阅读说明：本技术 一种用于mRNA疫苗靶向递送的聚氨基酸及其制备方法和用途 (Polyamino acid for mRNA vaccine targeted delivery and preparation method and application thereof ) 是由 王海龙 于 2021-10-08 设计创作，主要内容包括：本发明提供了一种用于mRNA疫苗靶向递送的的聚氨基酸及其制备方法和用途,所述聚氨基酸为侧链修饰的聚氨基酸,通过在聚氨基酸的侧链中引入亲水性成分和疏水性成分,能够形成聚氨基酸胶束,所述胶束用于高效地递送核酸分子。本发明的所述侧链修饰的聚氨基酸在侧链上引入在还原性环境中裂解的双硫键,能促进胶束对胞浆中还原性环境和低pH值响应,帮助mRNA疫苗从“溶酶体陷阱”中主动逃离进入胞浆。相较于被动地渗透压主导的逃离过程,纳米载体的主动裂解能更高效地释放mRNA。(The invention provides a polyamino acid for targeted delivery of an mRNA vaccine, a preparation method and application thereof. The side chain modified polyamino acid of the invention introduces disulfide bond cracked in reducing environment on the side chain, which can promote the response of micelle to reducing environment and low pH value in cytoplasm, and help mRNA vaccine actively escape from 'lysosome trap' and enter into cytoplasm. Active cleavage of the nanocarriers can release mRNA more efficiently than passive osmotic pressure dominated escape processes.)

1. A side-chain modified polyamino acid, wherein the polyamino acid comprises a backbone and a side chain; the main chain is polylysine or a copolymer formed by lysine and other amino acids; the other amino acids are selected from one or more of serine, threonine, tyrosine, arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, tryptophan, proline, asparagine and glutamine;

at least part of the side chains respectively contain a group A and a group G, wherein the group A is-L³-S-S-C_4-25Aliphatic hydrocarbonsBase, L³Is C_1-10An aliphatic alkylene radical, the group G being selected from the group G1 and/or the group G2, wherein the group G1 is terminated by a C_1-10The polyethylene glycol group of alkoxy, the group G2 is a polyethylene glycol group with a biological functional group connected with the end group;

the group a and the group G are linked to the amino acids in the backbone via a linking group Y.

2. The polyamino acid of claim 1, wherein the group G1 is-L¹-PEG-L²-Z¹-D, wherein PEG is a polyethylene glycol segment and D is C_1-10Alkoxy radical, Z¹Is a linking group, L¹、L²Is a direct bond or a spacer group;

the group G2 is-L¹-PEG-L²-Z²-E, wherein PEG is a polyethylene glycol segment, E is a biofunctional group, Z²Is a linking group, L¹、L²Is a direct bond or a spacer group.

3. The polyamino acid of claim 2, wherein L¹、L²Is a direct bond or C_1-10An alkyl group;

the linking group Z¹、Z²Comprises the following groups:

-CO-NH-、-CO-O-、-O-、-O-CH₂-CO-、-NH-、-N=CR₁-、-N-CHR₁-、-NH-CH₂-CO-、-S-S-、-S-CO-、-S-CH₂-CO-orAny one of a connecting group obtained by click reaction of azido and alkynyl and a connecting group obtained by click reaction of tetrazine and double bond;

the biological functional group is a bonder capable of being combined with protein, polypeptide and amino acid, and comprises a artificially synthesized bonder or natural biological molecules.

4. The polyamino acid of any one of claims 1 to 3, wherein the side chain modified polyamino acid may be one of the following: polyamino acids comprising group a and group G1, polyamino acids comprising group a and group G2, polyamino acids comprising group a, group G1 and group G2;

in the polyamino acid, the mole percentage of the number of side chains containing a group G1 and/or a group G2 to the total amount of all side chains is 2-98%, the mole percentage of the number of side chains containing a group A to the total amount of all side chains is 2-98%, and the mole percentage of the sum of the number of side chains containing a group A and the number of side chains containing a group G1 and/or a group G2 to the total amount of all side chains is 5-100%.

5. A side chain modified polyamino acid is prepared by the following preparation method:

the polyamino acid with side chain having active group is connected with one end of C_1-10Polyethylene glycol with reactive group x1 connected to the other end of alkoxy group and/or polyethylene glycol with biological functional group connected to one end and reactive group x2 connected to the other end, and C with reactive group x3 connected to the other end and activated disulfide bond_1-10Reacting aliphatic hydrocarbon; wherein the reactive groups x1, x2 and x3 react with the active groups on the side chain of the polyamino acid, such that the end group is provided with C_1-10Polyethylene glycol of alkoxy and/or biological functional group and aliphatic hydrocarbon containing activated disulfide bond group are connected to the side chain of polyamino acid; then the product is mixed with C_4-25And (3) reacting aliphatic hydrocarbon mercaptan to prepare the side chain modified polyamino acid.

6. The polyamino acid of claim 5, wherein the activated disulfide bond is-S-S-pyridyl.

7. A method for producing the side-chain-modified polyamino acid according to any one of claims 1 to 6, comprising the steps of:

the polyamino acid with side chain having active group is connected with one end of C_1-10Polyethylene glycol with reactive group x1 connected to the other end of alkoxyAnd/or polyethylene glycol with a biological functional group connected to one end and a reactive group x2 connected to the other end, and C with a reactive group x3 connected to one end and an activated disulfide bond connected to the other end_1-10Reacting aliphatic hydrocarbon; wherein the reactive groups x1, x2 and x3 react with the active groups on the side chain of the polyamino acid, such that the end group is provided with C_1-10Polyethylene glycol of alkoxy and/or biological functional group and aliphatic hydrocarbon with activated disulfide bond at the end group are connected to the side chain of polyamino acid; then the product is mixed with C_4-25And (3) reacting aliphatic hydrocarbon mercaptan to prepare the side chain modified polyamino acid.

8. A micelle comprising the side chain-modified polyamino acid of any one of claims 1 to 6.

9. A delivery system comprising the micelle of claim 8 and a nucleic acid molecule, the nucleic acid molecule being located within the micelle.

10. The delivery system of claim 9, wherein the nucleic acid molecule is an RNA vaccine, an RNA drug, a DNA vaccine.

Technical Field

The invention belongs to the field of polymer materials, and relates to a polyamino acid for mRNA vaccine targeted delivery, and a preparation method and application thereof.

Background

Viruses are a significant public health safety threat that endangers human health. The use of vaccines is an effective means for combating viruses in humans. Since the seed 'vaccinia' was used to overcome smallpox virus, vaccines have helped humans to effectively control and prevent a variety of viral diseases, including hepatitis a, hepatitis b, meningitis b, and influenza. The development of vaccines against the fatal Ebola (Ebola) virus and Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and 2019 coronavirus disease (COVID-19) caused by coronavirus is an urgent need to control the epidemic situation of virus infection. According to different technical routes, the types of vaccines include inactivated vaccines, recombinant protein vaccines, adenoviral vector vaccines, and nucleic acid vaccines. Wherein, ribonucleic acid (RNA) vaccine generates a protein segment of a binding region with a virus receptor, a virus capsid protein or other conserved regions in the cytoplasm of human cells as an antigen through a protein transcription-translation mechanism of the cells, induces an immune system to generate antibodies, and realizes the immunity to the virus.

Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are important genetic materials in life systems. According to the life center rule, genetic information is transcribed and translated into functional proteins through the process of DNA-RNA-protein. In this process, the cells use messenger rna (mrna) as a template to translate the protein. Messenger RNA (mrna) is a class of RNA molecules that stores genetic information in the base pair sequence of nucleotides a-U, G-C, transfers the genetic information from DNA to ribosomes, where it serves as a template for protein synthesis and expresses protein products. The mRNA vaccine does not need protein expression and purification, and mRNA is transferred to human cells to form immunity by synthesizing related sequence mRNA of the virus in vitro. mRNA vaccines thus have many advantages: 1) mRNA is easy to produce and purify in vitro, removes the complex process of preparing protein drugs and virus vectors, and can avoid host protein and virus-derived pollution; 2) the production process of the mRNA has strong universality, can be quickly applied to producing different target proteins, saves the time for developing the medicament and improves the efficiency; 3) mRNA can be translated into protein only by entering cytoplasm without entering cell nucleus, so that gene insertion and integration do not exist, and the safety of the medicine is improved; 4) the half-life can be altered by adjusting sequence modifications and delivery vectors. The mRNA vaccine has the characteristics of high biological safety, flexible design, rapid development and strong universality, so that the mRNA vaccine becomes an important way for vaccine development. The mRNA vaccine can not only prevent and inhibit the infection of the virus in organisms, but also treat tumors by using the mRNA vaccine, and for example, the mRNA vaccine developed aiming at melanoma and small cell lung cancer is reported to enter the clinical test stage.

In clinical trials, it has been found that the effectiveness of mRNA vaccines is limited by the following specific factors: 1) inability to pass freely through biological membranes; 2) are easily decomposed by RNA degrading enzymes (RNases) in plasma and tissues; 3) after entering the cell, the endosome is captured by endocytosomes (endosomes) and develops into "lysosomal traps" (lysosome trapping), which cannot function. Therefore mRNA immunization efficiency depends on improving the stability of mRNA vaccines in vivo and overcoming intracellular vesicle transport mechanisms. Due to the hydroxyl substitution at nucleotide position 2, RNA is less stable in physiological environments than DNA. To avoid hydrolytic and enzymatic consumption of RNA during circulation in the body, mRNA vaccines and drugs require encapsulation protection of the delivery system. However, the delivery efficiency of existing delivery systems is low due to the presence of "lysosomal traps," and efficient delivery of mRNA vaccines and drugs cannot be achieved.

