Amino acid polymer as well as preparation method and application thereof

Abstract

The invention provides an amino acid polymer with controllable molecular weight as well as a preparation method and application thereof. According to the preparation method disclosed by the invention, ring-opening polymerization of acid anhydrides in amino acid rings and derivatives of the acid anhydrides is catalyzed by introducing an acid and / or alkali catalyst in the presence of an initiator primary amine, so that polyamino acids with different molecular weights and narrow distribution are obtained. The invention also provides application of the amino acid polymer in the field of gene delivery, and compared with other amino acid polymers, the polytryptophan polypeptide material can effectively penetrate through the blood brain barrier to target the brain, is beneficial to treatment of cerebral diseases, and has a better application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical polymers, and in particular relates to an amino acid polymer and a preparation method and application thereof. Background Art

[0002] Polyamino acid materials, a class of biopolymers with excellent biocompatibility, degradability, and functional designability, hold a key position in fields such as drug delivery, tissue engineering, biosensing, and smart responsive materials. Their molecular structures mimic the sequence and conformation of natural proteins while offering the stability and controllability of synthetic materials, providing an ideal platform for the development of biomedical functional materials. However, the functional singularity and structural limitations of traditional polyamino acid materials (such as polylysine and polyglutamic acid) make it difficult to meet the demands of high-end applications for material specificity, dynamic responsiveness, and multi-dimensional functional integration.

[0003] In this context, tryptophan (Trp), a naturally encoded eukaryotic amino acid, has demonstrated significant value in a variety of fields due to its unique physicochemical properties. Its indole group combines hydrophobicity with diverse bonding capabilities (such as cation-π, π-π, and hydrophobic interactions), making it a core unit for the design of stable β-sheet structures, such as the Trpzip peptide. Research on polytryptophan-based self-assembled hydrogels, charge transfer properties, and biomedical applications (including antimicrobial, biotracing, cell delivery, bio-3D printing, and tumor nanotherapy) has attracted considerable attention.

[0004] The blood-brain barrier (BBB), composed of endothelial cells, allows only the passive diffusion of small, lipid-soluble molecules, while large molecules (such as proteins and nucleic acids) or hydrophilic drugs are virtually unable to pass through it. This limits the treatment of many brain diseases, such as brain tumors and neurodegenerative diseases. The low efficiency of BBB penetration remains a pressing issue that needs to be addressed.

[0005] Limited by the steric hindrance and conformational constraints of the tryptophan side chain, the high molecular weight (M n The controlled synthesis of polytryptophan has long been a challenge. In biosynthesis, tryptophan easily triggers ribosomal translation arrest. In chemical synthesis, solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL) have struggled to achieve lengths exceeding 20 repeating units.

[0006] In the past decade, the research on the controlled preparation of amino acids by ring-opening polymerization has been extensive, such as Chem.Soc. Rev., 2018, 47, 7401-7425.;Nature Chemistry, 2017, 9, 614–622.;CCSChemistry, 2024, 0:1–28.;Nature Communications, 2019, 10, 5470.;PNAS, 2019,116, 10658-10663.; ACS Macro Lett. 2019, 8, 1517−1521.;However, the research tends to focus on the ring-opening polymerization of L-glutamic acid benzyl ester N-carboxyl ring anhydride (BLG-NCA). It has been reported that the ring-opening polymerization of Trp-NCA is the most difficult due to the steric effect (Bulletin of the Chemical Society of Japan 2006, 69(3), 791.). The negative potential of the indole ring can shield the nucleophilic attack of the primary amine to a certain extent, and the short chain beta tendency makes it difficult to dissolve in some low-polarity solvents. Although studies have attempted to optimize the polymerization process through phenoxycarbonyl-functionalized α-amino acid (NPCA) systems, sugar-initiated systems, or Lewis acid-base pair copolymerization strategies, controlled polymerization methodologies are still imperfect. The synthesis of high-molecular-weight peptides containing tryptophan (and its derivatives) still faces challenges, limiting their application in high-end fields such as nanomedicine.

[0007] To address the above bottlenecks, the present invention proposes an innovative polymerization strategy: enhancing the helical conformational stability through low-polarity mixed solvents such as DCM and THF, and increasing the polymer solubility through DMF. The synergistic effect of the two solvents improves solubility and polymerization controllability. Unlike the classic base-induced activated monomer mechanism (AMM mechanism) (J. Am. Chem. Soc. 2024, 146, 35, 24189–24208), tryptophan is shielded by the base due to the weak acidity of the indole ring, resulting in a kinetic curve and product characteristics that are different from those of conventional BLG-NCA. For BLG-NCA, it is difficult to control the molecular weight when alkali is added to initiate polymerization, and the side chains tend to hydrolyze, resulting in an uncontrollable polymerization process (Chem. Commun. 2003, 23, 2944– 2945, Polym. Chem. 2013, 4 (11), 3182– 3186, Chem. Cmmun. 2015, 51(86), 15645–15648). This technology aims to achieve the controllable preparation of high molecular weight polytryptophan, precisely control the molecular weight and narrow distribution of products, and at the same time enhance the chemical reactivity and "label-free" fluorescence properties of the material, providing technical support for the development of a new generation of biomedical functional materials. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a polyamino acid polymer and a preparation method and application thereof. The synthesis method can effectively control the number average molecular weight of the synthesized polymer, and the molecular weight distribution of the product is narrow, which is used to solve the problems in the prior art that the molecular weight of the product is uncontrollable and the polymerization conditions are harsh and need to be carried out in an inert atmosphere.

[0009] To achieve the above-mentioned purpose and other related purposes, the present invention is obtained by including the following technical solutions.

[0010] In a first aspect, the present invention provides an amino acid polymer, wherein the amino acid polymer comprises a homopolymer or copolymer of an N-carboxyl cyclic anhydride of an amino acid or a derivative thereof.

[0011] In some embodiments of the present invention, the α-carbon configuration of the amino acid is one or more of L-type, D-type and racemic DL-type; and the amino acid includes an amino acid or a derivative thereof.

[0012] In some embodiments of the present invention, the N-carboxyl intracyclic anhydride monomer of tryptophan or its derivative has a structure of formula I;

[0013] Wherein R1, R3, R4, R5, and R6 are each independently one of a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a hydroxyl group or an acetonitrile group; and R2 is one of a hydrogen atom, a trifluoromethyl group, a methyl group, an isopropyl group, a tert-butyl group or a tert-butyloxycarbonyl group.

[0014] In some embodiments of the present invention, the amino acid N-carboxyl intracyclic anhydride further comprises a hydrophilic amino acid N-carboxyl intracyclic anhydride.

[0015] In some embodiments of the present invention, in order to improve hydrophilicity and facilitate drug administration, the polymer for gene delivery needs to provide a hydrophilic amino acid N-carboxyl ring anhydride monomer for ring-opening copolymerization in addition to the indole hydrophobic amino acid N-carboxyl ring anhydride (such as the N-carboxyl ring anhydride of tryptophan or its derivatives), wherein the hydrophilic amino acids include but are not limited to glutamic acid, lysine, aspartic acid, sarcosine, serine, threonine, cysteine, glutamine, glycine, and tyrosine; for example, the hydrophilic amino acid N-carboxyl ring anhydride monomer can be glutamic acid benzyl ester N-carboxyl ring anhydride, N6-Cbz-L-lysine N-carboxyl ring anhydride, aspartic acid benzyl ester N-carboxyl ring anhydride, and sarcosine N-carboxyl ring anhydride.

