into the liposome compound according to the proportion in the formula table, mixing uniformly and incubating for 5 min.

Particle size potential and PDI measurements of the encapsulated composites prepared in example 1

The prepared sample was diluted 10 times with purified water, and then particle size, potential and PDI were measured with a malvern laser particle sizer, with the results shown in table 2.

TABLE 2

In this example, 500. mu.L of sodium alginate Solution (SA) was added to 1ml of liposome complex (DOTAP-mRNA or DOTAP/CHOL-mRNA), mixed well and incubated for 5min to prepare an encapsulated complex. In formulas 5 to 8, the particle size of the preparation significantly increased with the addition of SA, the potential change was insignificant, and PDI significantly increased, indicating that a stable and uniform preparation could not be formed, and it is presumed that the addition of CHOL was not favorable for the formation of a liposome by SA. The detection results in the prescriptions 1 to 4 show that stable encapsulated compounds can be prepared by adopting different mass ratios of mRNA to SA, the charges of the encapsulated compounds gradually change into negative charges along with the increase of the dosage of SA, when the mass ratio of the mRNA to the SA reaches 1:0.5, the encapsulated compounds with the negative charges are prepared, and the PDI of the encapsulated compounds is gradually reduced along with the addition of the SA in a certain range; in the prescription 9-prescription 25, SA is found to be capable of wrapping the compound loaded with different mRNAs, DNAs, siRNAs, microRNAs, lncRNAs, sarRNAs, ASOs, plasmids, proteins, polypeptides and small-molecule chemical drugs; in prescriptions 26-31, SA was found to encapsulate liposome complexes prepared from a variety of cationic materials.

Example 2 preparation of hyaluronic acid HA-Encapsulated DOTAP-mRNA

TABLE 3 prescription composition

The liposome complexes were prepared according to the recipe in Table 3 by the method for preparing liposome complexes in example 1.

The preparation method of the encapsulated composite comprises the following steps: adding the sodium hyaluronate solution into the liposome complex according to the proportion in the formula table, uniformly mixing, and incubating for 5 min.

Particle size potential and PDI detection of the encapsulated composites prepared in example 2

The prepared sample was diluted 10 times with purified water, and then subjected to particle size, potential and PDI detection using a malvern laser particle sizer, with the results shown in table 4.

TABLE 4

Prescription	Particle size (nm)	Electric potential (mV)	PDI
				Prescription
1	70	39	0.05
				Prescription 2	125	30	0.27
Prescription 3	198	26	0.28
				Prescription 4	290	22	0.29
Prescription 5	155	26	0.25
				Prescription 6	150	22	0.23
Prescription 7	149	25	0.22
				Prescription 8	160	27	0.26
Prescription 9	156	22	0.28
				Prescription 10	134	26	0.22
Prescription 11	145	28	0.35
				Prescription 12	176	18	0.27
Prescription 13	167	24	0.31
				Prescription 14	155	28	0.25
Prescription 15	165	26	0.29
				Prescription 16	147	22	0.22
Prescription 17	139	23	0.21
				Prescription 18	172	24	0.23
Prescription 19	139	19	0.30

In the embodiment, a natural anionic compound sodium Hyaluronate (HA) is used to wrap a liposome complex, and formulas 2 to 4 show that after HA is added, the particle size of the complex is obviously increased, and the more the HA is added, the larger the particle size is, the potential of the complex gradually decreases and is stabilized between 15 mV and 30 mV; prescription 4-prescription 9 show that HA can form good package for liposome complexes loaded with different mRNA; prescription 10-prescription 19 respectively show that HA can complex and encapsulate liposomes loaded with different mRNAs, DNAs, siRNAs, microRNAs, lncRNAs, sarRNAs, ASOs, plasmids, proteins, polypeptides and small chemical drugs, and finally form a nanocomposite with particle size less than 300nm and charge of 15-30 mV.

Example 3 functional verification of various anion-packing layers

TABLE 5 prescription composition

The preparation method of the encapsulated composite comprises the following steps: preparing the liposome complex by the preparation method of the liposome complex in the embodiment 1 according to the mixture ratio in the formula table, adding corresponding anion solution into the liposome complex, uniformly mixing and incubating for 5 min.