In order to allow efficient entry of the vaccine into cells and release from "lysosomal traps" into the cytosol through a delivery system, the manner in which the mRNA vaccine is administered and the mode of delivery play a key role in the control of pharmacokinetics and dosage. The prior art uses liposomes, lipid nanoparticles as delivery systems to make nanocomposites of mRNA vaccines and drugs. Lipid nanoparticles containing positively charged lipid molecules can achieve good loading of negatively charged nucleic acid molecules by electrostatic interactions. In order to efficiently release mRNA vaccines and drugs from "lysosomal traps" into the cytosol, optimization of the stability, environmental response and targeted delivery of the delivery system is required. The environmental response includes changes in the microenvironment of the cells and tissues, such as pH, oxidation-reduction environment, and changes in hydrophilic-hydrophobic environment caused by contact with membrane structures. The target delivery capability refers to the capability of performing biological functionalization on a nucleic acid delivery system and obtaining target delivery capability on target cells (such as cancer cells, B cells and T cells of the immune system) and tissues (such as lymphoid tissues, lung, small intestine and other organs) by using biological functional ligands and antibodies.

Micelles are a form of nano aggregates formed spontaneously in an aqueous solution, are mostly spherical and also can be columnar. Surfactants containing both hydrophilic and lipophilic groups spontaneously associate into micelles when they reach and exceed a Critical Micelle Concentration (CMC) in solution. The micelle is in dynamic balance in the solution, can wrap and release the effective components, and is suitable for serving as a delivery system. The hydrophobic core of the micelle makes it possible to encapsulate poorly soluble hydrophobic compounds in the aqueous phase. The micelle having a negatively or positively charged component may be loaded with an active ingredient having a positive or negative charge. The micelle with biocompatibility can maintain the activity of the contacted biomolecules, ensure low cytotoxicity, and can be biodegraded in vivo or harmlessly discharged in vitro. The prior art reports the formation of high molecular micelles by structural design of block copolymers. For example under the trade name Pluronic^®A class of polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) triblock polymeric materials of F-127. In addition, it has been reported that a graft dendrite copolymer having a chitin backbone forms a micellar nanomaterial.

Disclosure of Invention

In order to overcome the low delivery efficiency of existing nucleic acid molecules (such as mRNA vaccines) due to "lysosomal traps", the present invention aims to provide a polyamino acid micellar delivery system that is sensitive to oxidation-reduction environment and/or pH changes in cells and tissue structures. Specifically, the invention provides a polyamino acid for delivering nucleic acid molecules, and a preparation method and application thereof. Polyamino acids are a class of backbone types that are completely biodegradable, and the backbone of polyamino acids is more biologically stable in the in vivo environment than ester linkages based on amide linkages.

The purpose of the invention is realized by the following technical scheme:

a side-chain modified polyamino acid, wherein the polyamino acid comprises a backbone and a side chain; the main chain is polylysine or a copolymer formed by lysine and other amino acids; the other amino acids are selected from one or more of serine, threonine, tyrosine, and arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, tryptophan, proline, asparagine, and glutamine;

at least part of the side chains respectively contain a group A and a group G, wherein the group A is-L³-S-S-C_4-25Aliphatic hydrocarbon radical, L³Is C_1-10An aliphatic alkylene radical, the group G being selected from the group G1 and/or the group G2, wherein the group G1 is terminated by a C_1-10The polyethylene glycol group of alkoxy, the group G2 is a polyethylene glycol group with a biological functional group connected with the end group;

the group a or the group G is linked to an amino acid in the backbone via a linking group Y.

In one embodiment, said C_4-25The aliphatic hydrocarbon group is C_4-25Alkyl radical, C_4-25Alkenyl radical, C_4-25Alkynyl.

In one embodiment, L³Is C_1-6An alkyl group.

In one embodiment, the linking group Y is the following group: -NH-CO-, -NH-, -N = CR₁-、-N-CHR₁-、-NH-CH₂-CO-、-O-CO-、-O-、-O-CH₂-any one of CO-. Wherein R is₁Independently selected from H, C_1-6An alkyl group. The first amino group or oxygen from the left in the above-mentioned linking group Y is derived from a reactive group such as an amino group or a hydroxyl group on the side chain of the polyamino acid.

In one embodiment, the linking group Y may be a direct bond (direct link) to the group a, or any spacer group may be present.

In one embodiment, the linking group Y may be a direct bond (direct link) to group G, or any spacer group may be present.

In one embodiment, the group G1 is-L¹-PEG-L²-Z¹-D, wherein PEG is a polyethylene glycol segment and D is C_1-10Alkoxy radical, Z¹Is a linking group, L¹、L²Is a direct bond or a spacer group.

In one embodiment, the group G2 is-L¹-PEG-L²-Z²-E, wherein PEG is a polyethylene glycol segment, E is a biofunctional group, Z²Is a linking group, L¹、L²Is a direct bond or a spacer group.

In one embodiment, L¹、L²For example, a direct bond, C_1-10An alkyl group.

In one embodiment, the linking group Z¹、Z²Comprises the following groups:

-CO-NH-、-CO-O-、-O-、-O-CH₂-CO-、-NH-、-N=CR₁-、-N-CHR₁-、-NH-CH₂-CO-、-S-S-、-S-CO-、-S-CH₂-CO-orAny one of a connecting group obtained by click reaction of azido and alkynyl and a connecting group obtained by click reaction of tetrazine and double bond.

Wherein, the connecting group obtained by click reaction of the azido and the alkynyl is triazolyl or a derivative thereof; the connecting group obtained by click reaction of tetrazine and double bonds is diazacyclo or derivatives thereof.

In one embodiment, the number of repeating units of the polyethylene glycol segment is an integer between 1 and 600, preferably an integer between 2 and 300, and more preferably an integer between 4 and 200.

In one embodiment, the disulfide bond in the group a may be cleaved in a reducing environment.

In the present invention, the reducing environment comprises a reducing agent, such as one or more of a mercapto compound, a phosphino compound, a sulfite, a dithionite, and a thiosulfate; preferably selected from one or more of glutathione, cysteine, dithiothreitol, mercaptoethanol, cysteamine and tris- (2-carboxyethyl) phosphine.

In one embodiment, the alkoxy group in the group G1 is C_1-6Alkoxy radicals, such as methoxy, ethoxy.

In one embodiment, the biofunctional group is a linker capable of binding to a biomolecule such as a protein, polypeptide, amino acid, etc., including a synthetic linker or a natural biomolecule.

In one embodiment, the binder comprises biotin which can bind to avidin; linkers that can be attached to the SNAP protein, such as benzylguanine and derivatives thereof; linkers that can be attached to CLIP proteins, such as benzyl cytosine and derivatives thereof; linkers (HaloTag Ligand (HTL)) that can be attached to a HaloTag protein, such as 6-chlorohexane and derivatives thereof; the derivative may be an oligoethylene glycol derivative having an ethylene glycol repeating unit number of less than 46.

In one embodiment, the natural biomolecule comprises at least one of a carbohydrate molecule, an RNA segment, an amino acid sequence.

Wherein the saccharide molecules are saccharide molecules capable of combining with receptor proteins and comprise monosaccharide, disaccharide and oligosaccharide; the monosaccharide is at least one of dihydroxyacetone, erythrose, threose, arabinose, ribose, deoxyribose, xylose, lyxose, glucose, mannose, fructose, galactose and rhamnose. The disaccharide is at least one selected from sucrose, lactose and maltose; the oligosaccharide is selected from at least one of maltotriose, melezitose, raffinose, gentianose, mannotriose and rhamnose, and at least one of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and delta-cyclodextrin which can be combined with other small molecules.

Wherein the RNA segment is a nucleotide or deoxynucleotide sequence which can be complementary with the biomolecule to be detected, such as a nucleic acid aptamer sequence which is 5-80 nucleotides and can be combined with protein.

Wherein the amino acid sequence is an amino acid sequence capable of binding to a protein, such as an antibody, and is about 2 to 100 amino acid residues in length, including influenza hemagglutinin HA amino acid sequence, FLAG amino acid sequence, myc-tag amino acid sequence, "C-tag" comprising-EPEA amino acid sequence, and amino acid "ALFA-tag" comprising SRLEEELRRRL.

In the invention, the antibody is at least one of polyclonal antibody, monoclonal antibody and nano antibody, and the antibody is an antibody derived from animals or an antibody prepared by expression of recombinant protein.

In one embodiment, the side chain modified polyamino acid may be one of the following: polyamino acids comprising group a and group G1, polyamino acids comprising group a and group G2, polyamino acids comprising group a, group G1 and group G2. In the polyamino acid, a polyamino acid side chain that is not modified may be present.

In one embodiment, the polyamino acid has a molar percentage of side chains containing group G1 and/or group G2 to the total amount of side chains ranging from 2% to 98%, preferably from 5% to 90%, more preferably from 10% to 80%.

In one embodiment, the number of side chains containing group a in the polyamino acid is from 2% to 98%, preferably from 5% to 90%, more preferably from 10% to 80% by mole of the total number of side chains.

In one embodiment, the molar percentage of the sum of the number of side chains containing a group a and the number of side chains containing group G1 and/or group G2 in the total number of side chains in the polyamino acid is 5% to 100%, preferably 20% to 100%, more preferably 40% to 100%, and may be, for example, 30% to 98%, 35% to 95%, or 40% to 90%.

In one embodiment, arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, proline, tryptophan, asparagine, glutamine in the backbone can adjust the hydrophilicity and hydrophobicity, hydrogen bonding ability, and pH response of the polyamino acid based on the hydrophobic characteristics or molecular configuration, size, aromaticity, hydrogen bonding ability, pH response, etc. of its side chains.

In one embodiment, the backbone of the polyamino acid is poly-l-lysine.

In one embodiment, the backbone of the polyamino acid is artificially synthesized poly-d-lysine.

In one embodiment, the backbone of the polyamino acid is alpha-polylysine or epsilon-polylysine.

In one embodiment, the main chain of the polyamino acid is a copolymer of lysine and other amino acids, including any one of lysine-arginine copolymer, lysine-histidine copolymer, lysine-serine copolymer, lysine-threonine copolymer, lysine-tyrosine copolymer, lysine-serine-threonine copolymer, lysine-arginine-histidine copolymer, lysine-serine-threonine-tyrosine copolymer, lysine-glycine copolymer, lysine-leucine copolymer, lysine-isoleucine copolymer, lysine-valine copolymer, lysine-phenylalanine copolymer, and lysine-tryptophan copolymer.