[0016] In some embodiments of the present invention, the amino acid N-carboxyl ring anhydride monomer may also include other hydrophobic amino acid N-carboxyl ring anhydride monomers, such as leucine N-carboxyl ring anhydride monomer, isoleucine N-carboxyl ring anhydride monomer, valine N-carboxyl ring anhydride monomer, etc.

[0017] In some embodiments of the present invention, the degree of polymerization of the amino acid polymer is 5-1000; it can also be 5-20, 20-40, 40-60, 60-80, 80-100, 100-150, 150-200, 200-400, 400-600, 600-800 or 800-1000.

[0018] In some embodiments of the present invention, the amino acid polymer includes a copolymer formed by an N-carboxylic anhydride monomer of tryptophan or its derivative and an N-carboxylic anhydride (NCA) monomer of lysine or its derivative.

[0019] In some embodiments of the present invention, in the amino acid polymer, the molar ratio of N-carboxyl cyclic anhydride of tryptophan or its derivative to N-carboxyl cyclic anhydride (NCA) of lysine or its derivative is 0.01-20:1, and can also be 0.01-0.1:1, 0.1-1:1, 1-5:1, 5-10:1, 10-15:1, or 15-20:1.

[0020] In some embodiments of the present invention, the number average molecular weight of the amino acid polymer is 1000–200000 Da; it can also be 1000–5000 Da, 5000–15000 Da, 15000–35000 Da, 35000–70000 Da, 70000–100000 Da, 100000–130000 Da, 130000–160000 Da, 160000–200000 Da.

[0021] In some embodiments of the present invention, the molecular weight distribution PDI of the amino acid polymer is <1.3; preferably 1<PDI<1.3.

[0022] In some embodiments of the present invention, the copolymer is a random copolymer or a block copolymer.

[0023] In some embodiments of the present invention, the block copolymer is a block copolymer of 2-10 blocks, and can also be a copolymer of 2, 3, 4, 5, 6, 7, 8, 9 or 10 blocks.

[0024] In some embodiments of the present invention, the length of each block is 10-100 amino acids, and can also be 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 amino acids.

[0025] In some embodiments of the present invention, the amino acid polymer comprises a homopolymer or copolymer having the structure of Formula II;

[0026]

[0027] wherein R1, R3, R4, R5, and R6 are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a hydroxyl group, or an acetonitrile group; R2 is a hydrogen atom, a trifluoromethyl group, a methyl group, an isopropyl group, a tert-butyl group, or a tert-butyloxycarbonyl group; R is a methyl group, an ethyl group, a propyl group, a butyl group, a tert-butyl group, a hexyl group, a pentyl group, a benzyl group, or a propargyl group; and n is a degree of polymerization, which is 5-1000.

[0028] The second aspect of the present invention provides a method for preparing the above-mentioned amino acid polymer, comprising subjecting one or more amino acids or their derivatives to a ring-opening polymerization of anhydride monomers within the N-carboxyl ring to form the amino acid polymer.

[0029] In a preferred embodiment, the at least one amino acid N-carboxyl intracyclic anhydride is an N-carboxyl intracyclic anhydride of tryptophan or a derivative thereof.

[0030] In some preferred embodiments of the present invention, the plurality includes 2 to 10 types.

[0031] In some embodiments of the present invention, when preparing a random copolymer, one or more N-carboxyl ring anhydrides of amino acids or their derivatives are mixed and then subjected to a polymerization reaction to form an amino acid polymer, wherein at least one N-carboxyl ring anhydride of an amino acid or its derivative is an N-carboxyl ring anhydride of tryptophan or its derivative.

[0032] In some embodiments of the present invention, when preparing a block copolymer, one or more N-carboxyl ring anhydrides of amino acids or their derivatives are polymerized step by step to form an amino acid polymer, wherein at least one N-carboxyl ring anhydride of an amino acid or its derivative is an N-carboxyl ring anhydride of tryptophan or its derivative.

[0033] For example, when there are two amino acid N-carboxyl ring anhydrides, in the presence of an initiator, the N-carboxyl ring anhydride of one amino acid or its derivative is first subjected to a polymerization reaction; after the above polymerization reaction is completed, the N-carboxyl ring anhydride of another amino acid or its derivative is added to carry out a polymerization reaction, thereby forming a block amino acid copolymer; wherein the N-carboxyl ring anhydride of at least one amino acid or its derivative is the N-carboxyl ring anhydride of tryptophan or its derivative.

[0034] When there are three N-carboxyl ring anhydride monomers of amino acids or their derivatives: in the presence of an initiator, the N-carboxyl ring anhydride of one amino acid or its derivative is first subjected to a polymerization reaction; after the above polymerization reaction is completed, the N-carboxyl ring anhydride of another amino acid or its derivative is added to carry out a polymerization reaction, thereby forming a block amino acid copolymer; wherein at least one of the N-carboxyl ring anhydride of the amino acid or its derivative is the N-carboxyl ring anhydride of tryptophan or its derivative.

[0035] In some embodiments of the present invention, the preparation method of the N-carboxyl cyclic anhydride of the amino acid or its derivative is: dissolving the amino acid or its derivative in a first organic solvent, adding triphosgene and an acid binding agent to carry out a ring-closure reaction, and obtaining an NCA monomer after purification.

[0036] In some embodiments of the present invention, the first organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, 1,4-dioxane, ethyl acetate, and N-methylpyrrolidone.

[0037] In some embodiments of the present invention, the derivative is a derivative derived from an amino acid; the derivative comprises introducing or replacing a functional group in the main chain or side chain of the amino acid.

[0038] In some embodiments of the present invention, the functional group includes but is not limited to hydroxyl, alkyl, amino, carboxyl, protecting group, etc.; in some embodiments of the present invention, the protecting group includes but is not limited to benzoyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), etc.

[0039] In some embodiments of the present invention, the alkyl group includes but is not limited to methyl, ethyl, and the like.

[0040] In some embodiments of the present invention, the amino acids include tryptophan or its derivatives; and may also include: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, cysteine, proline, histidine, arginine or one or more of their derivatives.

[0041] In some embodiments of the present invention, the tryptophan derivatives include 5-hydroxytryptophan, 5-hydroxy-tryptophan, L-methyltryptophan, L-ethyltryptophan, Cbz-L-tryptophan, Fmoc-L-tryptophan, D-tryptophan, and DL-tryptophan.

[0042] In some embodiments of the present invention, the polymerization reaction of the N-carboxyl anhydride of the amino acid or its derivative specifically comprises the following steps:

[0043] The N-carboxyl ring anhydride of an amino acid or a derivative thereof, an initiator, a catalyst and a second organic solvent are mixed to obtain a mixture, and ring-opening polymerization is performed to obtain an amino acid polymer.

[0044] In some embodiments of the present invention, the N-carboxyl ring anhydride monomer of tryptophan or its derivatives includes one or more of tryptophan-N-carboxylic acid ring anhydride, 5-hydroxy-tryptophan-N-ring anhydride, methyltryptophan-N-ring anhydride, and 7-bromotryptophan-N-ring anhydride.