The transfection detection of the encapsulated complex prepared in example 3 is shown in fig. 16, and the experimental results show that various anionic compounds such as sodium carboxymethylcellulose (CMC), heparin sodium (NA), Chondroitin Sulfate (CS), Dermatan Sulfate (DS), Keratan Sulfate (KS), Heparan Sulfate (HS), Sodium Dodecyl Sulfate (SDS), and the like can successfully coat the gene nano vaccine and improve the transfection capability of the gene nano vaccine to a certain extent.

EXAMPLE 4 preparation of micelle/Complex

TABLE 6 prescription composition

1) Preparing micelle nanoparticles by solvent volatilization, adding DOTAP and an auxiliary carrier solution into a round-bottom flask, and drying under a vacuum condition to form a film.

2) RNase-free water was added, and the micelle solution was collected by stirring at 60 ℃.

3) In ice bath, 100W ultrasound for 3 minutes to obtain micelle without drug loading.

4) The nano-drug delivery system with different N/P and different mass ratios is prepared by adopting a method of co-incubation of micellar solution and main drugs, and the method comprises the following steps: taking a proper amount of micelle which is not loaded with drugs as A; adding the main drug into RNase-free water, and dissolving to obtain B; uniformly mixing B and A according to the formula ratio, and incubating at room temperature for 10min to obtain different micelle compounds.

5) The preparation method of the encapsulated compound comprises the following steps: and adding a sodium alginate solution into the micelle compound according to the mixture ratio in the formula table, uniformly mixing, and incubating for 5 min.

Particle size potential and PDI measurements of the encapsulated composites prepared in example 4

The prepared sample was diluted 10 times with purified water, and then subjected to particle size, potential, and PDI detection using a malvern laser particle sizer, with the results shown in table 7.

TABLE 7

In the embodiment, Sodium Alginate (SA), a natural anionic compound, is found to be capable of better wrapping the micelle complex, and the formula 2-formula 4 show that when the mass ratio of mRNA to SA is more than 1:1, the more SA is added, the larger the particle size is, and the potential is stabilized between 20mV and 40 mV; prescription 5-prescription 7 show that after SA is added to a certain amount, namely when the mass ratio of mRNA to SA is less than 1:1, the electrical property of the micelle compound can be reversed, and the charge amount is between-20 mV and-30 mV. The prescription 8-prescription 13 shows that SA can well wrap micellar complex nano preparations loaded with different types, the prescription 14-prescription 17 respectively shows that SA can wrap liposomes loaded with DNA, protein, siRNA and plasmids in a compounding way, and can form a nano complex which is uniform and stable in preparation and has the charge of-20 mV to-30 mV, and the above results show that SA can well wrap micellar complexes.

Example 5 preparation of Shell-core-nanoparticles

Table 8 prescription composition

1) PLGA nanoparticles are prepared by an emulsification-solvent volatilization method. Firstly, weighing a proper amount of PLGA, placing the PLGA in an EP tube, adding a proper amount of ethyl acetate, and dissolving in water bath and ultrasonic to prepare an oil phase. Meanwhile, a proper amount of PVA is weighed and put into an EP tube, RNase-free water is added for dissolving, and an aqueous phase solution with PVA concentration of 1% (w/v) is prepared. And (3) quickly injecting the oil phase into the water phase by using an injector, quickly placing the oil phase into an ice bath condition, quickly transferring the emulsified solution into an eggplant-shaped bottle after ultrasonic emulsification, and performing rotary evaporation under the water bath condition of 37 ℃ to obtain the PLGA nanoparticles.

2) And (3) after the prepared nano-particles are used for hydrating the DOTAP lipid membrane, placing the membrane under a probe for 3min by ultrasound, and obtaining the CS nano-particles without drug loading.

3) A method for preparing nano delivery systems with different N/P and different mass ratios by co-incubation of CS nano particles and main drugs is adopted, and the method comprises the following steps: taking a proper amount of CS nano-particles without medicine loading as A; adding main drugs with different masses into RNase-free water, and dissolving to obtain B; and uniformly mixing the B and the A, and incubating for 10min at room temperature to obtain different CS nanoparticle compounds.

The preparation method of the encapsulated composite comprises the following steps: and adding a sodium alginate solution into the CS nano-particle compound according to the proportion in the formula table, uniformly mixing, and incubating for 5 min.