In one embodiment, the molar percentage of lysine in the polyamino acid to the total amount of other amino acids and lysine is 0.1% to 100%, preferably 10% to 100%, more preferably 30% to 100%.

In one embodiment, the number of repeating units of the polyamino acid is an integer between 2 and 3000, preferably an integer between 3 and 1500, more preferably an integer between 4 and 1000, even more preferably an integer between 5 and 500.

In one embodiment, the side chain modified polyamino acid may be, for example, specifically:

。

the invention also provides a side chain modified polyamino acid, which is prepared by the following preparation method:

the polyamino acid with side chain having active group is connected with one end of C_1-10Polyethylene glycol with reactive group x1 connected to the other end of alkoxy group and/or polyethylene glycol with biological functional group connected to one end and reactive group x2 connected to the other end, and C with reactive group x3 connected to the other end and activated disulfide bond_1-10Reacting aliphatic hydrocarbon; wherein the reactive groups x1, x2 and x3 react with the active groups on the side chain of the polyamino acid, such that the end group is provided with C_1-10Polyethylene glycol of alkoxy and/or biological functional group and aliphatic hydrocarbon containing activated disulfide bond group are connected to the side chain of polyamino acid; then the product is mixed with C_4-25And (3) reacting aliphatic hydrocarbon mercaptan to prepare the side chain modified polyamino acid.

According to the invention, the activated disulfide bond is an-S-S-pyridyl group.

In one embodiment, said one end is connected to C_1-10The polyethylene glycol having the reactive group x1 attached to the other end of the alkoxy group may be obtained commercially or may be prepared by methods conventional in the art.

In one embodiment, the polyethylene glycol having a biofunctional group attached to one end and a reactive group x2 attached to the other end is commercially available, or can be prepared by methods conventional in the art, such as the following:

reacting polyethylene glycol having a reactive group x2 attached to one end thereof and a reactive group x4 attached to the other end thereof with a biofunctional substance having a reactive group x5 attached thereto, wherein the reactive group x4 reacts with the reactive group x5, thereby attaching the biofunctional group to one end of the polyethylene glycol.

In one embodiment, the reactive groups x4, x5 are selected from, for example, hydroxyl, amino, carboxyl, aldehyde, ketone, ester, thiol, maleimide, α -halocarbonyl, alkyne, alkene, azide, tetrazine groups. Wherein the reactive groups x4 and x5 are reactive groups with each other and can react.

For example, an amino group is condensed with a carboxyl group to obtain an amide linking group, or an amino group is reacted with an aldehyde group or a ketone group to obtain a schiff base linking group, or an amino group is reacted with an ester group to obtain an amide linking group, or a hydroxyl group is condensed with a carboxyl group to obtain an ester linking group, or a hydroxyl group is dehydrated and condensed with a hydroxyl group to obtain an ether linking group, or maleimide and a thiol group are subjected to an addition reaction, or a thiol group is subjected to a substitution reaction with an α -halocarbonyl group, or an alkynyl group is subjected to a click reaction with an azide group to obtain a linking group, or an olefinic bond is subjected to a click reaction with a tetrazine group to obtain a linking group. Among them, the click reaction of an alkynyl group with an azido group is a "click chemistry" reaction known in the art, such as: azide-alkyne cycloaddition catalyzed by metal ions (e.g., cu (i)) (Sharpless reaction, with the alkynyl group typically at the end group), or cyclotension catalyzed azide-alkyne cycloaddition (SPAAC reaction, with the alkynyl group in the middle of the strained ring). The click reaction of an olefinic bond with a tetrazine group is a reaction known in the art, for example the cycloaddition reaction of a cyclic olefin with a tetrazine group.

Illustratively, when x4 is amino, x5 is at least one of carboxyl, aldehyde, ketone, ester, α -halocarbonyl; when x4 is hydroxyl, x5 is at least one of carboxyl, hydroxyl and alpha-halogenated carbonyl; when x4 is sulfhydryl, x5 is at least one of sulfhydryl, maleimide and alpha-halocarbonyl; when x4 is alkynyl, x5 is azido; when x4 is an olefinic bond, x5 is a tetrazinyl group. The reverse is also true.

The attachment between the polyethylene glycol and the terminal reactive groups x1, x2, x4 or the biofunctional group may be either direct, i.e. as a capping group, or may be by any spacer group, depending on the introduction of the reactive or biofunctional group onto the polyethylene glycol by any particular conventional method in the art. The spacer group between the polyethylene glycol and the reactive group, such as C, may be any spacer group that does not interfere with the preparation of the polyamino acids of the invention_1-12Alkyl, ester, amide, ketone, and the like.

In one embodiment, in the above steps, the reaction is a conventional reaction step in the art, and the reaction temperature is, for example, 10 to 40 ℃.

In one embodiment, the reaction may be carried out under the promotion of a coupling agent. For example, an amino group may be condensed with a carboxyl group in the presence of a coupling agent to provide an amide linking group, or a hydroxyl group may be condensed with a carboxyl group in the presence of a coupling agent to provide an ester linking group. The coupling agent is, for example, a carbodiimide derivative selected from 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, or N, N-dicyclohexylcarbodiimide, and succinimide. The coupling agent further comprises a disulfide linkage structure, such as succinimide 3- (2-pyridyldithio) propionate, succinimide-oligo (ethylene glycol) -3- (2-pyridyldithio) propionate. The molar ratio of the coupling agent to the reactant is 1-500, preferably 1-100, more preferably 1-50, and still more preferably 1-20.

In one embodiment, the polyamino acid is polylysine, or a copolymer of lysine with other amino acids; the other amino acids are selected from one or more of serine, threonine, tyrosine, and arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, tryptophan, proline, asparagine, and glutamine.

In the present invention, the side chain of the lysine contains an amino group; the side chains of the serine, the threonine and the tyrosine contain hydroxyl; the above groups can be modified by side chain functionalization through covalent coupling reaction.

In the invention, the arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, proline, tryptophan, asparagine and glutamine can be used for adjusting the hydrophilicity and hydrophobicity, the hydrogen bonding capability and the pH value response of the polyamino acid according to the characteristics of the hydrophobic characteristics or molecular configuration, size, aromaticity, hydrogen bonding capability, pH value response and the like of the side chain of the arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, proline, tryptophan, asparagine and glutamine.

In one embodiment, the biofunctional group is as defined above.

In one embodiment, said one end is connected to C_1-10The structure of the polyethylene glycol with the other end of the alkoxy connected with a reactive group x1 is as follows: x1-L¹-PEG-L²-Z¹-D, wherein PEG is a polyethylene glycol segment and D is C_1-10Alkoxy radical, Z¹Is a linking group, L¹、L²Is a direct bond or a spacer group, and the reactive group x1 is a hydroxyl group, a carboxyl group, an aldehyde group, a ketone group, an ester group, a maleimide group, an alpha-halocarbonyl group.

In one embodiment, the polyethylene glycol having a biofunctional group attached to one end and a reactive group x2 attached to the other end has the structure: x2-L¹-PEG-L²-Z²-E, wherein PEG is a polyethylene glycol segment, E is a biofunctional group, Z²Is a linking group, L¹、L²Is a direct bond or a spacer group, and the reactive group x2 is a hydroxyl group, a carboxyl group, an aldehyde group, a ketone group, an ester group, a maleimide group, an alpha-halocarbonyl group.

In one embodiment, the aliphatic hydrocarbon having a reactive group attached at one end x3 and an activated disulfide bond attached at the other end has the structure: x3-L³-S-pyridyl, the reactive group x3 being a hydroxyl, carboxyl, aldehyde, ketone, ester, maleimide, α -halocarbonyl group. L is³Is C_1-10An aliphatic hydrocarbon group.

In one embodiment, the reactive groups x1, x2, x3 are selected from hydroxyl, carboxyl, aldehyde, ketone, ester, maleimide, α -halocarbonyl; the reactive groups x1, x2, x3 react with the active groups amino or hydroxyl on the side chain of the polyamino acid; specifically, an amino group and a carboxyl group are subjected to a condensation reaction to obtain an amide connecting group, or the amino group and an aldehyde group or a ketone group are subjected to a reaction to obtain a Schiff base connecting group, or the amino group and an ester group are subjected to a reaction to obtain an amide connecting group, or the amino group and an alpha-halocarbonyl group are subjected to a substitution reaction, or a hydroxyl group and a carboxyl group are subjected to a condensation reaction to obtain an ester connecting group, or the hydroxyl group and a hydroxyl group are dehydrated to be subjected to a condensation reaction to obtain an ether connecting group, or the hydroxyl group and an alpha-halocarbonyl group are subjected to a substitution reaction.

Illustratively, when the side chain of the polyamino acid contains an amino group, the reactive groups x1, x2, x3 are at least one of carboxyl, aldehyde, ketone, ester, α -halocarbonyl; when the side chain of the polyamino acid contains hydroxyl, the reactive groups x1, x2 and x3 are at least one of carboxyl, hydroxyl and alpha-halogenated carbonyl.

The invention also provides a preparation method of the side chain modified polyamino acid, which comprises the following steps:

the polyamino acid with side chain having active group is connected with one end of C_1-10Polyethylene glycol with reactive group x1 connected to the other end of alkoxy group and/or polyethylene glycol with biological functional group connected to one end and reactive group x2 connected to the other end, and C with reactive group x3 connected to the other end and activated disulfide bond_1-10Reacting aliphatic hydrocarbon; wherein the reactive groups x1, x2 and x3 react with the active groups on the side chain of the polyamino acid, such that the end group is provided with C_1-10Polyethylene glycol of alkoxy and/or biological functional group and aliphatic hydrocarbon with activated disulfide bond at the end group are connected to the side chain of polyamino acid; then the product is mixed with C_4-25And (3) reacting aliphatic hydrocarbon mercaptan to prepare the side chain modified polyamino acid.

The invention also provides the application of the side chain modified polyamino acid, which is used for preparing micelle.

The invention also provides a micelle, which comprises the side chain modified polyamino acid.

In one embodiment, the micelle is formed by dissolving the side chain-modified polyamino acid into a solvent.

Preferably, the side chain-modified polyamino acid is dissolved in water or a buffer solution having a pH of 3 to 8 so that the concentration thereof is 1 to 1000 times the critical micelle concentration to form the micelle.