[0045] In some embodiments of the present invention, the acid binding agent includes one or more of propylene oxide, epichlorohydrin or pinene.

[0046] In some embodiments of the present invention, the molar ratio of the amino acid or its derivative to triphosgene is 1:0.1-5; it can also be 1:0.1-1, 1:1-2, 1:2-3, 1:3-4 or 1:4-5.

[0047] In some embodiments of the present invention, the molar ratio of the amino acid or its derivative to the acid-binding agent is 1:5-30; it can also be 1:5-10, 1:10-20, or 1:10-30.

[0048] In some embodiments of the present invention, the ratio of the amino acid or its derivative to the first solvent is 1g:10~50mL; it can also be 1g:10~20mL, 1g:20~30mL, 1g:30~40mL, 1g:40~50mL.

[0049] In some embodiments of the present invention, the temperature of the ring-closure reaction is room temperature; preferably 10-35°C; and can also be 10-15°C, 15-25°C, or 25-35°C.

[0050] In some embodiments of the present invention, the ring-closure reaction time is 1-3 hours.

[0051] In some embodiments of the present invention, the purification comprises extraction, washing, drying, rotary evaporation, and recrystallization.

[0052] In some embodiments of the present invention, the extractant used in the extraction includes ethyl acetate, dichloromethane, toluene, diethyl ether, n-hexane, etc.

[0053] In some embodiments of the present invention, the washing comprises washing with at least one of saturated saline solution, saturated sodium bicarbonate solution, saturated sodium carbonate solution, saturated ammonium chloride solution, saturated sodium sulfate solution, and saturated magnesium sulfate solution.

[0054] In some embodiments of the present invention, the drying comprises drying with anhydrous sodium sulfate, anhydrous calcium chloride, or anhydrous magnesium sulfate.

[0055] In some embodiments of the present invention, the recrystallization comprises using n-hexane, tetrahydrofuran, ethyl acetate, or petroleum ether for recrystallization.

[0056] In some embodiments of the present invention, the second organic solvent is one or more of toluene, benzene, tetrahydrofuran, dimethylformamide, dichloromethane, and ethyl acetate.

[0057] In some embodiments of the present invention, the second organic solvent is preferably dimethylformamide or dichloromethane.

[0058] In some embodiments of the present invention, the initiator includes an amine compound; preferably a mono-, di-, tri-, or poly-primary amine with a C1-C10 linear, branched, or cyclic structure.

[0059] In some embodiments of the present invention, the monoprimary amine includes one or more of n-hexylamine, benzylamine, n-propylamine, n-butylamine, and tert-butylamine.

[0060] In some embodiments of the present invention, the primary diamine includes one or more of p-phenylenediamine, 1,6-hexanediamine, 1,3-propylenediamine, 1,12-dodecanediamine, triethylenetetramine, and diethylenetriamine.

[0061] In some embodiments of the present invention, the catalyst includes an acid and / or a base; the acid includes a Bronsted acid or a Lewis acid, such as acetic acid, propionic acid, valeric acid, butyric acid, benzoic acid, methanesulfonic acid, triethylaluminum, diphenylzinc, pentafluorophenylboron; the base includes 1,5,7-triazidobicyclo (4.4.0) dec-5-ene, 7-methyl-1,5,7-triazabicyclo [4.4.0] dec-5-ene, 1,4-diazabicyclo [2.2.2] octane, 4-dimethylaminopyridine, 1,8-diazabicyclo [5.4.0] undec-7-ene, spartine, tetramethylguanidine, triethylamine, N,N-diisopropylethylamine, tetramethylguanidine, potassium alkoxide or sodium alkoxide. Further preferably, the catalyst in the present application is a mixture of an acid and a base. The acid-base mixture has a synergistic catalytic effect. In some cases, when only acid is used, the ring-opening polymerization reaction will lead to side reactions and an increase in by-products; in some cases, when only base is used, the molecular weight of the final product is still uncontrollable.

[0062] In some embodiments of the present invention, the molar ratio of acid to base in the catalyst is 0.01-1, and may also be 0.01-0.1, 0.1-0.5, or 0.5-1.

[0063] In some embodiments of the present invention, the molar ratio of the catalyst to the N-carboxyl cyclic anhydride of the amino acid or its derivative is 1:10-1:1000; it can also be 1:10-50, 1:50-100, 1:100-300, 1:300-500, 1:500-700, 1:700-1000.

[0064] In some embodiments of the present invention, the molar ratio of the catalyst to the initiator is 1:0.1-50; it can also be 1:0.1-1, 1:1-5, 1:5-10, 1:10-20, 1:20-30, 1:30-40, 1:40-50.

[0065] In some embodiments of the present invention, the molar ratio of the initiator to the N-carboxyl cyclic anhydride of the amino acid or its derivative is 1:20-500; it can also be 1:20-50, 1:50-100, 1:1-0-200, 1:200-300, 1:300-400, 400-500.

[0066] In some embodiments of the present invention, in a mixed system for ring-opening polymerization, the volume proportion of the second solvent in the mixture is 10%-90%; it can also be 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%.

[0067] In some embodiments of the present invention, the temperature of the ring-opening polymerization is 0-100°C; it can also be 0-10°C, 10-20°C, 20-30°C, 30-40°C, 40-50°C, 50-60°C, 60-80°C, or 80-100°C.

[0068] In some embodiments of the present invention, the time of the ring-opening polymerization is 5 min-10 h; it can also be 5 min-1 h, 1 h-2 h, 2 h-4 h, 4 h-6 h, 6 h-8 h, or 8 h-10 h.

[0069] In some embodiments of the invention, if the N-carboxyl ring anhydride of an amino acid or its derivative includes a protecting group, the method for preparing the polyamino acid further includes a deprotection step after the polymerization reaction; the deprotection step effectively removes the protecting group and restores the functional groups on the amino acid side chain, enabling it to participate in further chemical reactions or exhibit the desired biological activity.

[0070] In some embodiments of the present invention, the deprotection method preferably uses a TFA / HBr acetic acid solution or Pd / C plus NaBH4, and the amount of the reagent added is 1 to 3 times the number of protecting groups (such as benzyloxycarbonyl and benzyl ester groups) in the monomer; however, it is not limited to this, and conventional deprotection methods in the art can achieve similar effects.

[0071] The third aspect of the present invention provides the use of the polyamino acid in any of the following:

[0072] (I) preparing a drug carrier;

[0073] (II) preparing brain therapeutic drugs;

[0074] (III) Preparation of brain targeting agents.

[0075] In some embodiments of the present invention, the drug comprises a nucleic acid, a peptide, a protein or a small molecule drug.

[0076] In some embodiments of the invention, the nucleic acid is selected from antisense oligonucleotides, siRNA, miRNA, mRNA, and gRNA.

[0077] In some embodiments of the present invention, the polypeptides or proteins include antibodies, complements, enzymes, transport proteins, cytokines, polypeptide hormones, and the like.

[0078] In a fourth aspect, the present invention provides a targeting agent, wherein the targeting agent comprises the polyamino acid polypeptide material as described above.

[0079] In some embodiments of the present invention, the targeting agent further comprises an imaging agent and / or a therapeutic agent.

[0080] In some embodiments of the present invention, the therapeutic agent includes but is not limited to a nucleic acid, a peptide, a protein or a small molecule drug.