Particle size potential and PDI measurements of the encapsulated composites prepared in example 5

Taking a prepared sample, diluting the sample by 10 times by using purified water, and detecting the granularity, the potential and PDI by using a Malvern laser particle sizer, wherein the result is as follows:

TABLE 9

Prescription	Particle size (nm)	Electric potential (mV)	PDI
				Prescription
1	120	38	0.39
				Prescription 2	125	32	0.28
Prescription 3	128	27	0.27
				Prescription 4	324	22	0.39
Prescription 5	130	26	0.27
				Prescription 6	133	26	0.25
Prescription 7	125	29	0.23
				Prescription 8	126	22	0.25
Prescription 9	139	23	0.28
				Prescription 10	132	28	0.25
Prescription 11	126	24	0.29
				Prescription 12	124	22	0.26
Prescription 13	129	26	0.29

In the embodiment, the natural anionic compound Sodium Alginate (SA) is found to be capable of better wrapping the core-shell nano-composite, and the formula 2 to the formula 3 show that in a certain range, the more the SA is added, the smaller the potential is, and the formula 4 shows that when the mass ratio of the SA to the main drug reaches 1:1.5, the particle size of the composite is obviously increased; within a certain range, the potential of the compound is reduced along with the increase of the addition amount of SA, and the potential of the compound is stabilized between 20mV and 40 mV; the prescription 5-prescription 9 shows that SA can well wrap the shell-core type nanometer preparation loaded with different kinds of mRNA, the prescription 10-prescription 13 respectively shows that SA can complex and wrap liposome loaded with DNA, protein, siRNA and plasmid, and can form a nanometer compound with uniform and stable particle size and 20 mV-30 mV charge, and the above results show that sodium alginate can well wrap the shell-core type compound.

Example 6 transfection assay, stability and gel electrophoresis experiments

1) Transfection assay

(1) A positive control is prepared by a method of co-incubation of a transfection reagent lipo-2k and a main drug, and the preparation method of the lipo-2k comprises the following steps: 3 μ L of non-loaded lipo-2k into an EP tube containing 50 μ L of opti-MEM medium was designated A; add 1. mu.g GFP-mRNA to an EP tube containing 50. mu.L of opti-MEM medium and record as B; and uniformly mixing the B and the A, and incubating for 10min at room temperature to obtain positive control lipo-2 k.

(2) Transfection experiments were performed on samples of formula 1 to formula 8 in example 1. DC2.4 cells were seeded in 24-well culture plates (1X 10 cells) containing 0.5mL of DMEM (containing 10% fetal bovine serum) ⁴ Cells/well) overnight. The old medium was then replaced with medium containing different concentrations of fetal bovine serum and liposomes/mRNA complexes containing 1. mu.g GFP-mRNA were added to each well and incubated for 24 h. Finally, the transfection effect was evaluated by inverted fluorescence microscopy (Nikon, Japan) and flow cytometry (FACS, BD Accuric6, USA).

Results as shown in fig. 3-6, SA not only significantly enhanced transfection of DOTAP-GFP, but also significantly higher transfection efficiency than liposomal DOTAP/CHOL-GFP prepared using the general recipe; on the other hand, however, the transfection efficiency of SA @ DOTAP/CHOL-GFP prepared by encapsulating commonly prepared liposome DOTAP/CHOL-GFP with SA is significantly reduced, and it is speculated that the presence of CHOL may be detrimental to the encapsulation of the liposome complex by SA.

2) SA @ DOTAP-mRNA storage stability examination

Specifically, the liposome complexes of formula 1, formula 4 and formula 5 in example 1 were stored at 4 ℃ and the stability of the liposome/mRNA complexes was tested by particle size, PDI and Zeta potential. As a result: as shown in FIGS. 11-12, SA @ DOTAP-GFP has more uniform properties than DOTAP-GFP and DOTAP/CHOL-GFP in the stability study, has smaller PDI, and compared with the unencapsulated liposome complex DOTAP-GFP, the SA @ DOTAP-mRNA has no phenomenon of significantly increasing PDI during storage, and the particle size and Zeta potential of the SA @ DOTAP-mRNA have no significant changes in the storage process, and the SA is presumed to have the effect of stabilizing cationic liposomes.

3) Gel electrophoresis experiment

As shown in FIG. 11, the results of examining the stability of formula 1, formula 4 and formula 5 in example 1 without finding mRNA leakage from the preparations DOTAP-GFP, SA @ DOTAP-GFP and DOTAP/CHOL-GFP show that the wrapping of SA does not affect the loading of cationic liposome on mRNA, and further show that SA @ DOTAP-mRNA has wide industrial production and clinical application prospects.

