CN112266483A

CN112266483A - Side chain modified polyamino acid and preparation method and application thereof

Info

Publication number: CN112266483A
Application number: CN202011530508.4A
Authority: CN
Inventors: 王晓
Original assignee: Tianjin Libo Biotechnology Co ltd
Current assignee: Tianjin Libo Biotechnology Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2021-01-26

Abstract

The invention provides a side chain modified polyamino acid and a preparation method thereof, wherein the side chain modified polyamino acid has the following advantages: (1) the main chain and side chain structures and the connection mode thereof can be flexibly selected, so that the prepared polymer micelle has good biocompatibility and targeted delivery efficiency, (2) the charge polarity of the main chain of the polyamino acid is electropositive, the method comprises the following steps of (1) promoting the pH value response of a micelle by adjusting the charge of a main chain, helping RNA escape from a 'lysosome trap' into cytoplasm, (3) controlling the hydrophobic part of a side chain by quantifying the chain length, saturation and the number of the fatty chain modified by the side chain, and accurately adjusting the volume and association strength of the hydrophobic part, (4) efficiently constructing a package and a delivery body due to the similarity of the RNA and the DNA in structure and electronegativity, and (5) introducing different biological functional groups by modifying the side chain of an amphiphilic functional polymer, so that the specific combination of a delivery system to target tissues and sites is realized, and the targeted delivery effect is improved.

Description

Side chain modified polyamino acid and preparation method and application thereof

Technical Field

The invention belongs to the field of polymers, and relates to a side chain modified polyamino acid, a preparation method and application thereof, in particular to a delivery nucleic acid molecule.

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' has been used to overcome smallpox virus, vaccines have helped humans 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 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 RNA vaccine has the characteristics of high biological safety, flexible design, rapid development and strong universality, so that the RNA vaccine becomes an important way for vaccine development (US 20200085852). The RNA vaccine can not only prevent and inhibit the infection of the virus in organisms, but also treat tumors by using the RNA vaccine, and for example, the RNA 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. In order to avoid hydrolytic and enzymatic consumption of RNA during circulation in the body, RNA vaccines and drugs require encapsulation protection of the delivery system. The vaccine is efficiently delivered into the cell and released from the "lysosomal trap" into the cytosol by a delivery system. The drug-forming and delivery modes of RNA vaccines 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 effects on negatively charged nucleic acid molecules by electrostatic interactions (WO 2017049245). In order to efficiently release RNA vaccines and drugs from "lysosomal traps" into the cytosol, optimization of the stability and pH response of the delivery system is required. There is also a need for a nucleic acid delivery system that is biologically functionalized to obtain the ability to target cells (e.g., cancer cells, B cells of the immune system, T cells) and tissues (e.g., organs such as lymphoid tissue, lung, small intestine, etc.) using biologically functional ligands and antibodies.

The micelle is a nano aggregate form formed by amphiphilic molecules in an aqueous solution spontaneously, is 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 amphiphilic 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 (WO 2013064648A) forms micelle-type nanomaterials. In the field of application of biological nano materials, polyamino acid is a completely biodegradable main chain type. The main chain of polyamino acid is based on amido bond, and the biological stability in the in vivo environment is superior to that of ester bond.

Disclosure of Invention

The invention aims to provide a side chain modified polyamino acid, a preparation method and application thereof, and application of the side chain modified polyamino acid in forming micelles for coating and delivering, in particular to coating and delivering of nucleic acid molecules such as RNA vaccines and the like.

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

a side chain-modified polyamino acid, wherein,

the polymer main chain is polylysine, or a copolymer formed by lysine and other amino acids; the other amino acids are any one or more of serine, threonine, tyrosine, cysteine, and arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, tryptophan, proline, methionine, asparagine, and glutamine.

At least part of the side chains of the polymer respectively contain a group A and a group G, wherein the group A is C_4-40An aliphatic hydrocarbon group, the group G is a group G1 and/or G2, G1 is a terminal group connected with C_1-10The polyethylene glycol group of alkoxy, G2 is the polyethylene glycol group with the end group connected with a biological functional group;

the group A and the group G are connected with the side chain of the polyamino acid through a connecting group.

In one embodiment, the linking group is as follows: -NH-CO-, -NH-, -N = CR₁-、-N-CHR₁-、-NH-CH₂-CO-、-O-CO-、-O-、-O-CH₂-CO-、-S-、-S-S-、-S-CO-、-S-CH₂-CO-or

Any one of them. Wherein R is₁Independently selected from H, C_1-6An alkyl group.

According to the invention, the amino, oxygen and sulfur in the connecting group are derived from active groups on the side chain of polyamino acid, such as amino, hydroxyl and sulfydryl;

the linking group may be a direct bond with the polyethylene glycol in group a or group G, or may be any spacer group.

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

In one embodiment, the group A is C_4-25Alkyl radical, C_4-25Alkenyl radical, C_4-25Alkynyl.

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

According to the invention, the biological functional group is a bonder capable of being combined with biological molecules such as protein, polypeptide, amino acid and the like, and comprises a synthetic bonder or natural biological molecules.

According to the invention, 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.