[0081] In some embodiments of the present invention, the nucleic acid is selected from antisense oligonucleotides, siRNA, miRNA, mRNA, and gRNA.

[0082] In some embodiments of the present invention, the polypeptide or protein includes antibodies, complement, enzymes, transport proteins, cytokines, polypeptide hormones, etc.

[0083] In some embodiments of the present invention, the pharmaceutically active ingredient is a pharmaceutically active ingredient that acts on the brain.

[0084] In some embodiments of the present invention, the imaging agent comprises at least one of a radionuclide, biotin, a fluorescent protein, an antibody, horseradish peroxidase, and alkaline phosphatase.

[0085] In some embodiments of the present invention, the drug further comprises excipients, including but not limited to physiologically, medically, pharmaceutically, and nutritionally acceptable excipients.

[0086] The "auxiliary materials" used herein are intended to include, but are not limited to, carriers, excipients, diluents, adjuvants, colorants, fragrances, preservatives, etc. that are considered to be used by those skilled in the art when formulating the compounds of the present invention to prepare pharmaceutical compositions.

[0087] In some embodiments of the present invention, the drug includes an excipient for improving the water solubility or hydrophilicity of the drug.

[0088] In some embodiments of the present invention, the dosage form of the drug includes but is not limited to at least one of a solution, powder, tablet, granule, capsule, pill, drop, emulsion, injection, ointment, spray, gel, aerosol, suspension, transdermal patch, suppository or implant.

[0089] In some embodiments of the present invention, the administration route of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, oral administration, sublingual administration, nasal administration, aerosol administration or transdermal administration.

[0090] Beneficial effects of the technical solution of this application:

[0091] This invention discloses a method for synthesizing polypeptide materials containing polytryptophan or its derivatives with controllable molecular weight and narrow molecular weight distribution. Specifically, the invention introduces an acid and / or base as a catalyst to catalyze the ring-opening polymerization of amino acid anhydrides and their derivatives in the presence of a primary amine initiator, yielding polytryptophan with varying number-average molecular weights. Furthermore, the polymers exhibit adjustable structure, controllable molecular weight, and narrow molecular weight distribution. The number-average molecular weight of the polytryptophan is 1,000–200,000 Da, with a molecular weight distribution (PDI) < 1.3.

[0092] The present invention provides a method for synthesizing polypeptide materials derived from polytryptophan or its derivatives with high efficiency, low dosage, simple synthesis, relatively mild reaction conditions, and no metal residue in the product. Application of the method for ring-opening polymerization offers advantages such as simplicity, low cost, high reaction rate, controllable process, and narrow product molecular weight distribution. Polymerization can be performed under a variety of solvent conditions.

[0093] The present invention also provides the application of the above-mentioned polytryptophan polypeptide material in the field of gene delivery. The present invention further found that compared with other amino acid polymers, polymers based on tryptophan cyclic anhydride have brain targeting, while polyamino acids prepared from other amino acid cyclic anhydrides do not have this property. The polymer polypeptide material constructed based on tryptophan cyclic anhydride can not only be used as a carrier to efficiently deliver nucleic acid drugs, but can also be used as a brain targeting agent to deliver drugs to the brain through the blood-brain barrier, which is beneficial for the treatment of brain diseases and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 1H NMR spectrum of L-tryptophan N-carboxyl ring anhydride and infrared spectrum of polytryptophan.

[0095] Figure 2 This is the MALDI-TOF image of polytryptophan in Example 7.

[0096] Figure 3 (a) is the circular dichroism spectra of L-type and D-type Trp-NCA, Figure 3 Middle (b) shows the circular dichroism spectra of L-type and D-type polytryptophan.

[0097] Figure 4 (a) is the GPC graph of polytryptophan initiated by different monomer and initiator molar ratios, and (b) is the relationship between the number average molecular weight (Mn), theoretical molecular weight (Mn,th.) and polydispersity index (PDI) obtained by GPC analysis and the molar ratio of monomer and initiator.

[0098] Figure 5This is the GPC chart of multi-block poly Cbz-L-lysine-b-polytryptophan.

[0099] Figure 6 This is a diagram of the cell experiment effect.

[0100] Figure 7 In vivo imaging of mice loaded with siRNA (time: 2h).

[0101] Figure 8 This is the distribution diagram of the brain of mice dissected 24 hours after tail vein injection of KW-type materials and materials loaded with siRNA.

[0102] Figure 9 for Figure 8 The Average Radiant Efficiency statistical chart in the imaging results.

[0103] Figure 10 Random copolymer polypeptide K 80 W 40 Materials Immunofluorescence staining of brain sections. DETAILED DESCRIPTION

[0104] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0105] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the specific embodiments described below. It should also be understood that the terminology used in the examples is intended to describe specific embodiments and is not intended to limit the scope of the present invention. The experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0106] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0107] Example 1

[0108] To a 50 mL thick-walled pressure bottle, add 1.0 g (4.9 mmol, 1.0 eq) of L-tryptophan and THF (15 mL). Propylene oxide (3.4 mL, 49 mmol, 10.0 eq) and triphosgene (749 mg, 2.5 mmol, 0.5 eq) were added sequentially under magnetic stirring. The vessel was sealed and stirred at 25°C for 1.5 h, then placed in an ice bath at 4°C. For safety reasons, excess triphosgene was quenched by adding 15 mL of 4°C cold water. After stirring for 1-3 minutes, the mixture was extracted twice with ethyl acetate (EA, 20 mL). The organic phases were combined, washed with saturated brine at room temperature, and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation under vacuum at 35°C, the crude product was recrystallized from tetrahydrofuran / n-hexane (1:5 v / v) to obtain single crystals of L-tryptophan-N-carboxylic acid cyclic anhydride (Trp-NCA) (1.0 g, yield 89%). The H NMR spectrum of Trp-NCA was shown in FIG. Figure 1 NCA was stored in a glove box filled with argon at -15°C.

[0109] Example 2

[0110] To a 50 mL thick-walled pressure bottle, 1.0 g (4.9 mmol, 1.0 eq) of D-tryptophan and 15 mL of THF were added. Propylene oxide (3.4 mL, 49 mmol, 10.0 eq) and triphosgene (749 mg, 2.5 mmol, 0.5 eq) were then added sequentially under magnetic stirring. The vessel was sealed and stirred at 25°C for 1.5 h before being placed in an ice bath at 4°C. For safety reasons, excess triphosgene was quenched by adding 15 mL of 4°C cold water. After stirring for 1-3 minutes, the mixture was extracted twice with ethyl acetate (EA, 20 mL) at room temperature. The combined organic phases were washed with saturated brine and dried over anhydrous Na₂SO₄. The solvent was removed by rotary evaporation in vacuo at 35°C. The crude product was recrystallized from tetrahydrofuran / n-hexane (1:5 v / v) to afford single crystals of D-tryptophan-N-carboxylic acid anhydride (0.99 g, 88% yield). NCA was stored in a glove box filled with argon at -15°C.