Example 7 formulation study and transfection experiments with different Nanolipids

As shown in fig. 3 to 6, specifically examining from formulation 1 to formulation 8 in example 1, it was found that the anionic compound SA was advantageous in improving the gene delivery ability of DOTAP liposome, and thus, different liposome complexes were prepared by replacing different cationic lipids and these liposome complexes were encapsulated by SA. A plurality of encapsulated complexes are prepared, and the results of examining the prescription 26 to the prescription 31 in example 1 are shown in FIGS. 22, 23 and 24, which show that the encapsulation of SA can promote the gene transfection capability of a plurality of cationic liposomes.

Examining the formulas 1-4 in example 2 and the formulas 1-8 in example 3, the application of different types of anionic compounds in improving the cationic liposome transfection ability was explored, and the results are shown in fig. 14-16, which show that anionic compounds such as HA and SDS can improve the gene transfection ability of the cationic liposome.

Examining the formulas 1 to 6 in example 4 and 1 to 4 in example 5, the encapsulation effect of SA on different types of gene delivery vectors is explored, and the experimental results are shown in fig. 18 and 22, which indicate that SA can encapsulate various types of nano-preparations, such as micelle, polymer, shell-core nanoparticle and other nano-preparations, and can improve the transfection efficiency of the encapsulated layer to a certain extent.

Example 8 mechanism of transfection

TABLE 10 prescription composition

1. Cationic liposomes (DOTAP liposome and DOTAP/CHOL liposome) not loaded with main drugs are prepared by a thin film hydration method respectively. The method comprises the following specific steps:

1) adding DOTAP and cholesterol solution into a round-bottomed bottle, and drying under a vacuum condition to prepare a lipid membrane;

2) adding RNase ultrapure water, and collecting a lipid membrane at 60 ℃;

3) performing 100W ultrasound for 3 minutes in an ice bath to obtain empty DOTAP liposome and empty DOTAP/CHOL liposome;

4) the method for preparing the polypeptide has the N/P of 3 by adopting a method of co-incubating liposome and mRNA molecules marked with CY5 fluorescent dye: 1, the nano-delivery system. Adding a proper amount of unloaded liposome into a certain amount of water, and marking the loaded liposome as A; adding different quality of main drugs into RNase-free water (10 μ L1.5 mM RNase-free NaCl solution), and mixing to obtain B; and uniformly mixing the B and the A, and incubating for 10min at room temperature to obtain different liposome composites.

2. The preparation method of the encapsulated compound (SA @ DOTAP-CY5) comprises the following steps: adding 500 μ L sodium alginate solution with concentration of 200 μ g/ml into 1ml liposome complex, mixing well, and incubating for 5 min.

3. Cellular uptake

Specifically, in examples 8, recipes 1 to 3, DC2.4 cells were inoculated into 24-well plates (1X 10) ⁵ Individual cells/well) incubated overnight in 0.5mL DMEM containing 10% fetal bovine serum. The medium was replaced with fresh DMEM containing 10% fetal bovine serum and liposomes/mRNA complexes containing 1. mu.g CY5-mRNA were added to each well. 6h, researching the uptake of the DC2.4 cells to DOTAP-CY5, DOTAP/CHOL-CY5 and SA @ DOTAP-CY5 by using confocal microscopy and flow cytometry; and collecting cells, washing the cells with PBS for 2 times, and researching the uptake of the DC2.4 cells to DOTAP-CY5, DOTAP/CHOL-CY5 and SA @ DOTAP-CY5 by using flow cytometry so as to determine whether the higher transfection efficiency of SA @ DOTAP-GFP is related to the cellular uptake. As a result: as shown in fig. 25-26, SA and CHOL both up-regulate endocytosis of DOTAP-CY5, but there was no significant difference in endocytosis of DOTAP/CHOL-CY5 and SA @ DOTAP-CY 5. Thus, pretreatment of cells with cellular uptake inhibitors explored whether the uptake mechanism of the nanoformulation by the cells occurredAnd (6) generating changes. DC2.4 cells were seeded in 24-well plates (1X 10^ s) ⁵ Individual cells/well) were incubated overnight in 0.5mL DMEM containing 10% fetal bovine serum. The medium was then replaced with 500 μ L DMEM containing different uptake inhibitors. (the amount of uptake inhibitor: chlorpromazine, 5. mu.g/well; cytochalasin D, 0.255. mu.g/well; nystatin, 5. mu.g /). After 0.5h incubation, liposomes/mRNA complexes containing 1. mu.g CY5-mRNA were added per well and incubated for 2 h. Finally, the cells were collected and the fluorescence intensity was measured by flow cytometry.