According to the invention, the natural biomolecules comprise: a carbohydrate molecule, a small RNA segment of about 5-80 nucleotides in length, and an amino acid sequence.

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

The small RNA segment is a nucleotide or deoxynucleotide sequence that is complementary to the biomolecule to be detected, for example a protein-binding aptamer sequence containing 10-60 nucleotides.

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

The antibody is a polyclonal antibody, a monoclonal antibody and a nano antibody, and the antibody is an antibody derived from animals or an antibody prepared by expression of recombinant protein.

According to the present invention, 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 polymer, there may be polyamino acid side chains that are not modified.

In one embodiment, the number of side chains containing the group G1 and/or G2 in the polymer is 2% to 98%, preferably 5% to 90%, more preferably 10% to 80% mole based on the total number of side chains.

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

In one embodiment, the G2 group is-PEG-T-B, where PEG is a polyethylene glycol segment, B is a biofunctional group, and T is a linker that links group B to the polyethylene glycol chain.

In one embodiment, the linking group T includes, for example, 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-or

Any 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.

Arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, proline, methionine, tryptophan, asparagine and glutamine in the main chain of the polymer can adjust the hydrophilcity and hydrophobicity, the hydrogen bonding capability and the pH value response of the polymer according to the characteristics of the hydrophobic characteristics or molecular configuration, size, aromaticity, the hydrogen bonding capability, the pH value response and the like of the side chain of the polymer.

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

In one embodiment, the backbone of the polymer is a synthetic poly-d-lysine.

In one embodiment, the backbone of the polymer is alpha-polylysine.

In one embodiment, the backbone of the polymer is epsilon-polylysine.

In one embodiment, the backbone of the polymer is an amino acid copolymer, including any one of a lysine-arginine copolymer, a lysine-histidine copolymer, a lysine-serine copolymer, a lysine-threonine copolymer, a lysine-tyrosine copolymer, a lysine-cysteine copolymer, a lysine-serine-threonine copolymer, a lysine-arginine-histidine copolymer, and a lysine-serine-threonine-tyrosine copolymer.

In one embodiment, the main chain of the polymer is an amino acid copolymer, and includes any one of lysine-glycine copolymer, lysine-leucine copolymer, lysine-isoleucine copolymer, lysine-valine copolymer, lysine-phenylalanine copolymer, and lysine-tryptophan copolymer.

In one embodiment, the side chain-modified polyamino acid has a mole percentage of lysine to the total amount of lysine of 0.1% to 100%, preferably 10% to 100%, more preferably 30% to 100%, of other amino acids.

The side chain-modified polyamino acid of the present invention may specifically be, for example:

。

the invention also provides a side chain modified polyamino acid, which is prepared by the following preparation method, including: linking polyamino acid to one end of C_1-10Polyethylene glycol with a reactive group x1 connected to the other end of the alkoxy group and/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_4-40Reacting aliphatic hydrocarbon; whereinThe reactive groups x1, x2 and x3 react with the active groups of amino, hydroxyl and sulfydryl on the side chain of the polyamino acid, and polyethylene glycol with alkoxy and/or biological functional groups at the end groups and aliphatic hydrocarbon groups are connected to the side chain of the polyamino acid.

The polyamino acid is polylysine, or a copolymer formed by lysine and other amino acids; the other amino acids are any one or more of serine, threonine, tyrosine, cysteine, and arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, tryptophan, proline, methionine, asparagine, and glutamine.

In the invention, the side chain of lysine contains amino, the side chains of serine, threonine and tyrosine contain hydroxyl, the side chain of cysteine contains sulfydryl, and the side chains of the groups can be subjected to side chain functional modification through covalent coupling reaction.

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

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

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

In one embodiment, the polylysine is an alpha-polylysine.

In one embodiment, the polylysine is epsilon-polylysine.

In one embodiment, the polyamino acid is an amino acid copolymer, including any one of a lysine-arginine copolymer, a lysine-histidine copolymer, a lysine-serine copolymer, a lysine-threonine copolymer, a lysine-tyrosine copolymer, a lysine-cysteine copolymer, a lysine-serine-threonine copolymer, a lysine-arginine-histidine copolymer, and a lysine-serine-threonine-tyrosine copolymer.

In one embodiment, the polyamino acid is an amino acid copolymer, including any one of lysine-glycine copolymer, lysine-leucine copolymer, lysine-isoleucine copolymer, lysine-valine copolymer, lysine-phenylalanine copolymer, and lysine-tryptophan copolymer.

The invention also provides a preparation method of the side chain modified polyamino acid, which comprises the following steps: linking polyamino acid to one end of C_1-10Polyethylene glycol with a reactive group x1 connected to the other end of the alkoxy group and/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_4-40Reacting aliphatic hydrocarbon; wherein, the reactive groups x1, x2 and x3 react with the active groups of amino, hydroxyl and sulfydryl on the side chain of the polyamino acid, and polyethylene glycol with alkoxy and/or biological functional groups at the end group and aliphatic hydrocarbon are connected to the side chain of the polyamino acid.

According to the method, the raw materials can be subjected to one-pot reaction or two-step or more reaction.

According to the invention, the reactive groups x1, x2, x3 are selected, for example, from hydroxyl, carboxyl, aldehyde, ketone, ester, thiol, maleimide, α -halocarbonyl.