[0111] Example 3

[0112] To a 50 mL thick-walled pressure flask, add 1.0 g (4.5 mmol, 1.0 eq) of L-5-hydroxytryptophan and THF (15 mL). Under magnetic stirring, add 3.55 mL (45 mmol, 10.0 eq) of epichlorohydrin and 835 mg (22.5 mmol, 0.5 eq) of triphosgene in that order. The vessel is sealed and stirred at 25°C for 1.5 h, then placed in an ice bath at 4°C. For safety reasons, excess triphosgene is quenched by adding 15 mL of 4°C cold water. After stirring for 1-3 minutes, the mixture is extracted twice with ethyl acetate (EA, 20 mL) at room temperature. The combined organic phases are washed with saturated brine and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation in vacuo at 35°C, the crude product was recrystallized from tetrahydrofuran / n-hexane (1:5 v / v) to obtain single crystals of L-5-hydroxy-tryptophan-N-carboxylic acid anhydride (1.0 g, 91% yield). NCA was stored in a -15°C refrigerator in an argon-filled glove box.

[0113] Example 4

[0114] To a 50 mL thick-walled pressure flask, add 1.0 g (4.5 mmol, 1.0 eq) of DL-5-hydroxy-tryptophan and THF (15 mL). Under magnetic stirring, add 3.55 mL (45 mmol, 10.0 eq) of epichlorohydrin and 835 mg (22.5 mmol, 0.5 eq) of triphosgene in that order. The vessel is sealed and stirred at 25°C for 1.5 h, then placed in an ice bath at 4°C. For safety reasons, excess triphosgene is quenched by adding 15 mL of 4°C cold water. After stirring for 1-3 minutes, the mixture is extracted twice with ethyl acetate (EA, 20 mL) at room temperature. The combined organic phases are washed with saturated brine and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation in vacuo at 35°C, the crude product was recrystallized from tetrahydrofuran / n-hexane (1:5 v / v) to obtain single crystals of DL-5-hydroxy-tryptophan-N-carboxylic acid anhydride (0.85 g, 77% yield). NCA was stored in a -15°C refrigerator in an argon-filled glove box.

[0115] Example 5

[0116] To a 50 mL thick-walled pressure bottle, 0.5 g (0.23 mmol, 1.0 eq) of L-methyltryptophan and 15 mL of THF were added. Propylene oxide (1.6 mL, 23 mmol, 10.0 eq) and triphosgene (340 mg, 23 mmol, 0.5 eq) were then added sequentially under magnetic stirring. The vessel was sealed and stirred at 25°C for 1.5 h before being placed in an ice bath at 4°C. The solvent was removed by rotary evaporation in vacuo at 35°C. The crude product was washed with diethyl ether / n-hexane (1:1 v / v) and recrystallized from tetrahydrofuran / n-hexane (1:5 v / v) to afford a single crystal of L-methyltryptophan-N-carboxylic acid anhydride (0.85 g, 77% yield). NCA was stored in an argon-filled glove box at -15°C.

[0117] The polymerization reaction in the technical solution is further described using specific examples, which are as follows.

[0118] Example 6

[0119] In a 4 mL vial, 8.69 μL of catalyst DMAP (0.1 M, 12.2 mg / mL, solvent 50% DMF (50% DMF contains DMF and DCM, the volume ratio of the two is 1:1) and 11.48 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF) were added, followed by 153.7 μL of mixed solvent DMF / DCM (1:1, v / v). After shaking, 43.5 μL of monomer L-tryptophan N-carboxyl ring anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF) was added. The reaction system after mixing had [M]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0=50 / 1 / 1, where M is the monomer tryptophan N-carboxyl ring anhydride and I is the initiator n-hexylamine. The reaction was controlled at 25°C for 4 h, then precipitated twice with cold anhydrous ether, and vacuum dried to constant weight to obtain the polymer.

[0120] Where Conv. = 99%, M n,GPC = 20.8 kg / mol, PDI = 1.18.

[0121] The conversion rate was calculated by NMR. The peak at 10.8 ppm is characteristic of the indole ring NH in the monomer, and the peak at 10.4 ppm is characteristic of the indole ring NH in the polymer. The conversion rate was calculated using the TMS internal standard method.

[0122] Example 7

[0123] In a 4 mL vial, 8.69 μL of catalyst DMAP (0.1 M, 12.2 mg / mL 50% DMF), 11.48 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 2.49 μL of acetic acid were added, followed by 151.2 μL of a mixed solvent of DMF / DCM (1:1, v / v). After shaking, 43.5 μL of monomer L-tryptophan N-carboxyl ring anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF) ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 50 / 1 / 1) was added. The reaction was controlled at 25°C for 3 h, and then the mixture was precipitated twice with cold anhydrous ether and dried under vacuum to constant weight to obtain polymer W. 50 ; Conv. = 99%, M n,GPC = 10.5 kg / mol, PDI = 1.21. The mass spectrometry results are detailed in Figure 2 ,Depend on Figure 2 The table shows that the structure and molecular weight of the final product are consistent with the theory.

[0124] The polymerization process of D-type Trp-NCA is similar. The raw material used is changed from L-type tryptophan N-carboxyl ring anhydride to D-type. The polymerization feed of methyl tryptophan can also refer to this embodiment. The detection process is to take a 1mm cuvette, add 400 ul of 0.1mg / ml tetrahydrofuran solution of polytryptophan, monitor the CD signal at 210 nm-260 nm, and convert the unit to molar value. The mdeg value measured in the experiment is multiplied by 1000, divided by the optical diameter (mm), and then divided by the concentration (mM). The value obtained is the molar ellipticity [θ] (unit: deg.cm 2 .dmol -1 ).

[0125] The circular dichroism (CD) spectrum results are detailed in Figure 3 CD results showed that the L-form had a distinct negative peak at 218 nm and an upward Trp-Trp stacking peak at 229 nm, indicating an α-helical secondary structure, while the D-form exhibited the opposite peak shape. The negative peak of methyltryptophan shifted toward 220 nm, indicating a more stable α-helical structure.

[0126] Example 8

[0127] In a 4 mL vial, 5.79 μL of catalyst DMAP (0.1 M, 12.2 mg / mL 50% DMF), 7.66 μL of n-hexylamine (7.6 mg / 1 ml 50% DMF), and 2.49 μL of acetic acid were added, followed by 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v). After shaking, 43.5 μL of monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF) ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 75 / 1 / 1) was added. The reaction was controlled at 25°C for 3 h, and then the mixture was precipitated twice with cold anhydrous ether and dried under vacuum to constant weight to obtain the polymer; Conv. = 99%, M n,GPC = 15.5 kg / mol, PDI = 1.21.

[0128] Example 9

[0129] In a 4 mL vial, 4.34 μL of catalyst DMAP (0.1 M, 12.2 mg / mL 50% DMF), 5.74 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 2.49 μL of acetic acid were added, followed by 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v). After shaking, 43.5 μL of monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF) ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 100 / 1 / 1) was added. The reaction was controlled at 25°C for 4 h, and then precipitated twice with cold anhydrous ether. The polymer was dried under vacuum to constant weight to obtain a polymer with a Conv. = 99%, M n,GPC = 19.8 kg / mol, PDI= 1.03.

[0130] Example 10

[0131] In a 4 mL vial, 2.90 μL of catalyst DMAP (0.1 M, 12.2 mg / 1 mL 50% DMF), 3.82 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 2.49 μL of acetic acid were added, followed by 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v). After shaking, 43.5 μL of monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF) ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 150 / 1 / 1) was added. The reaction was controlled at 25°C for 5 h, and then the mixture was precipitated twice with cold anhydrous ether and dried under vacuum to constant weight to obtain a polymer with Conv. = 99%, M n,GPC = 28.6 kg / mol, PDI = 1.02.