As a result: as shown in figure 27, cellular uptake of DOTAP/CHOL-CY5, DOTAP-CY5, and SA @ DOTAP-CY5 decreased after pretreatment with clathrin and the macropinocytostatic inhibitors chlorpromazine and cytochalasin D, while cellular uptake of SA @ DOTAP-CY5 was inhibited by the inhibitor nystatin. Indicating that SA @ DOTAP-mRNA can enter cells via a caveolin-mediated pathway.

Vesicle protein-dependent endocytosis can prevent entry of the agent into the lysosome. Vaccines based on vesicle protein-dependent endocytosis are ultimately transported to the endoplasmic reticulum, where ribosomes and Major Histocompatibility Complex (MHC) molecules provide a convenient route for mRNA translation and antigen presentation. Provides basis for the efficient translation and antigen presentation of SA @ DOTAP-mRNA, which is shown in detail in FIG. 27.

4. Lysosomal escape

Specifically, the lysosome escape abilities of formulas 1 to 3 in example 8 were examined by inoculating DC2.4 cells into a confocal culture dish (x 10) containing 2mL of DMEM medium (containing 10% fetal bovine serum) ^{^5} Cells/well) for 24 h. CY5-mRNA is used as a tracer, and LysoTracker is used as a lysosome marker. Each plate was loaded with liposomes/mRNA complexes of CY5-mRNA (CY5-mRNA final concentration 0.5. mu.g/plate) and incubated in the dark at 37 ℃ for 2 h. After the incubation time point was reached, the medium was discarded, the cells were washed 2 times with pre-cooled PBS, and the nanopreparative adsorbed on the cell surface was removed (1 h before termination of uptake, lysosome marker (final concentration of 75 μ M Lyso-Tracker) was added to label lysosomes). Cells were pre-cooled, rinsed twice with PBS, and lysosome escape capacity of DOTAP/CHOL-CY5, DOTAP-CY5, and SA @ DOTAP-CY5 were investigated using confocal laser microscopy.

As a result: as shown in FIG. 28, both DOTA-CY5 and DOTAP/CHOL-CY5 co-localized with lysosomes to varying degrees, whereas SA @ DOTAP-CY5 co-localized with lysosomes less. The data show that SA has certain anionic polymer characteristics, and the escape capacity of nano-drugs from lysosomes can be improved.

First, the SA side chain may be replaced by H ⁺ Neutralization, separation as pH decreases; secondly, a non-charged hydrophobic side chain is inserted into a hydrophobic part of an endosome membrane, so that the stability of the endosome is reduced, and the endosome escape is promoted; third, cationic lipids disrupt the lysosomal membrane by fusing with anionic phospholipids on the lysosomal membrane, thereby mediating escape of liposomes in the lysosome.

In the present invention, our formulation contains only mRNA, cationic lipid DOTAP and coating material SA. After SA is combined with hydrogen ions and falls off, DOTAP-mRNA is exposed, the stabilizing effect of CHOL on the lipid membrane of the DOTAP-mRNA is avoided, and DOTAP molecules are easier to fuse with a lysosome membrane, so that the lysosome escape effect is improved.

5. Antiserum transfection assay

Firstly, a transfection experiment is carried out on DC2.4 cells under the condition of high-concentration fetal bovine serum, and a formula 1, a formula 5 and a formula 7 in example 1 are specifically examined, so that whether SA @ DOTAP-GFP increases the transfection amount by reducing serum protein adsorption is verified.

As a result: as shown in FIG. 30, SA/DOTAP-GFP has higher transfection efficiency under the condition of 30% fetal bovine serum, while the transfection efficiencies of DOTAP-GFP and DOTAP/CHOL-GFP are obviously reduced.

Second, the antiserum abilities of the prescription 1, the prescription 4 and the prescription 5 in the example 1 are specifically considered, the aqueous solution containing 50% fetal calf serum is respectively incubated with the preparations of the prescription 1, the prescription 5 and the prescription 7 in the example 1, and the particle size change of the three liposome/mRNA complexes is measured.