For example, an amino group may be reacted with a carboxyl group to give an amide linking group, or an amino group may be reacted with an aldehyde or ketone group to give a Schiff base linking group, or an amino group may be reacted with an ester group to give an amide linking group, or a hydroxyl group may be reacted with a carboxyl group to give an ester linking group, or a hydroxyl group may be dehydrated to give an ether linking group, or a thiol group may be reacted with maleimide, or a thiol group may be reacted with an α -halocarbonyl group to give a substitution reaction, or a thiol group may be reacted with a thiol group.

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; when the side chain of the polyamino acid contains a sulfydryl, the reactive groups x1, x2 and x3 are at least one of sulfydryl, maleimide and alpha-halogenated carbonyl.

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

The polyethylene glycol having a reactive group at one end and an alkoxy group at the other end is commercially available. They may also be prepared by methods conventional in the art.

The polyethylene glycol having a reactive group at one end and a biofunctional group at the other end is commercially available or can be prepared by a conventional method in the art, for example, by the following method:

reacting polyethylene glycol having a reactive group x2 at one end and a reactive group x4 at one end with a biofunctional substance having a reactive group x5, the reactive group x4 reacting with the reactive group x5, thereby attaching the biofunctional group to one end of the polyethylene glycol.

According to the invention, the reactive groups x4, x5 are for example selected from 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. 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 polymer of the present invention_1-12Alkyl, ester, amide, ketone, and the like.

The polyamino acid is polylysine or is formed by polymerizing lysine and other amino acids; the other amino acids are any one or more of serine, threonine, tyrosine cysteine, and arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, tryptophan, proline, methionine, asparagine, glutamine. As examples, the polyamino acid is poly-l-lysine, and artificially synthesized poly-d-lysine, and the like.

According to the invention, the number of polyamino acid repeating units is an integer from 2 to 3000, preferably from 2 to 1500, more preferably from 2 to 1000, and still more preferably from 2 to 500.

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%.

According to the present invention, in the above steps, the reaction is a conventional reaction step in the art, and the reaction temperature is 10 to 40 ℃ for example.

According to the invention, the reaction can 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, the molar ratio of coupling agent to reactants being from 1 to 500, preferably from 1 to 100, more preferably from 1 to 50, still more preferably from 1 to 20.

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.

According to the present invention, 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 an aqueous solution so that the concentration thereof is 1 to 1000 times the critical micelle concentration.

According to the invention, the micelle is composed of the polyamino acid modified by the side chain, or is composed of more than two polyamino acids modified by the side chain. 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.

According to the present invention, the critical micelle concentration of the side chain-modified polyamino acid is measured by methods known in the art, including, for example, a conductance method, a surface tension method, a drop volume method, an ultrafiltration curve method, a single-point ultrafiltration method, a two-point ultrafiltration method, an ultraviolet spectrophotometry method, a dye adsorption method, a light scattering luminescence method, a fluorescence probe method, a solubility method.

According to the present invention, the micelle size average of the side chain modified polyamino acid is less than 300nm, preferably less than 200 nm, more preferably less than 100 nm, and even more preferably less than 80 nm.

According to the present invention, the micelle size of the side chain-modified polyamino acid 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.

According to the present invention, 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 further preferably, the concentration is 1 to 10 times the critical micelle concentration.

According to the invention, the solvent is, for example, an aqueous solution or a buffer at a pH of 3-8, for example an acetate buffer, a phosphate buffer.

The invention also provides application of the side chain modified polyamino acid micelle in coating and delivering nucleic acid.

The nucleic acid of the present invention includes RNA and DNA.

In one embodiment, the RNA of the invention is a single-stranded RNA.

In one embodiment, the RNA of the invention is a double-stranded RNA.

The nucleic acid can be RNA vaccine, RNA medicine and DNA vaccine.

The nucleic acid can be used as vaccine or medicine for treating or preventing infectious diseases, tumors, rare diseases, other protein deficiency diseases and the like, or 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.

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

According to the invention, the delivery system optionally comprises an adjuvant selected from the group consisting of a natural phospholipid molecule, cholesterol, an amino acid, a polypeptide, an ionic surfactant and a non-ionic surfactant.

According to the invention, optionally, the molar ratio of adjuvant to RNA strand is 0.001-8000, preferably 0.01-4000, more preferably 0.1-1000.

The invention also provides a preparation method of the delivery system, which comprises the steps of contacting the solution containing the micelle with nucleic acid, incubating the mixed solution, and preparing the delivery system.

The invention also provides a preparation method of the delivery system, which is characterized in that the delivery system is prepared by forming a mixed solution of the polyamino acid with the modified side chain and nucleic acid.

The delivery system may optionally be solvent evaporated to dryness to give a lyophilized powder. When used, re-dissolved in a solvent to re-form the delivery system.

According to the invention, the molar ratio of nucleic acids to side-chain-modified polyamino acids is from 0.001 to 1000, preferably from 0.01 to 100, more preferably from 0.1 to 10.

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

Wherein the solution containing the micelles may be, for example, an organic 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 "amphiphilic polyamino acid" in the present invention may be used interchangeably.