In one embodiment, the micelle is composed of one side chain-modified polyamino acid described above, or a combination of two or more side chain-modified polyamino acids described above. The combination may be a combination of binary, ternary, or multiple side chain-modified polyamino acids, and the percentage of each side chain-modified polyamino acid in the composition is not particularly limited as long as the composition is capable of forming micelles.

In one embodiment, the critical micelle concentration of the side chain-modified polyamino acid is determined by methods known in the art, including, for example, conductance, surface tension, drop volume, ultrafiltration curve, single-point ultrafiltration, double-point ultrafiltration, uv spectrophotometry, dye adsorption, light scattering, fluorescence probe, and solubility.

In one embodiment, the micelles have a mean particle size of less than 300nm, preferably less than 200nm, more preferably less than 100nm, and even more preferably less than 80 nm.

In one embodiment, the size of the micelles is measured by methods known in the art, including, for example, dynamic laser scattering, fluorescence correlation spectroscopy, and scanning electron microscopy imaging.

The invention also provides a preparation method of the micelle, which comprises the following steps:

the polyamino acid with the modified side chain is dissolved in a solvent, and the concentration of the polyamino acid is 1-1000 times of the critical micelle concentration.

In one embodiment, the concentration is 1 to 1000 times the critical micelle concentration, preferably the concentration is 1 to 200 times the critical micelle concentration, more preferably the concentration is 1 to 50 times the critical micelle concentration, and even more preferably the concentration is 1 to 10 times the critical micelle concentration.

In one embodiment, the solvent is, for example, an aqueous solution or a buffer with a pH of 3-8, such as acetate buffer, phosphate buffer.

The invention also provides the use of the micelle in the coating and delivery of nucleic acid molecules.

The invention also provides a delivery system comprising a micelle and a nucleic acid molecule, the nucleic acid molecule being located within the micelle.

In one embodiment, the nucleic acid molecule comprises RNA and DNA.

In one embodiment, the nucleic acid molecule may be a vaccine or a drug, i.e. the nucleic acid molecule is an RNA vaccine, an RNA drug, a DNA vaccine.

In one embodiment, the RNA is single-stranded RNA or double-stranded RNA.

The nucleic acid molecule of the invention is used for treating or preventing infectious diseases, tumors, rare diseases, other protein deficiency diseases and the like, or is used for beautifying, resisting aging and the like. The RNA target sequences for the treatment of infectious diseases according to the invention are the gene sequences of proteins or protein fragments of the receptor binding region of the virus, the capsid protein of the virus or other conserved regions of the virus.

In one embodiment, the molar ratio of the nucleic acid molecule to the micelle-forming side-chain modified polyamino acid is from 0.001 to 1000:1, preferably from 0.01 to 100:1, more preferably from 0.1 to 10: 1.

In one embodiment, the delivery system optionally comprises an adjuvant selected from at least one of a naturally occurring phospholipid molecule, cholesterol, an amino acid, a polypeptide, an ionic surfactant, and a non-ionic surfactant.

In one embodiment, the molar ratio of adjuvant to nucleic acid molecule is 0.001-8000:1, preferably 0.01-4000:1, more preferably 0.1-1000: 1.

The present invention also provides a method of preparing a delivery system, the method comprising the steps of:

and (3) contacting the solution containing the micelle with nucleic acid molecules, and incubating the mixed solution to prepare the delivery system.

Wherein mechanical stirring, oscillation, thermal refluxing or ultrasonic dispersion is used in the contact process.

The present invention also provides a method of preparing a delivery system, the method comprising the steps of:

and mixing the side chain modified polyamino acid with nucleic acid molecules to form a mixed solution, and preparing to obtain the delivery system.

The invention also provides freeze-dried powder which is obtained by evaporating the solvent in the delivery system to dryness.

In one embodiment, the lyophilized powder is reconstituted in a solvent to form a delivery system at the time of use.

Wherein the solution containing the micelles may be, for example, an organic solution containing micelles or an aqueous solution containing micelles, and the solvent of the organic solution is not particularly limited as long as it can dissolve the material, and may be optionally one of alcohol, ether, ketone, ester, amide, sulfoxide, alkane, cycloalkane, aromatic hydrocarbon, chloroalkane, or a mixture thereof.

[ terms and explanations ]

The terms "side chain-modified polyamino acid" and "polyamino acid" in the present invention are interchangeable.

The "aliphatic hydrocarbon" referred to herein may also be referred to as an aliphatic compound, and refers to a hydrocarbon having a structure not containing an aromatic ring, in which carbon atoms are arranged in a straight chain, a branched chain or a cyclic structure, and is referred to as a straight chain aliphatic hydrocarbon, a branched chain aliphatic hydrocarbon or an alicyclic hydrocarbon, respectively. The aliphatic compound may be an alkane, alkene or alkyne. The aliphatic hydrocarbon of the present invention may have 1 to 40 carbon atoms.

The alkyl group in the present invention represents a linear, branched or cyclic alkyl group having 1 to 40 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, etc.

The alkenyl group in the present invention represents a linear, branched or cyclic alkenyl group having 1 to 40 carbon atoms, for example, ethylene, propylene, isopropylene, butene, etc. Preferably, the number of double bonds is an integer from 1 to 6.

The alkynyl group in the present invention represents a linear, branched or cyclic alkynyl group having 1 to 40 carbon atoms, for example, acetylene, propyne, butyne and the like. Preferably, the number of acetylenic bonds is an integer from 1 to 6.

The amino group of the present invention represents the group-N (R)₂)₂Wherein R is₂Independently selected from H, C_1-6An alkyl group.

The ester group according to the invention represents the group-CO-OR₃Wherein R is₃Is C_1-6An alkyl group.

The term "reactive group" may also be referred to as a "reactive group," which refers to a functional group that can form a chemical bond with another "reactive group. Suitable chemical bonds are well known in the art and may be, for example: hydroxyl, amino, carboxyl, aldehyde, ketone, ester, sulfhydryl, maleimide, alpha-halocarbonyl, alkynyl, olefinic bond, azido and tetrazine.

The term "linking group" refers to a group that links any two groups together, which is a group formed by the reaction of two "reactive groups".

The term "spacer group" means a group which may be formed when a reactive group or the like is introduced into the end of a polyethylene glycol chain by a conventional reaction. The groups depend on the reagents used to introduce the groups.

The term "binder" refers to a substance capable of binding to a biological molecule such as a protein, polypeptide, amino acid, etc., by means of, for example, covalent bonds, non-covalent bonds, etc.

The term "linker" may also be referred to as a "Ligand linker," and in english Ligand, refers to a group that is covalently linked to a protein, amino acid, antibody, polypeptide, or the like.

The term "nucleic acid" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

The invention has the beneficial effects that:

the invention provides a polyamino acid for delivering nucleic acid molecules, a preparation method and application thereof. The side chain modified polyamino acid has the following advantages:

(1) the extensive presence of glutathione and sulfhydryl metabolites in the cytoplasm of cells makes a reducing environment ubiquitous in the cytoplasm. Lysosomes formed after endocytosis of the nanocarrier by the cell are in a typical reducing environment and at low pH. The main chain structure and the side chain modification of the polyamino acid micelle carrier are comprehensively optimized, and the disulfide bond cracked in a reducing environment is introduced into the side chain, so that the response of the micelle to the reducing environment and the low pH value in cytoplasm can be promoted, and the mRNA vaccine can be helped to actively escape from a 'lysosome trap' to enter the cytoplasm. Active cleavage of the nanocarriers can release mRNA more efficiently than passive osmotic pressure dominated escape processes.

(2) The main chain and side chain structures and the connection mode thereof can be flexibly selected, and the number and the types of hydrophilic groups, charge characteristics and hydrophobic groups are adjusted, so that the prepared polyamino acid micelle has good biocompatibility and targeted delivery efficiency.

(3) The charge polarity of the main chain of the polyamino acid is electropositive, and the reactive groups of the side chain can be amino or hydroxyl respectively. The charge distribution of the main chain is adjusted through the quantity and distribution of other copolymerized amino acids such as arginine, histidine, serine and the like, and the efficient and controllable wrapping of electronegative RNA is realized. Charge modulation on the backbone promotes the pH response of the micelle, helping RNA escape from the "lysosomal trap" into the cytosol.

(4) The hydrophobic part of the side chain is controlled by quantifying the chain length, the saturation and the number of the fatty chains modified by the side chain, the volume and the association strength of the hydrophobic part are accurately adjusted, the volume of the hydrophilic part is adjusted by selecting the chain length and the number of branches of the polyethylene glycol polymer, and the two are cooperated to realize the accurate adjustment and control of the size and the stability of the polyamino acid micelle. The polyamino acid micelle has small size and good tissue penetrability.

(5) Due to the similarity of RNA and DNA in structure and electronegativity, the basic charge, size and stability of the micelle are flexibly adjusted, and a packaging and delivery system is efficiently constructed, so that the efficient packaging and delivery of effective components of nucleic acids such as mRNA vaccines, RNA medicines, DNA vaccines and the like can be realized.

(6) A plurality of same biological functional groups are introduced through side chain modification of functional macromolecules, and the functional macromolecules comprise targeted binding effectors such as micromolecule binders, proteins and antibodies, and the high-efficiency and stable binding of a delivery system to target tissues and parts is realized by utilizing multivalent functionalization.

(7) Different biological functional groups are introduced through the modification of the side chains of functional macromolecules, the functional groups comprise targeted binding effectors such as micromolecule binders, proteins and antibodies, the specific binding of a delivery system to target tissues and parts is realized through a composite-synergistic action mechanism of different functional groups, and the targeted delivery effect is improved.

Drawings

FIG. 1 measurement result of critical micelle concentration of polyamino acid prepared in example 12.

FIG. 2 dynamic laser light scattering results for micelles formed by the polyamino acid prepared in example 12, with the size distribution shown in logarithmic scale.

Fig. 3 results of dynamic laser light scattering of polyamino acid micelles containing mRNA vaccine prepared in example 16.

Fig. 4 results of dynamic laser light scattering of polyamino acid micelles containing mRNA vaccine prepared in example 19.