As a result: as shown in FIG. 29, the particle size of SA @ DOTAP-GFP did not change much with incubation time compared to DOTAP-GFP and DOTAP/CHOL-GFP, indicating that SA significantly reduced the adsorption of DOTAP to FBS.

Example 9 Immunity Activity examination

Table 11 recipe compositions

1) In this example, SA @ DOTAP-OVA, DOTAP-OVA and DOTAP/CHOL-OVA, DOTAP-LUC, DOTAP/CHOL-LUC, SA @ DOTAP-LUC were prepared according to the formulation of example 1 using OVA-mRNA as the main agent and following the formulation preparation method of example 1.

2) The antigen presenting ability of SA @ DOTAP-OVA, DOTAP-OVA and DOTAP/CHOL-OVA samples in example 9 on BMDCs cells and the ability of promoting the maturation and activation of the BMDCs cells are specifically examined to verify whether the antigen presenting effect can be improved by the high transfected SA @ DOTAP-mRNA. In particular BMDCs (5 x 10^ s) ⁵ ) Inoculate 0.5h in medium containing 0.5mL RPMI-1640 (containing 10% FBS), then add 1 μ g OVA-mRNA complex to each well, after 24h incubation. BMDCs cells were harvested and stained with PE-anti-mouse CD11c, FITC-anti-mouse CD86, APC-anti-mouse SIINFEKL/H-2Kb 25-D1.16 flow antibody. The antigen presentation of each preparation on BMDCs cells was examined by flow cytometry.

As a result: as shown in FIGS. 30-32, SA @ DOTAP-OVA had a stronger antigen-presenting effect than the control groups DOTAP-OVA and DOTAP/CHOL-OVA.

3) Samples of DOTAP-LUC, DOTAP/CHOL-LUC, SA @ DOTAP-LUC from example 9 were used to study the expression of SA @ DOTAP-LUC in various organs of mice.

In vivo expression of DOTAP-LUC, DOTAP/CHOL-LUC and SA @ DOTAP-LUC was detected in 6-7 week-old male C57BL/6/C mice, 3 mice per group. LUC-mRNA was used as a tool mRNA and was administered at a dose of 30. mu.g/mouse. The mice are killed 6h after injection, and the in vivo expression conditions of the four groups of liposome/mRNA complexes of the heart, the liver, the spleen, the lung and the kidney of the mice are observed by in vitro imaging.

As a result: as shown in FIGS. 33-34, SA @ DOTAP-LUC was significantly higher in both lung and spleen than the control groups DOTAP-LUC and DOTAP/CHOL-LUC. The results of the detection of SA @ DOTAP-mRNA (FIG. 2) are combined to illustrate that changing the charge of the liposomes is a feasible way to change the cationic liposome distribution.

4) Samples of SA @ DOTAP-OVA, DOTAP-OVA and DOTAP/CHOL-OVA from example 9 were used to investigate whether SA @ DOTAP-OVA could better promote the proliferative differentiation of T cells into specific CTLs due to its high transfection efficiency and strong antigen-presenting ability.

Normal mice were immunized with samples of SA @ DOTAP-OVA, DOTAP-OVA and DOTAP/CHOL-OVA from example 9, mice were sacrificed at day25 and spleen cell suspensions were prepared from each group of mice by mouse immunotherapy (mRNA dose 10. mu.g/mouse) at day7, day10 and day15, respectively. Then 1. mu.L of FITC-anti-mouse CD3, 1. mu.L of APC-anti-mouse CD8A and 1. mu. L H-2kB OVA tetramer SIINFKL/H-2Kb was added to the flow tube and incubated in the dark at 4 ℃ for 40 min. Finally, OVA-specific T cells were detected by flow cytometry.

As a result: as shown in FIG. 35, there was no significant difference between the control group DOTAP-OVA and DOTAP/CHOL-OVA in promoting proliferation of OVA-specific CLTs, and SA @ DOTAP-OVA significantly promoted proliferation of OVA-specific CLTs. In addition, lymph nodes in the treated mice increased significantly, and higher levels of antigen presenting ability promoted proliferation of OVA-specific CTLs, providing potential for effective anti-tumor immune responses, as shown in fig. 37.

EXAMPLE 10 pharmacodynamic experiment

In vivo antitumor effect

Since TCR activation and IFN signaling may depend on the route of mRNA administration, some studies have shown that by intravenous mRNA vaccines can avoid the adverse effects of innate immunity presented by mRNA and promote CD8+ T cell responses. Therefore, this example uses intravenous administration to examine the in vivo efficacy.