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 side chain modified polyamino acid and a preparation method thereof, wherein the side chain modified polyamino acid can efficiently form a polymer micelle by introducing a hydrophilic component and a hydrophobic component. The side chain modified polyamino acid has the following advantages: (1) 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 polymer micelle has good biocompatibility and targeted delivery efficiency. (2) The charge polarity of the main chain of the polyamino acid is electropositive, and the reactive groups of the side chain can be respectively amino, hydroxyl and sulfydryl. 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. (3) 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 macromolecule, and the two are cooperated to realize the accurate adjustment and control of the size and the stability of the polymer micelle. The polymer micelle disclosed by the invention is small in size and has good tissue penetrability. (4) 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 wrapping and delivery system is efficiently constructed, so that the efficient wrapping and delivery of RNA vaccines, RNA medicaments and DNA vaccines can be realized. Through the main chain structure and the side chain modification under the dual-pipe condition, the pH value response of the micelle is promoted, and the ribonucleic acid is helped to escape from a 'lysosome trap' and enter cytoplasm. (5) Different biological functional groups are introduced through side chain modification of amphiphilic functional macromolecules, and the targeting binding effectors comprise micromolecule binders, proteins, antibodies and the like, so that specific binding of a delivery system to target tissues and parts is realized, and the targeting delivery effect is improved.

Drawings

FIG. 1 is a partial NMR spectrum of an amphiphilic polyamino acid prepared in example 6.

FIG. 2 is the result of measuring the critical micelle concentration of the amphiphilic polyamino acid prepared in example 12 using example 3.

FIG. 3 shows the results of dynamic laser light scattering of micelles formed by using the amphiphilic polyamino acid prepared in example 12, and the size distribution is expressed in logarithmic scale.

FIG. 4 shows the results of dynamic laser light scattering of micelles formed by using the amphiphilic polyamino acid prepared in example 13 in example 9, and the size distribution is expressed in logarithmic scale.

FIG. 5 shows the results of dynamic laser light scattering of amphiphilic polyamino acid micelles containing mRNA vaccines prepared in example 17, with the size distribution expressed in logarithmic coordinates.

Fig. 6 shows the results of dynamic laser light scattering of amphiphilic polyamino acid micelles containing the mRNA vaccine prepared in example 20, with the size distribution expressed in logarithmic coordinates.

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 amphiphilic polyamino acid PLL-OL25-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 the end group modified BIOTIN and succinimide carboxymethyl ester was weighed and dissolved in 150. mu.L of tetrahydrofuran (analytical reagent, Nanjing reagent Inc.) to obtain a solution (B). To (B) 4.1. mu.L of distilled purified oleic acid (OL) was added. The solution (A) was mixed with the solution (B) in a test tube at room temperature, and 35 mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was immediately added to the resulting mixed solution. And oscillating at room temperature overnight, reacting for 8 hours, adding 100 mu L of Tris buffer solution with pH of 8.0, and continuing oscillating 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 25 mg of freeze-dried powder. The freeze-dried powder is functionalized amphiphilic polyamino acid grafted and modified by 25% of oleic acid and 20% of polyethylene glycol 2000-Biotin, the polymer is named as PLL-OL25-PEG2000-Biotin20, and the specific structural formula is shown as follows.

Wherein the number of side chains to which no other component is bonded is represented by n. The number of side chains of polyethylene glycol connected to the main chain of the polyamino acid is represented by p, the number of repeating units of polyethylene glycol is represented by q, and the number of side chains of the polyamino acid connected to the hydrophobic component is represented by m

EXAMPLE 2 preparation of the amphiphilic polyamino acid PLL-OL25-PEG3500-Biotin20

10 mg of PLL hydrobromide (average molecular weight 22500 daltons) was weighed into a2 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, purchased from Kyoto Keka science and technology Co., Ltd.) with the end group modified BIOTIN and succinimide carboxymethyl ester was weighed and dissolved in 150. mu.L of tetrahydrofuran to obtain a solution (B). To (B) 4.1. mu.L of distilled purified oleic acid (OL) was added. The reaction product was prepared according to the method of example 1 and lyophilized to obtain 31 mg of lyophilized powder. The white powder is functionalized amphiphilic polyamino acid grafted and modified by 25% of oleic acid and 20% of polyethylene glycol 3500-Biotin, and the polymer is named as PLL-OL25-PEG3500-Biotin 20.

EXAMPLE 3 preparation of the amphiphilic polyamino acid PLL-NO25-PEG3500-Biotin20

Similar to example 2, except that 35 mg of white lyophilized powder was prepared using 2.5 μ L of n-octanoic acid (NO), which is a functionalized amphiphilic polyamino acid graft-modified with 25% n-octanoic acid and 20% polyethylene glycol 3500-Biotin, the polymer was named PLL-NO25-PEG3500-Biotin 20.

Example 4 preparation of amphiphilic Polyaminoacid PLL-OL50-PEG3500-Biotin50

Similar to example 2, except that 82 mg of terminal group-modified succinimide carboxymethyl ester and biotin polyethylene glycol 3500 were weighed and dissolved in 150 μ L of tetrahydrofuran to obtain a solution (B). Adding 7.9 muL of oleic acid (OL) into (B), preparing white freeze-dried powder which is functionalized amphiphilic polyamino acid grafted and modified by 50% of oleic acid and 50% of polyethylene glycol 3500-Biotin and named as PLL-OL50-PEG3500-Biotin50 according to the method of example 1.