FIG. 5 results of dynamic laser light scattering experiments before and after loading of Kjeldahl targeting antibodies for multivalent targeting vaccine H7N9-BT21 (A) and monovalent targeting vaccine H7N9-BT1 (B).

FIG. 6 gel electrophoresis chromatogram of reverse transcription-PCR analysis of IL-6 receptor stable transfected cells of HEK 293T. Wherein, 1 is the cell added with the multivalent targeted H7N9-BT21 mRNA vaccine, 2 is the cell added with the monovalent targeted H7N9-BT1 mRNA vaccine, and 3 is the control group without vaccine.

FIG. 7 results of dynamic laser light scattering experiments on the oxidation-reduction conditional response of the binary polypeptide complex biofunctionalized polyamino acid mRNA vaccine of example 12. The solid line and the black squares show the particle size distribution of the polyamino acid mRNA vaccine, and the dashed line and the grey squares are the particle sizes of the polyamino acid mRNA vaccine in a 5mM DTT reducing environment.

Detailed Description

The preparation method of the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

The buffers used in the examples described below were commonly used buffer solutions including phosphate buffer, HEPES buffer, and Tris buffer, water was sterilized in milliq deionized water with a conductivity of 18.2 ohms cm.

The percentages (%) used in the examples below are in mol%.

Optionally indicating the presence or absence of the stated feature, and also indicating that the stated feature must be present, although the particular choice may be arbitrary.

Example 1 preparation of the polyamino acid PLL-SSMO25-PEG2000-Biotin20

10 mg of a hydrobromide salt of α -poly-L-lysine (a hydrobromide salt of PLL, average molecular weight 22500 Dalton) was weighed and dissolved in 150 μ L of phosphate buffer solution (pH 7.4) to obtain a solution (A). 20 mg of polyethylene glycol 2000 (BIOTIN-PEG 2000-SCM, polyethylene glycol average molecular weight 2000 Dalton, available from Kyoto Kai technologies, Inc.) with end group modified BIOTIN and succinimide carboxymethyl ester was weighed and dissolved in 150 μ L of tetrahydrofuran (analytical reagent, Nanjing reagent, Inc.). To the resulting solution was added 3.8 mg of succinimide 3- (2-pyridyldithio) propionate (SPDP, molecular weight 312, semenfei) coupling agent to obtain a mixture solution (B). The solution (A) and the solution (B) were mixed in a test tube, and reacted for 4 hours at room temperature with shaking, after which 2.5. mu.L of n-octylmercaptan (MO, analytically pure, molecular weight 146, density 0.84 g/L, Beijing Bailingwei) of 1.2 times reaction equivalent was added to the resulting mixed solution. Shaking at room temperature overnight, adding 100 mu L of Tris buffer solution with pH of 8.0, and continuing to shake for 1 hour to terminate the reaction. The reaction solution was dialyzed against deionized water for 24 hours (MWCO 50 ten thousand daltons). The obtained solution is frozen and then placed in a freeze dryer to prepare 19 mg of freeze-dried powder. The freeze-dried powder is 25% of disulfide bond n-octane and 20% of polyethylene glycol 2000-Biotin graft-modified functional polyamino acid, the polyamino acid is named as PLL-SSMO25-PEG2000-Biotin20, and the specific structural formula is shown as follows.

The number of side chains of a polyamino acid to which no other component is bonded is represented by n, the number of side chains of a polyamino acid to which polyethylene glycol is bonded is represented by p, the number of repeating units of polyethylene glycol is represented by q, and the number of side chains of a polyamino acid to which a disulfide bond-containing hydrophobic component is bonded is represented by m, i.e., m/(m + n + p) =0.25, and p/(m + n + p) = 0.2.

Example 2 preparation of the polyamino acid PLL-SSMO25-PEG3500-Biotin20

10 mg of PLL hydrobromide (average molecular weight 22500 daltons) was weighed into a 2 ml centrifuge tube and dissolved in 150. mu.L phosphate buffer (pH 7.4) to obtain solution (A). 33 mg of polyethylene glycol 3500 (BIOTIN-PEG 3500-SCM, polyethylene glycol average molecular weight 3500 Dalton, available from Kyoto Keka science and technology Co., Ltd., Beijing) with the end group-modified BIOTIN and succinimide carboxymethyl ester was weighed and dissolved in 150. mu.L of tetrahydrofuran. To the resulting solution was added 3.8 mg of succinimide 3- (2-pyridyldithio) propionate (SPDP, molecular weight 312, semenfei) coupling agent to obtain a mixture solution (B). The solution (A) and the solution (B) were mixed in a test tube, and reacted for 4 hours at room temperature with shaking, after which 2.5. mu.L of n-octylmercaptan (MO, analytically pure, molecular weight 146, density 0.84 g/L, Beijing Bailingwei) of 1.2 times reaction equivalent was added to the resulting mixed solution. The reaction product was prepared according to the method of example 1 and lyophilized to obtain 23 mg of lyophilized powder. The white powder is functionalized polyamino acid grafted and modified by 25 percent of disulfide bond n-octane and 20 percent of polyethylene glycol 3500-Biotin, and the polyamino acid is named as PLL-SSMO25-PEG3500-Biotin 20.

Example 3 preparation of the polyamino acids PLL-SSDT25-PEG3500-Biotin20

The same as example 2, except that 27 mg of lyophilized powder was prepared using 3.0 μ L of n-decanethiol (DT, molecular weight 174, Sigma Aldrich Co.). The white powder is 25% disulfide bond n-decane and 20% polyethylene glycol 3500-Biotin graft modified functionalized polyamino acid which is named as PLL-SSDT25-PEG3500-Biotin 20.

The number of side chains of a polyamino acid to which no other component is bonded is represented by n, the number of side chains of a polyamino acid to which polyethylene glycol is bonded is represented by p, the number of repeating units of polyethylene glycol is represented by q, and the number of side chains of a polyamino acid to which a disulfide bond-containing hydrophobic component is bonded is represented by m, i.e., m/(m + n + p) =0.25, and p/(m + n + p) = 0.2.

Example 4 preparation of the polyamino acid PLL-SSMO50-PEG3500-Biotin50

The same as in example 2, except that 82 mg of terminal group-modified succinimide carboxymethyl ester and biotin polyethylene glycol 3500 were weighed, dissolved in 150. mu.L of tetrahydrofuran, and 7.6 mg of succinimide 3- (2-pyridyldithio) propionate coupling agent was added to obtain a solution (B). Adding 5.0 muL of n-octanethiol (MO) into the mixture (B), and preparing white freeze-dried powder which is functionalized polyamino acid grafted and modified by 50% of disulfide bond n-octane and 50% of polyethylene glycol 3500-Biotin and is named as PLL-SSMO50-PEG3500-Biotin50 according to the method in the example 1.

Example 5 preparation of the polyamino acids ε PLL-SSMO25-PEG5000-Biotin30

10 mg of hydrobromide of ε -poly-L-lysine (hereinafter referred to as hydrobromide of ε PLL, average molecular weight 50000 daltons) was weighed in a 2 ml centrifuge tube and dissolved in 150. mu.L of phosphate buffer to obtain a solution (A). 60 mg of polyethylene glycol 5000 (BIOTIN-PEG 5000-SCM, average molecular weight 5000 Dalton, available from Kyoto Kai technologies, Ltd.) with end group-modified BIOTIN and succinimide carboxy methyl ester as terminal groups was weighed and dissolved in 150 μ L of tetrahydrofuran. To the resulting solution was added 3.8 mg of succinimide 3- (2-pyridyldithio) propionate (SPDP, molecular weight 312, semenfei) coupling agent to obtain a mixture solution (B). The solution (A) and the solution (B) were mixed in a test tube, and the reaction was carried out with shaking at room temperature for 4 hours, after which 2.5. mu.L (MO, analytically pure, molecular weight 146) of n-octylthiol of 1.2 times reaction equivalent was added to the resulting mixed solution. After shaking overnight at room temperature for 8 hours, 100. mu.L of 20 mM Tris buffer pH 8.0 was added, and the reaction was terminated by continuing shaking for 1 hour. The reaction solution was dialyzed against deionized water for 24 hours (MWCO 50 ten thousand daltons). Freezing the obtained solution, and placing in a freeze dryer to obtain 39 mg of freeze-dried powder, and storing at-20 deg.C under sealed condition. The white powder is 25% of disulfide bond n-octane and 30% of polyethylene glycol 5000-Biotin graft modified functional polyamino acid epsilon PLL-SSMO25-PEG5000-Biotin30, and the structural formula is as follows.

The number of side chains of a polyamino acid to which no other component is bonded is represented by n, the number of side chains of a polyamino acid to which polyethylene glycol is bonded is represented by p, the number of repeating units of polyethylene glycol is represented by q, and the number of side chains of a polyamino acid to which a disulfide bond-containing hydrophobic component is bonded is represented by m, i.e., m/(m + n + p) =0.25, and p/(m + n + p) = 0.3.

Example 6 preparation of the polyamino acids ε PLL-SSMO25-PEG5000-N3

10 mg of Epsilon PLL (average molecular weight 50000 daltons) was weighed and dissolved in 150. mu.L of phosphate buffer to obtain a solution (A). 72 mg of polyethylene glycol 5000 (AZIDE-PEG 5000-SCM, average molecular weight 5000 Dalton, available from Kyoto Keyka science and technology Co., Ltd.) of terminal group-modified AZIDE salt and succinimide carboxymethyl ester was weighed and dissolved in 150 μ L of tetrahydrofuran. To the resulting solution was added 3.8 mg of succinimide 3- (2-pyridyldithio) propionate (SPDP, molecular weight 312, semenfei) coupling agent to obtain a mixture solution (B). The solution (A) and the solution (B) were mixed in a test tube, and the reaction was carried out for 4 hours at room temperature with shaking, after which 2.5. mu.L (MO, analytically pure, molecular weight 146) of n-octylthiol was added to the resulting mixed solution. The reaction solution was continued to be mixed in a shaker at room temperature. The product was prepared as described in example 5, lyophilized to 31 mg of lyophilized powder and stored sealed at-20 ℃. The white powder is polyamino acid grafted and modified by 25 percent of disulfide bond N-octane and 30 percent of polyethylene glycol 5000-azide, and the polyamino acid is named as epsilon PLL-SSMO25-PEG 5000-N3.