The therapeutic effect on tumor-bearing mice was examined using samples of SA @ DOTAP-OVA, DOTAP-OVA and DOTAP/CHOL-OVA and SA @ DOTAP-LUC in example 9, and EG7 tumor-bearing mice were treated by intravenous injection of mouse tail with DOTAP-OVA and DOTAP/CHOL-OVA as positive controls, SA @ DOTAP-LUC as negative controls, and SA @ DOTAP-OVA as the target treatment group. As shown in FIG. 24, after successful tumor inoculation, mice were treated with mouse immunotherapy (mRNA dose 10. mu.g/mouse) at day7, day10, day15, and the change in tumor volume was monitored every two days from the first administration, and was examined. Tumor growth in each group of mice is shown in FIG. 38.

Compared with the saline group, each group inhibited tumor growth by increasing antigen presentation, while SA @ DOTAP-LUC had a slight inhibition effect on tumor growth, which is probably due to the fact that DOTAP itself has a certain immunoadjuvant effect. The treatment effects of the DOTAP/CHOL-OVA and the DOTAP-OVA are not obviously different, and the antitumor effect of the SA @ DOTAP-OVA is obviously higher than that of the DOTAP-OVA.

As shown in FIG. 39 and FIG. 40-A, although there was no significant difference between tumor volume and mouse tumor weight, there was a significant regression of tumor growth trend in the SA @ DOTAP-OVA group after the last administration.

As shown in FIG. 40-B, the tumor inhibition rate of SA @ DOTAP-OVA was 56%, the tumor inhibition rate of DOTAP/CHOL-OVA was 37%, and the tumor inhibition rate of SA @ DOTAP-OVA was significantly higher than that of DOTAP/CHOL-OVA.

As shown in FIG. 40 and FIG. 41, the tumor-infiltrating OVA-specific CTL in the SA @ DOTAP-OVA group was significantly higher than that in the control group DOTAP/CHOL-OVA, i.e., the anti-tumor effect of SA @ DOTAP-OVA was stronger than that of DOTAP/CHOL-OVA.

Example 11 evaluation of safety and toxicity of enveloped mRNA vaccines

1) Whether SA reduces the toxicity of cationic liposomes was tested by CCK8 to test the cytotoxic effects of SA @ DOTAP-OVA, DOTAP-OVA and DOTAP/CHOL-OVA formulations in example 9.

DC2.4 cells were seeded onto 96-well plates (cell density 1X 10) ⁴ Cells/well), 24h later, different amounts of nano-vaccine were added to each well. After 24h incubation, 101. mu.L of CCK8 was added to each well, incubated at 37 ℃ for 4h, and cell viability was measured using a microplate reader.

As a result: as shown in FIG. 42, formulation 3 (SA @ DOTAP-OVA) in example 9 was less toxic than the control group DOTAP-OVA and DOTAP/CHOL-OVA, and had no abnormal changes in intermediate weights during treatment.

2) The effect of samples of SA @ DOTAP-OVA, DOTAP-OVA, DOTAP/CHOL-OVA, and SA @ DOTAP-LUC from example 9 on the physiological safety of mice was further investigated by H & E staining and biochemical analysis.

In example 10, after the treatment of the mice, 1 mouse was randomly selected from each group, and vital organs thereof were taken, and pathological tissue sections of the organs were prepared and stained. And observing the stained tissues and taking pictures on a pathological section scanner, and observing pathological changes of important tissues and organs such as liver, kidney, spleen, lung and the like of the mouse.

As a result: as shown in FIGS. 43 to 45, no significant pulmonary toxicity was found in mice by H & E staining for DOTAP/CHOL-OVA. However, biochemical indexes show that the DOTAP-OVA and the DOTAP/CHOL-OVA cause damage to main organs of mice such as liver and kidney, and the SA @ DOTAP-OVA does not obviously damage the organs of the mice. This shows that the addition of SA can reduce the damage of DOTAP-OVA to the functions of the important organs of mice, i.e. the toxicity and inflammatory reaction of the tissues of the mice induced by the DOTAP-OVA can be reduced by adding SA, i.e. a safer liposome/mRNA compound for gene therapy can be established by modifying SA.

In conclusion, the invention obtains an encapsulated nano preparation with simplified prescription, high stability and easy preparation by encapsulating the anionic material polymer on the active ingredient-containing nano composite with positive charge, has obvious effect on improving the in vivo and in vitro stability and delivery efficiency, and provides a method for improving the existing nano medicament by using the anionic compound.