Example 5 preparation of the amphiphilic polyamino acid ε PLL-LA25-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 a2 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, purchased from Kyoto Kai technologies, Ltd.) with an end group modified BIOTIN and succinimide carboxy methyl ester was weighed and dissolved in 150. mu.L of tetrahydrofuran to obtain a solution (B). 2.8. mu.L of Lauric Acid (LA) was added to (B), and the solution (A) and the solution (B) were mixed in a test tube at room temperature, and 35 mg of EDC was immediately added to the resulting mixed solution to perform a coupling reaction. 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 43 mg of lyophilized powder, and storing at-20 deg.C under sealed condition. The white powder is a functionalized amphiphilic polyamino acid epsilon PLL-LA25-PEG5000-Biotin30 grafted and modified by 25% of lauric acid and 30% of polyethylene glycol 5000-Biotin, and the structural formula is as follows.

Wherein the number of side chains to which no other component is bonded is represented by n. The number of side chains of polyethylene glycol connected to the main chain of the polyamino acid is represented by p, the number of repeating units of polyethylene glycol is represented by q, and the number of side chains of the polyamino acid connected to the hydrophobic component is represented by m.

EXAMPLE 6 preparation of the amphiphilic polyamino acids ε PLL-OL25-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 (g) of terminal-modified azide salt and succinimide carboxymethyl esterAZIDE-PEG5000-SCM with an average molecular weight of 5000 daltons, purchased from Kyoto Keyka technologies, Inc., was dissolved in 150. mu.L of tetrahydrofuran to obtain a solution (B). To (B) 4.1. mu.L oleic acid (OL) was added, and the solution (A) and the solution (B) were mixed in a test tube at room temperature with a shaker. The product was prepared as described in example 5, lyophilized to obtain 38 mg of lyophilized powder, and stored sealed at-20 ℃. The white powder was 25% oleic acid and 30% polyethylene glycol 5000-azide graft modified amphiphilic polyamino acid, designated ε PLL-OL25-PEG 5000-N3. The proton nuclear magnetic resonance spectrum of the product is shown in FIG. 1 (¹H-NMR，500 MHz, CDCl3）。

Example 7 preparation of the amphiphilic polyamino acid PLL-LA25PEG2000-OMe37

10 mg of a PLL hydrobromide salt (average molecular weight 22500 daltons) was weighed into a2 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. mu.L of tetrahydrofuran to obtain a solution (B). To (B) 2.8. mu.L of lauric acid was added, and solution (A) was mixed with solution (B) in a test tube at room temperature with a shaker. In analogy to example 1, 30 mg of white lyophilized powder were obtained by EDC coupling. The white powder is 25% of lauric acid and 37% of polyethylene glycol 2000-methoxy graft modified amphiphilic polyamino acid, and the polymer is named as PLL-LA25-PEG2000-OMe 37. The specific structural formula is shown as follows.

EXAMPLE 8 preparation of the amphiphilic polyamino acid PLL-LA25-PEG2000-OMe75

Similar to example 7, except that 73 mg of polyethylene glycol (M-PEG 2000-SCM, average molecular weight 2000 Dalton, available from Kyork scientific Co., Ltd.) having an end group-modified methoxy group and succinimide carboxy methyl ester was weighed out and dissolved in 150. mu.L of tetrahydrofuran to obtain a solution (B). 59 mg of a white lyophilized powder was obtained, which was 25% oleic acid and 75% polyethylene glycol 2000-methoxy graft-modified amphiphilic polyamino acid, which was named PLL-LA25-PEG2000-OMe 75.

Example 9 preparation of the amphiphilic polyamino acid PLL-NO25-PEG2000-OMe37

Similar to example 7, except that 2.5 μ L of octanoic acid (NO) was taken in a glass vial, and placed in 150 μ L of tetrahydrofuran to obtain a solution (C). 22 mg of a white lyophilized powder was obtained, which was 25% n-octanoic acid and 37% polyethylene glycol 2000-methoxy graft-modified amphiphilic polyamino acid, which was named PLL-NO25-PEG2000-OMe 37.

Example 10 preparation of a biofunctionalized amphiphilic polyamino acid PLL-OL25-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 5 mM 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, available from Kyork science and technology Co., Ltd., Beijing) with methoxy and succinimidyl esters at both ends were weighed in a small glass vial, dissolved in 150. mu.L of tetrahydrofuran, and 114. mu.L of the above prepared tetrahydrofuran solution of COOH-PEG2000-BG was added to obtain a solution (B). To the solution (B), 3.2. mu.L of oleic acid was added. The solutions (A) and (B) were mixed, 28 mg of EDC coupling reagent was immediately added to the resulting mixed solution, shaken overnight, and 100. mu.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 29 mg lyophilized powder. The freeze-dried powder is amphiphilic polyamino acid grafted and modified by 25% of oleic acid, 30% of polyethylene glycol 2000-methoxyl and 30% of ligand linker of polyethylene glycol 2000-SNAPTag protein, and the polymer is named as PLL-OL25-PEG2000-OMe30-PEG2000-BG 30. The specific structural formula is shown as follows.