The number of side chains of a polyamino acid to which no other component is bonded is represented by n, the number of side chains of a polyamino acid to which polyethylene glycol is bonded is represented by p, the number of repeating units of polyethylene glycol is represented by q, and the number of side chains of a polyamino acid to which a disulfide bond-containing hydrophobic component is bonded is represented by m, i.e., m/(m + n + p) =0.25, and p/(m + n + p) = 0.3.

Example 7 preparation of the polyamino acid PLL-SSDDT25-PEG2000-OMe37

10 mg of a PLL hydrobromide salt (average molecular weight 22500 daltons) was weighed into a 2 ml centrifuge tube and dissolved in 150. mu.L of phosphate buffer to obtain a solution (A). 36 mg of polyethylene glycol (M-PEG 2000-SCM, average molecular weight 2000 Dalton, available from Kyork technology Co., Ltd.) with terminal group-modified methoxy group and succinimide carboxymethyl ester was weighed and dissolved in 150 μ L of tetrahydrofuran. To the resulting solution was added 3.8 mg of succinimide 3- (2-pyridyldithio) propionate (SPDP, molecular weight 312, semenfei) coupling agent to obtain a mixture solution (B). The solution (A) and the solution (B) were mixed in a test tube, and the reaction was carried out with shaking at room temperature for 4 hours, after which 3.5. mu.L of n-dodecylmercaptan (DDT, molecular weight 202, Sigma Aldrich Co.) was added to the resulting mixed solution, and the reaction solution was continued to be mixed overnight with shaking in a test tube at room temperature. In a similar manner to example 1, 26 mg of a white lyophilized powder was obtained. The white powder was 25% disulfide-bonded n-dodecane and 37% polyethylene glycol 2000-methoxy graft-modified polyamino acid, which was named PLL-SSDDT25-PEG2000-OMe 37. The specific structural formula is shown as follows.

The number of side chains of a polyamino acid to which no other component is bonded is represented by n, the number of side chains of a polyamino acid to which polyethylene glycol is bonded is represented by p, the number of repeating units of polyethylene glycol is represented by q, and the number of side chains of a polyamino acid to which a disulfide bond-containing hydrophobic component is bonded is represented by m, i.e., m/(m + n + p) =0.25, and p/(m + n + p) = 0.37.

Example 8 preparation of the polyamino acid PLL-SSDDT25-PEG2000-OMe75

The same as example 7, except that 73 mg of polyethylene glycol (M-PEG 2000-SCM, average molecular weight 2000 Dalton, available from Kyoto Kamiiki technologies, Inc.) having a terminal-modified methoxy group and a succinimide carboxy methyl ester was weighed out. 47 mg of a white lyophilized powder was obtained which was 25% disulfide-bonded n-dodecane and 75% polyethylene glycol 2000-methoxy graft-modified polyamino acid, designated PLL-SSDDT25-PEG2000-OMe 75.

Example 9 preparation of the polyamino acid PLL-SSDT25-PEG2000-OMe37

Same as example 7, except that 3.0 μ L of n-decanethiol (DT, molecular weight 174, SigmaAldrich Co.) was used, 20 mg of a white lyophilized powder was prepared which was 25% disulfide n-decane and 37% polyethylene glycol 2000-methoxy graft modified polyamino acid designated PLL-SSDT25-PEG2000-OMe 37.

Example 10 preparation of the polyamino acid PLL-SSMO25-PEG2000-OMe30-PEG2000-BG30

Weighing 96 mg of polyethylene glycol (SCM-PEG 2000-SCM, PEG average molecular weight 2000 Dalton, purchased from Kyork science and technology Co., Ltd.) with an end group modified by bis-succinimide carboxymethyl ester and a linker O6- [4- (aminomethyl) benzyl ] guanine (abbreviated as BG, purchased from Santa Cruz Biotechnology Co., Ltd.) of 10 mg of SNAPTag protein, dissolving the mixture in anhydrous 200 muL tetrahydrofuran, adding 4 muL triethylamine, reacting for 4 hours under nitrogen protection at room temperature, adding deionized water for 30 minutes, and terminating the reaction. The mixture was placed in a vacuum oven and the organic solvent was removed at 60 ℃. The reaction product was separated on a G200 silica gel chromatography plate with the mobile phase dichloromethane: and collecting a product strip at an Rf value of 0.25 of ethyl acetate (80: 20 by volume) to obtain 41 mg of oily substance COOH-PEG2000-BG, dissolving the oily substance COOH-PEG2000-BG in 200 mu L of tetrahydrofuran, and storing the oily substance away from light.

7.5 mg of poly-L-lysine hydrobromide (PLL hydrobromide, average molecular weight 22500 Dalton) was weighed out and dissolved in 100. mu.L of 5mM HEPES buffer solution (pH 7.4) to obtain solution (A). 24 mg of polyethylene glycol (M-PEG 2000-SCM, polyethylene glycol average molecular weight 2000 Dalton, purchased from Kyork science and technology Co., Ltd.) having methoxy and succinimide ester at both ends thereof were weighed in a small glass vial, dissolved in 150. mu.L of tetrahydrofuran, 114. mu.L of the above prepared tetrahydrofuran solution of COOH-PEG2000-BG was added, and 3.8 mg of succinimide 3- (2-pyridyldithio) propionate (SPDP, molecular weight 312, Saimer fly) coupling agent was added to the obtained solution to obtain a mixture solution (B). The solution (A) and the solution (B) were mixed in a test tube, and the reaction was carried out for 4 hours at room temperature with shaking, after which 2.5. mu.L of n-octylmercaptan (MO, analytically pure, molecular weight 146, density 0.84 g/L, Bailingwei, Beijing) was added to the resulting mixed solution. The reaction solution was continued to be shaken overnight in a shaker at room temperature in a test tube, and 100 μ L of deionized water was added for 30 minutes to terminate the reaction. The reaction solution was dialyzed for 24 hours (MWCO 50 ten thousand daltons). Lyophilizing to obtain 31 mg lyophilized powder. The freeze-dried powder is polyamino acid graft-modified by 25% of disulfide bond n-octane, 30% of polyethylene glycol 2000-methoxyl and 30% of ligand linker of polyethylene glycol 2000-SNAPTag protein, and the polyamino acid is named as PLL-SSMO25-PEG2000-OMe30-PEG2000-BG 30. The specific structural formula is shown as follows.

Wherein the number of side chains of the polyamino acid to which no other component is attached is represented by n, the number of side chains of the polyamino acid to which polyethylene glycol-methoxy groups are attached is represented by p, the number of repeating units of polyethylene glycol is represented by q, the number of side chains of the polyamino acid to which polyethylene glycol-BG functional groups are attached is represented by r, the number of repeating units of polyethylene glycol is represented by t, and the number of side chains of the polyamino acid to which disulfide bond-containing hydrophobic components are attached is represented by m, that is, m/(m + n + p + r) =0.25, p/(m + n + p + r) =0.3, and r/(m + n + p + r) = 0.3.

Example 11 preparation of the polyamino acid PLL-SSMO25-PEG3500-HA15-PEG3500-ALFAtag15 containing a Complex functionalized group of a binary polypeptide

7.5 mg of poly-L-lysine (PLL, average molecular weight 22500 daltons) was weighed out in a plastic vial and dissolved in 200 μ L of HEPES buffer (100 mM, pH 7.4) to give solution (A). 42 mg of polyethylene glycol (MAL-PEG 3500-SCM, PEG average molecular weight 2000 Dalton, purchased from Kyork science and technology Co., Ltd.) with end group modification of maleimide and succinimide carboxymethyl ester respectively, 6.5 mg of polypeptide lyophilized powder (HA-tag, N-terminal acetyl modification, molecular weight 1.3 kDa) with amino acid sequence of CYPYDVPDYA, and 8.5 mg of polypeptide lyophilized powder (ALFA-tag, N-terminal acetyl modification, molecular weight 1.9 kDa) with amino acid sequence of CPSRLEEELRRRLTE were weighed. This was dissolved in 200 μ L of HEPES buffer solution (5 mM HEPES) to adjust the pH of the solution to 6.0, to obtain a solution (B). And (3) oscillating for 10 minutes at room temperature to obtain polyethylene glycol solutions with end groups respectively containing HA polypeptide, ALFA-tag polypeptide binary composite functional groups and succinimide carboxymethyl ester, adding the polyethylene glycol solutions into the solution (A), and oscillating for 4 hours at room temperature. 3.8 mg of a succinimide 3- (2-pyridyldithio) propionate (SPDP, molecular weight 312, Sammerfei) coupling agent was added to the resulting mixed solution, and the reaction was carried out at room temperature for 4 hours with shaking, after which 2.5. mu.L (MO, analytical purity, molecular weight 146, density 0.84 g/L, Beijing Bailingwei) of n-octylmercaptan was added to the resulting mixed solution. The reaction solution was continued to be shaken overnight in a shaker at room temperature in a test tube, and 100 μ L of deionized water was added for 30 minutes to terminate the reaction. The reaction solution was dialyzed for 24 hours (MWCO 50 ten thousand daltons). Lyophilizing to obtain 37 mg lyophilized powder. The freeze-dried powder is polyamino acid subjected to binary composite functional graft modification by 25% of disulfide bond n-octane, 15% of polyethylene glycol 3500-HA-Tag and 15% of polyethylene glycol 3500-ALFA-Tag, and the polyamino acid is named as PLL-SSMO25-PEG3500-HA15-PEG3500-ALFAtag 15.

Wherein the number of side chains of the polyamino acid to which no other component is linked is represented by n, the number of side chains of the polyamino acid to which a side chain of polyethylene glycol-HA-tag is linked is represented by p, the number of repeating units of polyethylene glycol is represented by q, the number of side chains of the polyamino acid to which a side chain of polyethylene glycol-AFLA-tag is linked is represented by r, the number of repeating units of polyethylene glycol is represented by t, and the number of side chains of the polyamino acid to which a disulfide bond-containing hydrophobic component is linked is represented by m, that is, m/(m + n + p + r) =0.25, p/(m + n + p + r) =0.15, and r/(m + n + p + r) = 0.15.