[0132] Example 11

[0133] In a 4 mL vial, 2.17 μL of catalyst DMAP (0.1 M, 12.2 mg / 1 mL 50% DMF), 2.87 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 2.49 μL of acetic acid were added, followed by 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v). After shaking, 43.5 μL of monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF) ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 200 / 1 / 1) was added. The reaction was controlled at 25°C for 7 h, and then precipitated twice with cold anhydrous ether. The polymer was dried under vacuum to constant weight to obtain a polymer with a Conv. = 99%, M n,GPC = 32.2 kg / mol, PDI = 1.03; its infrared characterization results are detailed in Figure 1 .

[0134] In this invention, GPC characterization was performed by gel permeation chromatography (GPC) to characterize the molecular weight of the polymers using a 5 mg / mL solution (0.1 M LiBr in DMF). The molecular weight of the polymers was determined based on the dn / dc values of each polymer sample using an internal calibration system using ASTRA software (version 8.12, Wyatt Technology, Santa Barbara, CA, USA). For polytryptophan, the dn / dc value was 0.1250.

[0135] In addition, the GPC diagrams of the polytryptophan prepared in Examples 7-10 and 11 are shown in Figure 4 It can be seen that the molecular weight of polytryptophan prepared by different monomer and initiator molar ratios is controllable and has a narrow distribution, and the degree of polymerization is consistent with the theoretical value.

[0136] Example 12

[0137] In a 4 mL vial, 2.17 μL of catalyst DMAP (0.1 M, 12.2 mg / 1 mL 20% DMF), 4.35 μL of PEG2k-NH2 (100 mg / 1 mL 50% DMF), and 4.98 μL of acetic acid were added, followed by 151.2 μL of a mixed solvent DMF / DCM (2:8, v / v). After shaking, 43.5 μL of monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF) ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 200 / 1 / 1) was added. The reaction was controlled at 25°C for 7 h, and then precipitated twice with cold anhydrous ether. The polymer was dried under vacuum to constant weight to obtain the polymer, where Conv. = 99%, M n,GPC = 35.2 kg / mol, PDI = 1.05.

[0138] Example 13

[0139] In a 4 mL vial, 34.8 μL of catalyst DMAP (0.1 M, 12.2 mg / mL 50% DMF), 45.9 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 9.96 μL of acetic acid were added, followed by 348 μL of a mixed solvent DMF / DCM (1:1, v / v). After shaking, 173.9 μL of monomer N6-benzyloxycarbonyl-L-lysine cyclic anhydride (ZLL-NCA, 1 M, 306.3 mg / 1 mL 50% DMF) was added ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 50 / 1 / 1). The reaction was controlled at 25°C for 1 h, and infrared spectroscopy was used to monitor the reaction at 1850 cm -1 and 1790 cm -1 The disappearance of the peak proves that the monomer is complete, and then it is precipitated twice with cold anhydrous ether and vacuum dried to constant weight to obtain polymer K. 50 , where Conv. = 99%, M n,GPC = 12.1 kg / mol, PDI = 1.16, PZLL 50 See the GPC diagram for Figure 5 .

[0140] Example 14

[0141] In a 4 mL vial, 34.8 μL of catalyst DMAP (0.1 M, 12.2 mg / 1 mL 50% DMF), 45.9 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 9.96 μL of acetic acid were added. 348 μL of the mixed solvent DMF / DCM (1:1, v / v) was then added. After shaking, 173.9 μL of the pre-mixed monomer N6-benzyloxycarbonyl-L-lysine cyclic anhydride (1 M) and 173.9 μL of the monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL) were added. The temperature was controlled at 25°C for 4 h. The reaction was monitored by infrared spectroscopy at 1850 cm -1 and 1790 cm -1 The disappearance of the peak proves that the monomer is complete. Then, it is precipitated twice with cold anhydrous ether and vacuum dried to constant weight to obtain the random polymer PZLL. 50 -ran-PLW 50 , where Conv. = 99%, M n,GPC =23.0 kg / mol, PDI = 1.05.

[0142] In the present invention, PZLL represents poly N6-benzyloxycarbonyl-L-lysine, PLW represents poly L-tryptophan N-carboxyl ring anhydride, and ran represents random copolymer. 20 -ran-PLW 10 , PZLL 15 -ran-PLW 15 , PZLL 30 -ran-PLW 30 , PZLL 80 -ran-PLW 40 The same method as this case can be used to synthesize all the materials. By changing the monomer feed ratio, the material can be obtained within 8 hours.

[0143] Example 15

[0144] In a 4 mL vial, 34.8 μL of catalyst DMAP (0.1 M, 12.2 mg / 1 mL 50% DMF), 45.9 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 9.96 μL of acetic acid were added. Then, 348 μL of the mixed solvent DMF / DCM (1:1, v / v) was added. After shaking, 278 μL of the pre-mixed monomer N6-benzyloxycarbonyl-L-lysine cyclic anhydride (1 M) and 139 μL of the monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL) were added. The temperature was controlled at 25°C for 4 h. The reaction was monitored by infrared spectroscopy at 1850 cm -1 and 1790 cm -1 The disappearance of the peak proves that the monomer is complete. Then, it is precipitated twice with cold anhydrous ether and vacuum dried to constant weight to obtain the random polymer PZLL. 80 -ran-PLW 40 , where Conv. = 99%, M n,GPC = 25.0kg / mol, PDI = 1.05.

[0145] Example 16

[0146] Synthesis of block copolymer polypeptides In a 4 mL vial, 34.8 μL of catalyst DMAP (0.1 M, 12.2 mg / 1 mL 50% DMF), 45.9 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 9.96 μL of acetic acid were added, followed by 348 μL of a mixed solvent DMF / DCM (1:1, v / v). After shaking, 173.9 μL of monomer N6-benzyloxycarbonyl-L-lysine cyclic anhydride (ZLL-NCA, 1 M, 306.3 mg / 1 mL 50% DMF) was added ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 50 / 1 / 1) and the temperature was controlled at 25°C for 1 h. The disappearance of the 1850 and 1790 peaks by infrared monitoring indicated that the monomer was complete, and then 173.9 μL of the second monomer was added. The reaction of L-tryptophan N-carboxycyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL) was carried out at 25°C for 4 h. The IR spectrum at 1850 cm -1 and 1790 cm -1 The disappearance of the peak proves that the monomer is complete. Then, it is precipitated twice with cold anhydrous ether and vacuum dried to constant weight to obtain the polymer PZLL. 50 -b-PLW 50 , where Conv. = 99%, M n,GPC= 23.8 kg / mol, PDI = 1.05, PZLL 50 -b-PLW 50 The GPC diagram is shown in Figure 5 .

[0147] PZLL stands for poly N6-benzyloxycarbonyl-L-lysine, PLW stands for poly L-tryptophan N-carboxyl ring anhydride, b stands for block, and 50 stands for the degree of polymerization of each segment. 20 -b-PLW 10 , PZLL 15 -b-PLW 15 , PZLL 30 -b-PLW 30 , PZLL 80 -b-PLW 40 The same method as this case can be used to synthesize all the materials. By changing the monomer feed ratio, the material can be obtained within 8 hours.