Wherein the number of side chains to which no other component is bonded is represented by n. The number of side chains to which the polyethylene glycol-methoxy group is attached is denoted by p, and the number of repeating units of polyethylene glycol is denoted by q. The number of side chains to which polyethylene glycol-BG is attached is denoted by r, and the number of repeating units of polyethylene glycol is denoted by t. The number of side chains to which the hydrophobic moiety is attached is represented by m.

EXAMPLE 11 preparation of the polypeptide Bio-functionalized amphiphilic polyamino acid PLL-OL25-PEG3500-HA30

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). In addition, 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 and 13 mg of polypeptide lyophilized powder (HA-tag, N-end modified by acetyl group) with amino acid sequence CYPYDVPDYA were weighed and dissolved in 200 μ L of HEPES buffer solution (5 mM HEPES) to adjust the pH of the solution to 6.0, thereby obtaining a solution (B). After shaking for 10 minutes at room temperature, polyethylene glycol solutions with the terminal groups of the HA polypeptide sequence conjugate and the succinimide carboxymethyl ester are obtained, and the polyethylene glycol solutions are added into the solution (A) and shaken for 4 hours at room temperature. The mixed solution was added to 400 μ L of tetrahydrofuran solution containing 3.2 μ L of oleic acid. The solution was transferred to a glass vial, and 28 mg of EDC coupling reagent was immediately added to the resulting mixed solution, shaken overnight, and 100. mu.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 32 mg lyophilized powder. The freeze-dried powder is 25% oleic acid and 30% polyethylene glycol 3500-HA-Tag graft modified amphiphilic polyamino acid, and the polymer is named as PLL-OL25-PEG3500-HA 30.

Example 12 preparation and characterization of amphiphilic polyamino acid micelles

This example uses the bio-functionalized n-octanoic acid-polyethylene glycol graft modified polylysine PLL-NO25-PEG3500-Biotin20 prepared in example 3 as an example, and illustrates that critical micelle concentration CMC of amphiphilic 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 light 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-NO25-PEG3500-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 and a slit width set to excitation: 5 nm, emission: 2.5 nm and detector bias at 700V. Experiment temperatureDegree 22 + -1 deg.C.

FIG. 2 shows the fluorescence emission spectrum change of pyrene in different concentrations of PLL-NO25-PEG3500-Biotin20 aqueous solution. The ratio (I) of the fluorescence intensity of the first peak (373 nm) to the third peak (384 nm) of pyrene₁/ I₃FIG. 2A) reflects the polarity change of the microenvironment around the pyrene molecule. The ratio decreased with increasing concentration of amphiphilic polyamino acid PLL-NO25-PEG3500-Biotin20 in aqueous solution (FIG. 2B). When the concentration of the amphiphilic polyamino acid is increased to a certain value, the curve is mutated, the mutation shows that the polarity of the environment where pyrene is located is changed, and PLL-NO25-PEG3500-Biotin20 micelles are formed. Thus, the first mutation point corresponds to the CMC value of PLL-NO25-PEG3500-Biotin 20. The critical micelle concentration of PLL-NO25-PEG3500-Biotin20 was measured by this method to be 1.6 mg/mL. When the concentration of PLL-NO25-PEG3500-Biotin20 in the aqueous solution is more than the value, micelle can be formed.

PLL-NO25-PEG3500-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 93. + -.36 nm using Zetasizer Nano ZS ZEN3600 from Malvern Instruments, UK, and the results are shown in FIG. 3.

Example 13 preparation and characterization of amphiphilic polyamino acid micelles

Using the caprylic acid-polyethylene glycol grafted polylysine PLL-NO25-PEG2000-OMe37 prepared in example 9 as an example, the critical micelle concentration of the amphiphilic polyamino acid micelle determined by a probe steady state fluorescence emission method and the average hydration diameter of the micelle measured by a dynamic laser light scattering method are less than 80 nm. According to the pyrene fluorescence probe method described in example 12, the ratio (I) of the fluorescence intensity of the first peak (373 nm) to the third peak (384 nm) of pyrene was measured₁/ I₃) The first mutation point of (2.3 mg/mL). The Critical Micelle Concentration (CMC) here corresponding to PLL-NO25-PEG2000-OMe37 was 2.3 mg/mL. PLL-NO25-PEG2000-OMe37 was prepared as a 5.0 mg/mL phosphate buffer solution (pH 7.4), and 1mL of the solution was used for dynamic laser light scattering experiments. The hydrated diameter of the micelles was measured using Zetasizer Nano ZS ZEN3600 (Malvern Instruments, Inc.) in the United kingdomThe particle size was 75. + -.28 nm, and the results are shown in FIG. 4.

Example 14 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, a MeGAclear transfer clear-Up Kit (purchased from Sonofugreek) was usedDepartment, Waltham, MA, USA). The resulting sample can be used immediately to form micelle encapsulated formulations or lyophilized for storage.