Claims

1. An encapsulated composite for reducing serum adsorption and/or resisting adhesion of non-target cells, wherein the encapsulated composite is composed of an outer encapsulating layer and an inner composite, and the inner composite is composed of a nano preparation which is easily adsorbed by serum and/or adhered by non-target cells; the external wrapping layer is anionic compounds such as sodium hyaluronate, sodium alginate, sodium carboxymethylcellulose, sodium heparin, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, sodium dodecyl sulfate and the like and related derivatives thereof.

2. The encapsulated complex of claim 1, wherein said nanoformulation is useful for loading nucleic acid drugs, protein polypeptide drugs, or small molecule drugs, and mixtures thereof.

3. The encapsulated complex of claim 1, wherein said nano-formulation is a cationic formulation including, but not limited to: liposome, nanogel, core-shell nanoparticles, HDL nanoparticles, lipid nanoparticles, solid lipid nanoparticles, polymer micelle lipid nanoparticles, polymer nanoparticles, micelles and emulsions.

4. The encapsulated complex of claim 3, wherein said cationic liposome comprises one or more of DOTAP, DOTMA, DOSPA, DTAB, DDAB, DC-CHOL lipid material.

5. The encapsulated composite of claim 1, wherein the surface charge of the composite is between 40 and-30 mV.

6. The compound of claim 2, wherein the mass ratio of the coating layer to the nucleic acid drug, the protein polypeptide drug or the small molecule drug is 1: 0.25-1: 4.

7. The encapsidated complex of claim 2, wherein the mRNA includes, but is not limited to, encoded viral antigen proteins, gene editing tool proteins, protein-complement therapy-associated proteins, cytokines. In particular, the protein can be used for encoding antigen proteins of EBV, HPV, HBV, SARS-Cov-2, antigen proteins of malaria, syncytial virus, dengue, Zika, rabies and influenza virus, encoding tumor-associated antigen proteins such as Mucin1, KRAS and the like, encoding gene edited Cas9, encoding cytokine IL 12.

8. The encapsulated complex of any of claims 1-7, wherein said complex stabilizes the lipid molecular layer of the liposome without the need for a membrane stabilizing agent.

9. The coated nano preparation is characterized by consisting of a nano preparation and a coating layer, wherein the coating layer is an anionic compound or a related anionic mixture; the nano preparation is prepared from a material with at least one positive cation center, the nano preparation material is a pharmaceutically acceptable carrier material for preparing micelles, emulsions, nanogels, core-shell nanoparticles, HDL nanoparticles, solid lipid nanoparticles, liposomes and/or polymeric micelles, and the nano preparation is prepared by a pharmaceutically acceptable method.

10. The coated nano-formulation according to claim 9, wherein the coated nano-formulation comprises:

(c) a non-cationic lipid comprising a mixture of phospholipids and or other carriers, in an amount of 10-50% by weight of the total lipid in the mixture;

(d) an anionic compound or a derivative thereof.

11. Use of the encapsulated complex of claim 1 or the encapsulated nano-formulation of claim 9 for the preparation of a vaccine.

12. The use according to claim 11, wherein the diseases for which the vaccine is used for treatment include, but are not limited to nasopharyngeal carcinoma, cervical carcinoma, head and neck squamous carcinoma, liver carcinoma, breast carcinoma, ovarian carcinoma, pancreatic carcinoma, gastric carcinoma, viral infections and atherosclerosis.

13. The method for preparing the inclusion complex according to claim 1 or the inclusion nano-formulation according to claim 9, which comprises the following steps:

2) adding the anionic compound or the anionic mixture to be adsorbed by the coating layer to obtain the coating type compound or the nano preparation.

14. When the mRNA vaccine is prepared, the anionic compound or the anionic mixture is modified/coated on the surface of the nano preparation, so that the gene transfection efficiency of the nano preparation is improved.

15. In the preparation of mRNA vaccines, the anionic compound or the anionic mixture is used to alter endocytic pathways and/or enhance lysosomal escape capacity by modification/encapsulation on the nanoformulation surface.

16. Use of said anionic compound or said anionic mixture as an in vivo expression enhancer, spleen targeting agent, antigen presenting ability enhancer or proliferation inducer of CTL in the preparation of mRNA vaccine.