Example 12 preparation and characterization of polyamino acid micelles

Using polylysine PLL-SSMO25-PEG2000-Biotin20 prepared in example 1 as an example, the critical micelle concentration CMC of polyamino acid micelles is determined by a probe steady state fluorescence emission method and the hydration diameter of the micelles is measured by a dynamic laser scattering method.

Probe pyrene (product of Sigma, gold tag, without further purification) was dissolved in anhydrous methanol to prepare a solution of 1.0X 10^-4The mol/L solution is ready for use. Putting 5 mu L of pyrene methanol solution into a series of 5 mL volumetric flasks, introducing nitrogen to blow the methanol for drying, sequentially adding 5 mL of PLL-SSMO25-PEG2000-Biotin20 aqueous solution with different concentrations, putting the aqueous solution into an ultrasonic bath for dispersing for 1 hour, and taking 1mL of sample solution for measuring the fluorescence emission spectrum of pyrene. The fluorescence spectrum was measured with a Hitachi F-4500 fluorescence spectrophotometer, Japan, with an excitation wavelength of 335 nm, a slit width of 5 nm, an emission wavelength of 2.5 nm, a detector bias of 700V, and an experimental temperature of 22. + -. 1 ℃.

FIG. 1 shows the fluorescence emission spectrum change of pyrene in different concentrations of PLL-SSMO25-PEG2000-Biotin20 aqueous solution. The ratio (I) of the fluorescence intensity of the first peak (373 nm) to the third peak (384 nm) of pyrene₁/ I₃A) in FIG. 1 reflects the polarity change of the microenvironment around the pyrene molecule.

This ratio decreases successively with increasing concentration of the polyamino acid PLL-SSMO25-PEG2000-Biotin20 in aqueous solution (B in FIG. 1). When the concentration of the polyamino acid is increased to a certain value, the curve has mutation, the mutation indicates the change of the polarity of the environment where pyrene is located, and PLL-SSMO25-PEG2000-Biotin20 micelles are formed. Thus, the first mutation point corresponds to the CMC value of PLL-SSMO25-PEG2000-Biotin 20. The critical micelle concentration of PLL-SSMO25-PEG2000-Biotin20 was determined to be 2.1 mg/mL by this method. When the concentration of PLL-SSMO25-PEG2000-Biotin20 in aqueous solution is more than the value, micelle can be formed.

PLL-SSMO25-PEG2000-Biotin20 aqueous solution (concentration is more than CMC) with concentration of 3.0 mg/mL is prepared, and dynamic laser scattering experiment is carried out on 1mL solution. The hydrated diameter of the micelles was measured to be 97. + -.35 nm using Zetasizer Nano ZS ZEN3600 from Malvern Instruments, UK, and the results are shown in FIG. 2.

Example 13 preparation of avian influenza virus H7N9 subtype mRNA vaccine

In the first step, a DNA template is synthesized. To prepare an mRNA vaccine, DNA sequences are first synthesized for subsequent in vitro transcription. The gene sequence segment of avian influenza virus A/Shanghai/02/2013(H7N9) polymerase PB2 was selected as the target antigen sequence (gene sequence-1). The target antigen sequence is optimized by GC content (the optimization method is shown in mol. ther. 23, 1456-1464 (2015)), and an untranslated region regulatory element (i) 5 'UTR (beta-globin-1) is added at the 5' end:

CAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC。

adding an untranslated region regulatory element (ii) at the 3 'end of the protein, (2 beta-globin) 3' UTR:

AGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC

the sequences in (i) the 5 ' UTR can inhibit 5 ' -exonucleolytic degradation, and (ii) the 3 ' UTR can inhibit mRNA noradenylation to enhance mRNA stability and translation efficiency. (ii) further adding 70 repeated adenosines and 30 repeated cytosines at the 3' end to form (iii) Poly-a and Poly-C tails to enhance mRNA stability:

TTAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTCTAGACAATTGGAATT

and (3) synthesizing all the DNA sequences into a linear template according to the assembly sequence of 5 'UTR-target sequence-3' UTR-PolyA-PolyC, and carrying out in-vitro transcription of the next step.

Second, in vitro transcription. In the CAP analog (m)⁷GpppG) was prepared in vitro using T7 polymerase to transcribe the prepared DNA template to produce a "capped" mRNA. Subsequently, mRNA was purified using the MEGAclear Transcription clear-Up Kit (purchased from Sonofibrino corporation, Waltham, MA, USA). The resulting sample can be used immediately to form micelle encapsulated formulations or lyophilized for storage.

Example 14 preparation of mRNA vaccine for influenza A virus H1N1

In the first step, a DNA template is synthesized. The gene sequence of HA (Gene sequence-2) of hemagglutinin of influenza virus A/Puerto Rico/8/1934H 1N1 was used as the DNA sequence of the antigen of interest. After the GC content of the target gene sequence is optimized (see mol. ther. 23, 1456-1464 (2015)), an untranslated region regulatory element (i) 5 'UTR (beta-globin-2) is added at the 5' end:

AGAGCGGCCGCTTTTTCAGCAAGATTAAGCCCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC

3 '-end addition of untranslated region regulatory element (ii) 3' UTR (2 β -globin):

AGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC

(ii) further adding (iii) a Poly-A tail and a Poly-C tail comprising repeated adenosine and repeated cytosine sequences:

TTAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTCTAGACAATTGGAATT

and (3) synthesizing all the DNA sequences into a linear template according to the assembly sequence of 5 'UTR-target sequence-3' UTR-PolyA-PolyC, and carrying out in-vitro transcription of the next step.

Second, in vitro transcription. In the CAP analog (m)⁷GpppG) was prepared as a "capped" mRNA by in vitro transcription of the prepared DNA template using T7 polymerase. Subsequently, mRNA was purified using the MEGAclear Transcription clear-Up Kit (purchased from Sonofibrino corporation, Waltham, MA, USA). The mRNA vaccine of influenza A virus H1N1 is obtained by the in vitro transcription, capping and purification, and is used for forming micelle coating preparation or freeze-drying preservation.

Example 15 preparation of mRNA vaccine for coronavirus SARS-Cov-2

The DNA template was synthesized according to the procedure described in example 14, first step. The Gene sequence of the surface spike protein receptor binding region of SARS-CoV-2 coronavirus (NIH Gene ID: 43740568, Gene sequence-3) was used as the DNA sequence of the antigen of interest. The target gene sequence is optimized by GC content, and a linear template is synthesized by adding an untranslated region regulatory element (i) 5 'UTR (beta-globin-2), (ii) 3' UTR (2 beta-globin), and (iii) Poly-A tail and Poly-C tail of repeated adenosine and cytosine. And secondly, the obtained DNA template is subjected to in vitro transcription, capping and purification to obtain the mRNA vaccine of the coronavirus SARS-Cov-2, and the mRNA vaccine is used for forming a micelle encapsulated preparation or freeze-drying storage.

Example 16 preparation of polyamino acid micelles comprising avian influenza virus mRNA vaccine

In a 10 ml round bottom flask, 10 mg of PLL-SSMO25-PEG2000-Biotin20 prepared in example 1 was dissolved in 300. mu.l of ethanol, and 200. mu.l of an aqueous solution containing 3 mg of avian influenza virus H7N9 subtype mRNA vaccine prepared in example 13 was rapidly added to the polyamino acid solution (water was autoclaved to ensure no active nucleolytic enzyme, pH was adjusted to 4 with hydrochloric acid). Then, the solvent was purged with nitrogen gas by a syringe to form a transparent film. 2 mL of purified water was added, and vortexed at 10 ℃ for 15 minutes in a cold room. At this volume, the concentration of PLL-SSMO25-PEG2000-Biotin20 was greater than the critical colloidal concentration (see CMC measured in example 12), and the polyamino acids and mRNA formed micellar solutions. A200 mu L sample is diluted 5 times in pure water, the solution is subjected to a dynamic laser scattering experiment (see example 12 for the method), and the hydration diameter of the micelle is measured to be 161 +/-37 nm, and the result is shown in FIG. 3. The solution was equally divided into 100 μ L samples, each containing 30 μ g of mRNA vaccine, and stored in a 4 ℃ freezer or liquid nitrogen snap-freeze.

Example 17 preparation of polyamino acid micelles comprising coronavirus mRNA vaccine

Polyamino acid micelles containing an mRNA vaccine containing the protein sequence of interest as the surface spike protein receptor binding region were prepared as described in example 16, except that 200 microliters of an aqueous mRNA vaccine solution containing 2mg of the coronavirus SARS-Cov-2 prepared in example 15 was rapidly added to the polyamino acid solution.

Example 18 preparation of a Biofunctionalized polyamino acid nanomaterial comprising an avian influenza Virus mRNA vaccine

2 ml of polyamino acid micelles containing the mRNA vaccine were prepared according to the method of example 16, and 100. mu.M streptomycin phosphate buffer was added to the sample solution until the final concentration of streptomycin was 100 nanomolar (nM). The resulting sample solution was transferred to a semipermeable membrane with a cut-off molecular weight of 1 million daltons and dialyzed in 1 liter of phosphate buffer solution of pH 6 (autoclave sterilization treatment) for 24 hours. And adding 10 mg of recombinant human serum albumin (hSA) into the dialyzed solution, freezing the obtained mixed solution, and putting the frozen mixed solution into a freeze dryer to obtain freeze-dried powder. Sealing and storing at-20 deg.C. The freeze-dried powder sample is a streptomycin functionalized poly-amino acid nano material containing mRNA vaccine.

The nano material freeze-dried powder can be re-dissolved in pure water and is used for being combined with a biotin-modified small molecule ligand and an antibody protein (such as a PD-L1 antibody, a T cell receptor antibody, an interleukin 6 receptor antibody Kevlar (sarilumab)) and the like, so that the targeted delivery of target cells and tissues is improved.