Example 15 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):

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

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 16 preparation of mRNA vaccine for coronavirus SARS-Cov-2

A DNA template was synthesized according to the procedure described in example 15, 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 17 preparation of amphiphilic polyamino acid micelles comprising mRNA vaccine

In a 10 ml round bottom flask, 10 mg of PLL-NO25-PEG3500-Biotin20 prepared in example 3 was dissolved in 300. mu.l of ethanol, and 200. mu.l of an aqueous solution containing 3 mg of the avian influenza virus H7N9 subtype mRNA vaccine prepared in example 14 was rapidly added to the amphiphilic 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-NO25-PEG3500-Biotin20 was greater than the critical colloidal concentration (see CMC measured in example 12), and the polyamino acid and mRNA formed a micellar solution. 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 determined to be 144 +/-34 nm, and the result is shown in FIG. 5. The solution was equally divided into 100 μ L samples, each containing 30 μ g of mRNA vaccine, stored in a refrigerator at 4 ℃ or stored by liquid nitrogen snap freezing.

Example 18 preparation of amphiphilic polyamino acid micelles comprising mRNA vaccine

Amphiphilic polyamino acid micelles containing an mRNA vaccine were prepared as described in example 17, except that 200 microliters of an aqueous mRNA vaccine solution containing 2 mg of the coronavirus SARS-Cov-2 prepared in example 16, which contained a target protein sequence of the surface spike protein receptor binding region, was rapidly added to the polyamino acid solution.

Example 19 preparation of biofunctionalized amphiphilic polyamino acid nanomaterials comprising mRNA vaccines

2 ml of amphiphilic polyamino acid micelles containing the mRNA vaccine were prepared according to the method of example 17, 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 amphiphilic polyamino 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. The interleukin 6 receptor antibody Kevlar functionalized mRNA vaccine can be used for patients with the coronavirus COVID-19 with the increased interleukin 6 level.

Example 20 preparation of amphiphilic polyamino acid-adjuvant mixtures comprising mRNA vaccines

10 mg of PLL-NO25-PEG3500-Biotin20 prepared in example 3 and 0.5 mg of cholesterol as adjuvant and 3 mg of lecithin were dissolved together in 300. mu.L of ethanol in a 10 ml round-bottomed flask, and 200. mu.L of an aqueous solution containing 3 mg of 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-NO25-PEG3500-Biotin20 was greater than the critical colloidal concentration (see CMC measured in example 12), and the polyamino acid and mRNA formed a micellar solution. And (3) taking 200 muL of sample to dilute the sample by 5 times in pure water, and carrying out a dynamic laser scattering experiment on the solution to obtain that the hydration diameter of the micelle is 176 +/-42 nm, wherein the result is shown in FIG. 6. 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 21 preparation of amphiphilic polyamino acid-adjuvant mixture nanomaterial containing mRNA vaccine

2 mL of an amphiphilic polyamino acid-adjuvant mixture containing mRNA vaccine was prepared according to the method of example 20, and 100. mu.M (micromolar) of streptomycin phosphate buffer was added to the sample solution until the final concentration of streptomycin was 100 nM (nanomolar). Dialyzing and freeze-drying according to the method of example 19 to obtain freeze-dried powder, and storing at-20 deg.C under sealed condition. The freeze-dried powder sample is a streptomycin functionalized amphiphilic 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.

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.

Claims

1. The side chain modified polyamino acid is characterized in that the main chain of the polyamino acid is polylysine or a copolymer formed by lysine and other amino acids; the other amino acid is any one or more of serine, threonine, tyrosine, cysteine, arginine, histidine, glycine, leucine, isoleucine, valine, phenylalanine, tryptophan, proline, methionine, asparagine, and glutamine;

at least part of the side chains of the polymer respectively contain a group A and a group G, wherein the group A is C_4-40An aliphatic hydrocarbon group, the group G is a group G1 and/or G2, G1 is a terminal group connected with C_1-10Poly (alkylene oxide) sThe G2 is a polyethylene glycol group with a biological functional group connected with the end group.

2. The side-chain modified polyamino acid according to claim 1, wherein the group a and the group G are linked to the side chain of the polyamino acid by a linking group selected from the group consisting of: -NH-CO-, -NH-, -N = CR₁-、-N-CHR₁-、-NH-CH₂-CO-、-O-CO-、-O-、-O-CH₂-CO-、-S-、-S-S-、-S-CO-、-S-CH₂-CO-or

Wherein R is₁Independently selected from H, C_1-6An alkyl group;

3. The side-chain-modified polyamino acid according to claim 1, wherein the number of repeating units of the polyethylene glycol is an integer between 1 and 600;

said group A is C_4-25Alkyl radical, C_4-25Alkenyl radical, C_4-25An alkynyl group;

the alkoxy group in the group G1 is C_1-6An alkoxy group;

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 side-chain modified polyamino acid of claim 3, wherein the binder comprises: biotin which can bind to avidin; a linker that can be linked to a SNAP protein; a linker that can be linked to a CLIP protein; a linker that can be linked to a HaloTag protein;

the natural biomolecules include: saccharide molecule, RNA segment with length of 5-80 nucleotides, and amino acid sequence.