[0148] Example 17 Synthesis of block copolymer polypeptides

[0149] In a 4 mL vial, 34.8 μL of catalyst DMAP (0.1 M, 12.2 mg / 1 mL 50% DMF), 45.9 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), and 9.96 μL of acetic acid were added. Then, 348 μL of a mixed solvent of DMF / DCM (1:1, v / v) was added. After shaking, 173.9 μL of monomer N6-benzyloxycarbonyl-L-lysine cyclic anhydride (ZLL-NCA, 1 M, 306.3 mg / 1 mL 50% DMF) was added ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 50 / 1 / 1). The reaction was controlled at 25°C for 1 h. The reaction was monitored by infrared spectroscopy at 1850 cm -1 and 1790 cm -1 The disappearance of the peak indicated that the monomer was completely formed. Then, 173.9 μL of the second monomer, L-tryptophan N-carboxyl ring anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF), was added. The temperature was controlled at 25°C for 4 h. The reaction was monitored by infrared spectroscopy at 1850 cm -1 and 1790 cm -1The disappearance of the peak indicated that the monomer was completely formed. Then, 173.9 μL of the third monomer, N6-benzyloxycarbonyl-L-lysine cyclic anhydride (ZLL-NCA, 1 M, 306.3 mg / 1 mL) ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 50 / 1 / 1) was added. The temperature was controlled at 25°C for 1 h, and the mixture was precipitated twice with cold anhydrous ether and dried under vacuum to constant weight to obtain block copolypeptide K. 50 -bW 50 -bK 50 , where Conv. = 99%, M n,GPC = 33.0 kg / mol, PDI = 1.05, PZLL 50 -b-PLW 50 -b-PZLL 50 The GPC diagram is shown in Figure 5 .

[0150] Example 18 Polypeptide Deprotection Step

[0151] 3.0 g of the copolypeptide was dissolved in 18.0 mL of trifluoroacetic acid, and 9.0 mL of a mixed solution of hydrogen bromide and acetic acid was slowly added. 9 mL of diethyl phosphite was added to protect the tryptophan side indole. The reaction was stirred at 30°C for 2 h, and then precipitated in excess ether, washed, filtered, and vacuum-dried. The resulting solid was dissolved in a small amount of water, placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in distilled water for purification, and freeze-dried to obtain a white solid (1.5 g, yield: 50%).

[0152] The deprotected material is defined as K-ran-W or KbW, where K represents polylysine, W represents polytryptophan, ran represents the product of random copolymerization, and b represents the product of block copolymerization.

[0153] The cytotoxicity experiments in this application are as follows:

[0154] The CCK-8 method was used to detect the cytotoxicity of different preparations. The brief steps are as follows: 293T-luc cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated overnight to allow the cells to adhere. Subsequently, blank preparations and siLUC vector preparations with different N / P ratios (10, 15, 20, 25, 30) were added and cultured for 48 hours. Then, CCK-8 reagent was added to each well and incubated for another hour. Finally, the absorbance value was measured at a wavelength of 450 nm using an enzyme reader. The results of the cytotoxicity experiment are shown in Figure 2. Figure 6As shown, the results show that pure poly-lysine is highly toxic, while it is almost non-toxic after forming random and block copolymers with poly-tryptophan, indicating that the material is safe and reliable. The transfection experiment in this application is as follows: 5000 293T-luc cells were seeded in a 96-well plate and cultured for 48 hours. The original culture medium was discarded and replaced with fresh culture medium containing 10% fetal bovine serum (FBS), which contained 200 ngLUC siRNA and complexes formed with different preparations at N / P ratios (10, 15, 20, 25, 30). Continue to culture until the detection time point, and use a luciferase detection kit to measure the fluorescence intensity using a microplate reader. The Lipofectamine 2000 transfection group was used as a positive control. The data are expressed as mean ± standard deviation (n=3). The transfection experiment results are shown in Figure 6 As shown, the results show that the transfection effect of KW series materials after loading siRNA is comparable to that of Lipofectamine 2000, indicating its effectiveness as a carrier.

[0155] Example 19 Polypeptide fluorescent labeling (K 50 -W 50 -Cy5)

[0156] Dissolve 9 μL of 1 mg / mL Cy5-NHS ester in 100 μL of DMSO and add it to the solution containing peptide K. 50 -ran-W 50 A 10 mg peptide (Cy5-NHS ester) was added to 400 μL of DMSO solution at a 1:50 ratio. 0.5 μL of triethylamine (10 μL / 1 mL) was then added to the mixture. The reaction mixture was stirred overnight at room temperature in the dark. Water was then added and the solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed against distilled water and freeze-dried to obtain a blue solid (7.1 mg, yield: 71%).

[0157] The same method can be used to label other peptides with Cy5, by adding corresponding proportions of Cy5-NHS.

[0158] Example 20 Preparation of a gene delivery system (N / P = 10)

[0159] (1) Preparation of polylysine-polytryptophan (K 80 -ran-W 40 , KW) in a nuclease-free DEPC aqueous solution (1 mg / mL).

[0160] (2) Take 6.14 μL of the above solution, add 587 μL of nuclease-free water and 7 μL of Cy5-siRNA (0.2 μg / μL), mix well, mix for 30 seconds, mechanically pipette and mix 10 times, and incubate at 37°C for 15 minutes. Mix well, and the mass ratio of the polynucleotide to the target gene in the mixture is 6.14 μg:1.4 μg. (siRNA sequence S: GCGACGACUGCCUAAGAUdTdT, AS: AUCUUAGGCAGAUCGUCGCdTdT)

[0161] Example 21 Preparation of a Gene Delivery System (N / P = 15)

[0162] (1) Preparation of polylysine-polytryptophan (K 80 -ran-W 40 , KW) in a nuclease-free DEPC aqueous solution (1 mg / mL).

[0163] (2) Take 9.21 μL of the above solution, add 584 μL of nuclease-free water and 7 μL of Cy5-siRNA (0.2 μg / μL), mix well, mix for 30 seconds, and mechanically pipette and mix 10 times at 25°C; then incubate at 37°C for 15 minutes; mix well, and the mass ratio of the polypeptide to the target gene in the mixture is 9.21 μg:1.4 μg. (siRNA sequence S: GCGACGACUGCCUAAGAUdTdT, AS: AUCUUAGGCAGAUCGUCGCdTdT)

[0164] Example 22 Preparation of a Drug Delivery System (N / P = 20)

[0165] (1) Preparation of polylysine-polytryptophan (K 80 -ran-W 40 , KW) in a nuclease-free DEPC aqueous solution (1 mg / mL).

[0166] (2) Take 12.3 μL of the above solution, add 581 μL of nuclease-free water and 7 μL of Cy5-siRNA (0.2 μg / μL), mix well, mix for 30 seconds, mechanically pipette and mix 10 times, and incubate at 37°C for 15 minutes. Mix well, and the mass ratio of the polynucleotide to the target gene in the mixture is 12.3 μg:1.4 μg. (siRNA sequence S: GCGACGACUGCCUAAGAUdTdT, AS: AUCUUAGGCAGAUCGUCGCdTdT)

[0167] The complexation method of block copolypeptides and genes is similar, and the amount of siRNA added is adjusted based on the actual polymerization degree and N / P ratio.