Example 19 preparation of polyamino acid-adjuvant mixture comprising influenza A mRNA vaccine

10 mg of PLL-SSMO25-PEG2000-Biotin20 prepared in example 1 and 0.5 mg of cholesterol as adjuvant, 3 mg of lecithin were co-dissolved in 300. mu.L of ethanol in a 10 ml round bottom flask, and 200. mu.L of an aqueous solution containing 3 mg of influenza A virus mRNA prepared in example 14 was rapidly added to the solution of the polyamino acid (water was autoclaved to ensure inactive nucleolytic enzyme, pH was adjusted to 4 with hydrochloric acid). Then, the solvent was purged with nitrogen gas by a syringe to form a transparent film. 2 mL of purified water was added, and vortexed at 10 ℃ for 15 minutes in a cold room. At this volume, the concentration of PLL-SSMO25-PEG2000-Biotin20 was greater than the critical colloidal concentration (see CMC measured in example 12), and the polyamino acids and mRNA formed micellar solutions. And (3) taking 200 mu L of sample, diluting the sample by 5 times in pure water, and carrying out a dynamic laser scattering experiment on the solution to obtain the hydration diameter of the micelle which is 187 +/-45 nm, wherein the result is shown in FIG. 4. The solution was equally divided into 100 μ L samples, each containing 60 μ g mRNA, and stored in a refrigerator at 4 ℃ or snap frozen in liquid nitrogen.

Example 20 preparation of polyamino acid-adjuvant mixture nanomaterial containing influenza A virus mRNA vaccine

2 mL of polyamino acid-adjuvant mixture containing mRNA vaccine was prepared according to the method of example 19, and 100. mu.M (micromolar) of streptomycin phosphate buffer was added to the sample solution until the final concentration of streptomycin was 100nM (nanomolar). Dialyzing and freeze-drying according to the method of example 18 to obtain freeze-dried powder, and storing at-20 deg.C under sealed condition. The freeze-dried powder sample is a streptomycin functionalized polyamino acid-adjuvant mixture nano material containing mRNA vaccine. When in use, the nano material is re-dissolved in pure water, and is combined with biotin-modified small molecule ligands and antibody proteins (such as PD-L1 antibody, T cell receptor antibody and the like), so that the targeted delivery to target cells and tissues is improved.

Example 21 polyamino acid nanoparticles of avian influenza virus mRNA vaccine loaded with multivalent Kjeldahl targeting antibodies

The polyamino acid nano-material lyophilized powder of the streptomycin functionalized avian influenza virus H7N9 subtype-containing mRNA vaccine prepared in example 18 was redissolved in pure water, and mixed with biotin-modified interleukin 6 receptor antibody Kevlar (sarilumab) to obtain multivalent Kevlar targeting antibody-loaded polyamino acid nanoparticles containing the mRNA vaccine.

In this example, 20% of the polyamino acid with the side chain modified with the terminal biotin-polyethylene glycol prepared in example 1 was used, and according to the average molecular weight (22.5 kDa) of poly-l-lysine and the modification ratio of 20% of biotin side chain grafting, the polyamino acid nanoparticles containing the mRNA vaccine obtained in this example are a multivalent targeted delivery system with an average of 21 biotin/streptomycin/kevlar per particle. The polyamino acid nanoparticle containing the mRNA vaccine is named as H7N9-BT21 mRNA vaccine. The change of the hydration diameter of the polyamino acid nanoparticles containing the mRNA vaccine before and after loading the kevlar targeting antibody was measured by dynamic laser scattering experiments, as shown in a in fig. 5. The results showed that the peak values of the particle sizes were 140 nm (before) and 180nm (after), respectively, i.e., the average particle size increased by 40nm upon loading the targeting antibody.

As a control experiment for a monovalent targeted mRNA vaccine, the present example prepared amphiphilic polyamino acid micellar nanoparticles comprising the avian influenza virus subtype H7N9mRNA vaccine prepared in example 13 using the polyamino acid PLL-SSDDT25-PEG2000-OMe75 of example 8. The polyamino acid side chain PLL-SSDDT25-PEG2000-OMe75 of example 8 does not contain biotin functional groups and does not have the ability to load multivalent targeting antibodies. With the coupling agent 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), only the carboxyl groups of the biotin molecule can be reacted with the amine groups at the N-terminus of the backbone of the polyamino acid PLL-SSDDT25-PEG2000-OMe75 molecule. Polyamino acid nanoparticles carrying monovalent kevlar targeting antibodies containing mRNA vaccines were then prepared according to the method described above. Based on the structural characteristics of the main chain N-terminal modification, the obtained polyamino acid nanoparticles containing the mRNA vaccine are monovalent delivery systems with single biotin/streptomycin/Kjeldahl on average in single particles. The hydrated diameter of the polyamino acid nanoparticles containing the mRNA vaccine before and after loading of the kevlar targeting antibody was not changed by dynamic laser scattering experiments (130 nm, shown as B in fig. 5). It was named H7N9-BT1 mRNA vaccine. This result clearly demonstrates that the monovalent targeted H7N9-BT1 mRNA vaccine is significantly less efficient in antibody loading than the multivalent targeted vaccine, comparing a in fig. 5.

Example 22 Effect of multivalent Targeted vaccines

To verify the efficacy of the multivalent targeting vaccine, this example used the multivalent and monovalent targeting polyamino acid nanoparticles containing avian influenza virus H7N9mRNA vaccine prepared in example 21 to transfect the interleukin 6 receptor stable cell line of HEK293T, and reverse transcription-PCR quantification of the transfection efficacy was performed. The experimental procedure and results are as follows (see fig. 6).

The interleukin 6 receptor of HEK293T stably transfected cells at 37 ℃ and 5% CO₂Suspension culture was carried out at 130rpm under the conditions. Diluting the cells with activity greater than 95% to 1.0 × 10⁶cell/mL, seeded in six well plates at 1.6X 10 per well⁵Cells in MEM +10FBS at 37 ℃ in 5% CO₂The cells were cultured in a cell incubator overnight. 2mg of H7N9-BT21 mRNA vaccine freeze-dried powder is re-dissolved in 200 muL of MEM +10FBS culture solution, 100 muL of the culture solution is added into two culture wells respectively, and the cells are continuously cultured overnight. Similarly, 2mg of H7N9-BT1 mRNA vaccine was added to each of the two wells and the cells were allowed to continue to culture overnight. The six-well plate was removed, trypsinized, and all cells were collected by centrifugation. Cells added with the H7N9-BT21 mRNA vaccine were collected as a group, designated 1. Cells added with the H7N9-BT1 mRNA vaccine were collected as a group, designated 2. The group without added vaccine is labeled 3. Reverse transcription-PCR analysis was performed on 1, 2 and 3 groups of cells, respectively, to quantify the target mRNA content of cells transfected with monovalent and multivalent targeting shoots. The Qiagen reverse transcription-PCR Kit Oligotex mRNA Mini Kit is used and the operation is carried out according to the instruction, wherein the sequence of an upstream primer is atacatcaggaagacaggagaaga, the sequence of a downstream primer is tcaatgcacctgcatcctttccaagc, and the length of the target mRNA is 2000 bp. Gel electrophoresis experimentThe results are shown in FIG. 6. The group 1 cell sample showed a significant band at the 2000 bp position. The result shows that the target mRNA in the cells added with the H7N9-BT21 mRNA vaccine is efficiently expressed, and the multivalent targeting H7N9-BT21 mRNA can efficiently transfect an HEK293T interleukin 6 receptor stable cell line. The target mRNA expression level in the cells added with the monovalent H7N9-BT1 mRNA vaccine is the same as that in the untransfected control group, and no target mRNA band can be detected. As can be seen, the interleukin 6 receptor stable cell line of H7N9-BT1 mRNA transfected HEK293T targeted univalently is far inferior to the multivalent targeted H7N9-BT21 mRNA vaccine.

Example 23 Oxidation-reduction Condition response of binary polypeptide Complex Biofunctionalized polyamino acid mRNA vaccines

10 mg of PLL-SSMO25-PEG3500-HA15-PEG3500-ALFAtag15 prepared in example 11 was dissolved in 300. mu.L of ethanol, and 200. mu.L of an aqueous mRNA sequence solution containing 3 mg of influenza A virus H1N1 prepared in example 14 was rapidly added to the binary polypeptide complex biofunctionalized polyamino acid solution (water was autoclaved to ensure inactive nuclease, pH was adjusted to 4 with hydrochloric acid). Then, the solvent was purged with nitrogen gas by a syringe to form a transparent film. 2 mL of purified water was added and vortexed in a cold chamber at 10 ℃ for 15 minutes to allow formation of a micellar solution of the PLL-SSMO25-PEG3500-HA15-PEG3500-ALFAtag15 polyamino acid and mRNA. A200 mu L sample is diluted by 5 times in pure water, and the solution is subjected to a dynamic laser scattering experiment, so that the hydration diameter of the micelle is measured to be 195 +/-45 nm, and the result is shown in FIG. 7 (solid line + black square).

To observe the oxidation-reduction condition response of the binary polypeptide composite biologically functionalized polyamino acid mRNA vaccine, another 200 μ L of the sample was weighed, 500mM Dithiothreitol (DTT) phosphate buffer was added to make the final concentration of DTT in the sample solution 5mM, and the sample was incubated at room temperature for 60 minutes. The sample was diluted to 1mL with pure water, and the solution was subjected to a dynamic laser light scattering experiment, and the hydrated diameter of the micelle in the DTT reducing environment was found to be 95 ± 65 nm, as shown in fig. 7 (dotted line + grey squares). The increased distribution width and particle size peak shift to a small particle size of 100nm, indicating that the reducing conditions cause cleavage of disulfide bonds in the polyamino acids, resulting in disruption of the intact micelle structure, which is beneficial for mRNA vaccine release.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Tianjin Libo Biotechnology Ltd

<120> polyamino acid for mRNA vaccine targeted delivery, preparation method and application thereof

<141> 2021-10-01

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ttattttcat tgcagctcgc tttcttgctg tccaatttct attaaaggtt cctttgttcc 180

ctaagtccaa ctactaaact gggggatatt atgaagggcc ttgagcatct ggattctgcc 240

taataaaaaa catttatttt cattgc 266

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