5. The side-chain-modified polyamino acid according to claim 4, wherein the linker to which the SNAP protein can be linked is benzylguanine and derivatives thereof; the linker capable of being connected with the CLIP protein is benzylcytosine and derivatives thereof; the linker capable of being connected with the HaloTag protein is 6-chloro-n-hexane and derivatives thereof;

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

the RNA chain segment is a nucleic acid sequence which contains 10-60 nucleotides and can be combined with protein;

the amino acid sequence is an amino acid sequence capable of binding to a protein, including influenza hemagglutinin HA amino acid sequence, FLAG amino acid sequence, myc-tag amino acid sequence, and "C-tag" comprising the-EPEA amino acid sequence, and amino acid "ALFA-tag" comprising SRLEEELRRRL.

6. The side-chain-modified polyamino acid according to claim 1, wherein the number of side chains containing group G1 and/or G2 in the polymer is 2 to 98% by mole based on the total amount of side chains;

in the polymer, the mole percentage of the number of side chains containing the group A to the total amount of all the side chains is 2-98%;

in the polymer, the mole percentage of the sum of the number of side chains containing the group A and the number of side chains containing the groups G1 and/or G2 in the total amount of all side chains is 5-100%;

in the side chain modified polyamino acid, the mole percentage of lysine in the main chain accounts for 0.1-100% of the total amount of other amino acids and lysine.

7. The side-chain-modified polyamino acid according to claim 1, wherein the backbone of the polymer is α -polylysine or is e-polylysine; or

The main chain of the polymer is an amino acid copolymer, and comprises any one of lysine-arginine copolymer, lysine-histidine copolymer, lysine-serine copolymer, lysine-threonine copolymer, lysine-tyrosine copolymer, lysine-cysteine 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;

the group G2 is-PEG-T-B, wherein PEG is a polyethylene glycol chain segment, B is a biological functional group, and T is a connecting group.

8. The side-chain-modified polyamino acid according to claim 1, wherein the side-chain-modified polyamino acid is:

wherein the number of side chains to which no other component is bonded is represented by n, the number of side chains to which polyethylene glycol is bonded is represented by p or r, the number of repeating units of polyethylene glycol is represented by q or t, and the number of side chains to which a hydrophobic group is bonded is represented by m.

9. A side chain modified polyamino acid, which is prepared by the following preparation method, comprising the following steps: linking polyamino acid to one end of C_1-10Polyethylene glycol with a reactive group x1 connected to the other end of the alkoxy group and/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_4-40Reacting aliphatic hydrocarbon; wherein, the reactive groups x1, x2 and x3 react with the active groups of amino, hydroxyl and sulfydryl on the side chain of the polyamino acid, and polyethylene glycol with alkoxy and/or biological functional groups at the end group and aliphatic hydrocarbon are connected to the side chain of the polyamino acid;

10. The method for producing a side-chain-modified polyamino acid according to any one of claims 1 to 9, comprisingThe method comprises the following steps: linking polyamino acid to one end of C_1-10Polyethylene glycol with a reactive group x1 connected to the other end of the alkoxy group and/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_4-40Reacting aliphatic hydrocarbon; wherein, the reactive groups x1, x2 and x3 react with the active groups of amino, hydroxyl and sulfydryl on the side chain of the polyamino acid, and polyethylene glycol with alkoxy and/or biological functional groups at the end group and aliphatic hydrocarbon are connected to the side chain of the polyamino acid.

11. The method of claim 10, wherein the reactive groups x1, x2, x3 are selected from hydroxyl, carboxyl, aldehyde, ketone, ester, thiol, maleimide, α -halocarbonyl;

the number of the polyamino acid repeating units is an integer of 2-3000; the lysine accounts for 0.1 to 100 percent of the total mole percentage of other amino acids and lysine.

12. Use of the side-chain modified polyamino acid of any one of claims 1 to 9 for the preparation of micelles.

13. A micelle consisting of one or more side chain modified polyamino acids according to any one of claims 1 to 9.

14. A micelle according to claim 13, wherein the micelle is formed by dissolving a side chain modified polyamino acid according to any one of claims 1-9 in a solvent, the side chain modified polyamino acid having a micelle average particle size of less than 300 nm.

15. The method for preparing micelles of claim 13, comprising the steps of:

16. Use of the micelle of claim 13 in the coating and delivery of nucleic acid molecules.

17. A delivery system comprising the micelle of claim 13 and a nucleic acid, wherein the nucleic acid is located within the micelle.

18. The delivery system according to claim 17, wherein the nucleic acid is an RNA vaccine, an RNA drug, a DNA vaccine.

19. The delivery system according to claim 17, wherein the nucleic acid is a vaccine or a drug for the treatment or prevention of infectious diseases, tumors, rare diseases, and other protein-deficient diseases, or for cosmetic and anti-aging purposes.

20. The delivery system according to claim 17, wherein the delivery system comprises an adjuvant selected from the group consisting of a naturally occurring phospholipid molecule, cholesterol, an amino acid, a polypeptide, an ionic surfactant, and a non-ionic surfactant.

21. A method of making a delivery system, comprising: contacting the solution containing the micelle with nucleic acid, and incubating the mixed solution to prepare the delivery system; or,

and forming a mixed solution of the side chain modified polyamino acid and nucleic acid to prepare the delivery system.