[0168] Example 23 In vivo imaging of the brain-targeted delivery platform KW and KW@siRNA

[0169] The prepared labeled Cy5 random copolymer K 20 W 10 , K 15 W 15 , K 40 W 20 , K 30 W 30 , K 80 W 40 Random copolymer K containing siRNA and Cy5 80 W 40 (N / P = 20), Cy5, and Angiopep-2 (positive control) were injected into C57BL / 6 mice via tail vein injection (125 μL, 1 mg / ml). In vivo imaging of the small animals was performed along a time gradient to test their in vivo targeting ability. Twenty-four hours later, the mice were anesthetized, the thorax was opened along the manubrium, the pericardium was cut, and the heart was exposed. The left ventricle was opened at an angle of 30-45° to the apex. A perfusion needle was inserted through the left ventricle into the aorta, the needle was fixed, and the right atrial appendage was opened. The blood was then rapidly flushed with normal saline (20-40 ml for mice) until the outflowing fluid was pale and essentially clear (rapid whitening of the liver, eyes, and paws is an effective visual indicator of blood removal). The mice experienced twitching of their limbs and tail, and perfusion was completed when the liver turned white and the internal organs bulged. The brain and internal organs were then removed, dissected, and imaged. The results showed that the brain-targeted delivery platform obtained in this example can rapidly and accurately target the brain tissue surrounding the mouse.

[0170] Figure 7-Figure 9 The results showed that there was obvious Cy5 fluorescence in the mouse brain, which was significantly stronger than that of the Cy5 group and the positive control Angiopep-2 commercial brain-targeting peptide group; this shows that the brain-targeted delivery platform KW series polypeptides obtained in this example can quickly and accurately target the surrounding brain tissue in mice. Moreover, the degree of brain enrichment showed a positive correlation with the increase of polymerization degree and tryptophan content, which indicates the necessity of long-chain polytryptophan with a high tryptophan ratio. Figure 9 It can also be seen that the enrichment degree of brain material is positively correlated with the tryptophan content, and the higher the degree of polymerization, the better.

[0171] Example 24

[0172] Based on Example 23, the distribution of the brain-targeted delivery platform KW in brain tissue was further studied. The frozen section and immunofluorescence staining steps were as follows:

[0173] (1) Fixation of frozen sections: Bake the frozen sections in a 37°C oven for 10-20 minutes to dry them. Place them in fixative for 30 minutes and then wash them three times in PBS (pH 7.4) on a decolorizing shaker for 5 minutes each time.

[0174] (2) Antigen retrieval: During the retrieval process, prevent excessive evaporation of the buffer and do not allow the slides to dry out. After retrieval, allow the slides to cool naturally. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each.

[0175] (3) Circle serum blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue, add BSA (if the primary antibody is from goat, use 10% donkey serum to block, if the primary antibody is from other sources, use 3% BSA to block), and block for 30 minutes.

[0176] (4) Add primary antibody: Add the prepared primary antibody dropwise and incubate the slices flat in a humidified chamber at 4°C overnight.

[0177] (5) Adding secondary antibody: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 minutes each time. Add the corresponding secondary antibody and incubate at room temperature in the dark for 50 minutes.

[0178] (6) DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 minutes each time. Add DAPI staining solution and incubate at room temperature for 10 minutes in the dark.

[0179] (7) Quenching tissue autofluorescence: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 minutes each time. Add autofluorescence quencher B solution for 5 minutes and rinse with running water for 10 minutes.

[0180] (8) Sealing: Seal the slides with anti-fluorescence quenching sealing medium.

[0181] (9) Image acquisition: DAPI excitation wavelength 330-380 nm, emission wavelength 420 nm; 488 excitation wavelength 465-495 nm, emission wavelength 515-555 nm; CY5 excitation wavelength 608-648 nm, emission wavelength 672-712 nm.

[0182] Figure 10 The results showed that random copolymerization of polypeptide K 80 W 40 It is distributed in the cerebral cortex, hippocampus, prefrontal lobe, cerebellum and other brain regions, with the majority being in the cerebellum.

[0183] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be encompassed by the present invention.

Claims

1. A method for preparing an amino acid polymer with controllable molecular weight, characterized in that: The N-carboxyl anhydride of one or more amino acids or their derivatives is subjected to ring-opening polymerization to form an amino acid polymer; the ring-opening polymerization adopts a primary amine initiator and an acid and / or base catalyst.

2. The preparation method according to claim 1, characterized in that The acid catalyst includes Bronsted acid or Lewis acid. Preferably, the acid catalyst is selected from one or more of acetic acid, propionic acid, valeric acid, butyric acid, benzoic acid, methanesulfonic acid, triethylaluminum, diphenylzinc, and pentafluorophenylboron; and / or, the base catalyst is selected from one or more of 1,5,7-triazidobicyclo(4.4.0)dec-5-ene, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 1,4-diazabicyclo[2.2.2]octane, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, spartine, tetramethylguanidine, triethylamine, N,N-diisopropylethylamine, tetramethylguanidine, potassium alkoxide or sodium alkoxide; And / or, the primary amine initiator is selected from one or more of mono-, di-, tri- and poly-primary amines with a C1-C10 linear, branched or cyclic structure; more preferably, the mono-primary amine includes one or more of n-hexylamine, benzylamine, n-propylamine, n-butylamine and tert-butylamine, and the di-primary amine includes one or more of p-phenylenediamine, 1,6-hexanediamine, 1,3-propylenediamine, 1,12-dodecanediamine, triethylenetetramine and diethylenetriamine.

3. The preparation method according to claim 1, characterized in that The N-carboxyl ring anhydride of at least one amino acid or a derivative thereof is an N-carboxyl ring anhydride of tryptophan or a derivative thereof; And / or, the ring-opening polymerization reaction is carried out in a second organic solvent, and the second organic solvent is selected from one or more of toluene, benzene, tetrahydrofuran, dimethylformamide, dichloromethane, and ethyl acetate.

4. The preparation method according to claim 1, characterized in that The preparation method of the N-carboxyl ring anhydride of an amino acid or its derivative comprises the following steps: dissolving the amino acid or its derivative in a first organic solvent, performing a ring-closure reaction with triphosgene and an acid-binding agent, and obtaining the anhydride after purification.

5. The preparation method according to claim 4, characterized in that The acid binding agent includes one or more of propylene oxide, epichlorohydrin or pinene; and / or the first organic solvent is selected from one or more of tetrahydrofuran, acetonitrile, 1,4-dioxane, ethyl acetate and N-methylpyrrolidone.

6. A polyamino acid obtained by the preparation method according to any one of claims 1 to 5.

7. The polyamino acid according to claim 6, characterized in that The number average molecular weight of the amino acid polymer is 1000-200000 Da; and / or the molecular weight distribution PDI of the amino acid polymer is <1.

3.

8. Use of the polyamino acid according to claim 6 or 7 in any of the following items; 1) Preparation of drug carriers; 2) Preparation of brain therapeutic drugs; 3) Preparation of brain-targeting agents.

9. A targeting agent, comprising the amino acid polymer according to claim 6 or 7.

10. The targeting agent according to claim 9, characterized in that The targeting agent also includes a therapeutic agent and / or an imaging agent.

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