An amino acid polymer, its preparation method and application

Abstract

This invention provides a molecular weight controllable amino acid polymer, its preparation method, and its applications. The preparation method involves introducing an acid and / or base catalyst in the presence of a primary amine initiator to catalyze the ring-opening polymerization of intracyclic anhydrides and their derivatives of amino acids, yielding polyamino acids with different molecular weights and narrow distributions. This invention also provides applications of the above-mentioned amino acid polymer in gene delivery. This invention reveals that, compared to other amino acid polymers, polytryptophan peptide materials can effectively penetrate the blood-brain barrier and target the brain, which is beneficial for the treatment of brain diseases and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymers, and in particular relates to an amino acid polymer, its preparation method, and its application. Background Technology

[0002] Polyamino acid materials, as a class of biopolymers with excellent biocompatibility, biodegradability, and functional designability, occupy an important position in drug delivery, tissue engineering, biosensing, and smart responsive materials. Their molecular structures can mimic the sequence and conformation of natural proteins, while also possessing the stability and tunability of synthetic materials, providing an ideal platform for developing biomedical functional materials. However, the functional limitations and structural constraints 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] Against this backdrop, tryptophan (Trp), as a naturally encoded eukaryotic amino acid, has demonstrated significant value in multiple fields due to its unique physicochemical properties. Its indole group possesses both hydrophobicity and diverse bonding capabilities (such as cation-π, π-π, and hydrophobic interactions), making it a core unit for designing stable β-sheet structures (such as Trpzip peptides). Research on polytryptophan-based self-assembled hydrogels, charge transfer properties, and biomedical applications (including antibacterial, biotracking, cell delivery, bioprinting, and tumor nanotherapy carriers) has attracted considerable attention.

[0004] The blood-brain barrier, composed of endothelial cells, allows only passive diffusion of small, lipid-soluble molecules, while large molecule drugs (such as protein and nucleic acid drugs) or hydrophilic drugs can hardly pass through, limiting the treatment of many brain diseases (such as brain tumors and neurodegenerative diseases). Low efficiency in penetrating the blood-brain barrier remains a pressing problem that needs to be addressed.

[0005] Due to the steric hindrance and conformational constraints of the tryptophan side chain, high molecular weight (M n The controlled synthesis of polytryptophan has long faced challenges. In biosynthesis, tryptophan easily triggers ribosome translation arrest; in chemical synthesis, solid-phase peptide synthesis (SPPS) and natural chemical linking (NCL) struggle to break through the 20 repeating units.

[0006] In the past decade or so, there has been extensive research on the methodologies for the controlled preparation of amino acids via ring-opening polymerization, as documented in publications 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, research has often focused on the ring-opening polymerization of L-glutamic acid benzyl ester N-carboxyl anhydride (BLG-NCA). Reports indicate that the ring-opening polymerization of Trp-NCA is the most challenging due to steric hindrance (Bulletin of the Chemical Society of Japan 2006). 69(3), 791.). The negative potential of the indole ring can shield against nucleophilic attack by primary amines to some extent, while the beta tendency of the short chain makes it difficult to dissolve in some low-polarity solvents. Although some 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, the controlled polymerization methodology is still imperfect, and the synthesis of high molecular weight peptides containing tryptophan (and its derivatives) remains challenging, limiting its application in high-end fields such as nanomedicine.

[0007] To address the aforementioned bottlenecks, this invention proposes an innovative polymerization strategy: enhancing the stability of the helical conformation through low-polarity mixed solvents such as DCM and THF, and increasing polymer solubility through DMF. The synergistic effect of the two solvents improves solubility and polymerization controllability. Unlike the classic base-initiated activated monomer mechanism (AMM mechanism) (J. Am. Chem. Soc. 2024, 146, 35, 24189–24208), tryptophan exhibits a shielding effect from bases due to the weak acidity of its indole ring, resulting in kinetic curves and product characteristics distinct from conventional BLG-NCA. For BLG-NCA, alkali initiation makes molecular weight control difficult, and the side chains tend to hydrolyze, leading to uncontrollable polymerization (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 regulate molecular weight and narrow distribution products, and enhance the chemical reactivity and "label-free" fluorescence properties of the material, providing technical support for the development of next-generation biomedical functional materials. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a polyamino acid polymer, its preparation method and application. This 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 solves the problems of uncontrollable product molecular weight and the need for harsh polymerization conditions under an inert atmosphere in the prior art.

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

[0010] In a first aspect, the present invention provides an amino acid polymer comprising homopolymers or copolymers of N-carboxycyclic anhydrides of amino acids or their derivatives.

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

[0012] In some embodiments of the present invention, the N-carboxyl ring anhydride monomer of the tryptophan or its derivatives has the structure of Formula I;

[0013]

[0014] R1, R3, R4, R5, and R6 are each independently one of hydrogen atom, fluorine atom, chlorine atom, bromine atom, iodine atom, hydroxyl group, or acetonitrile group; R2 is one of hydrogen atom, trifluoromethyl group, methyl group, isopropyl group, tert-butyl group, or tert-butyloxycarbonyl group.

[0015] In some embodiments of the present invention, the amino acid N-carboxyl ring anhydride further includes hydrophilic amino acid N-carboxyl ring anhydride.

[0016] In some embodiments of the present invention, in order to improve hydrophilicity for drug delivery, the polymer used for gene delivery, in addition to indole-type hydrophobic amino acid N-carboxyl ring anhydrides (such as N-carboxyl ring anhydrides of tryptophan or its derivatives), needs to provide hydrophilic amino acid N-carboxyl ring anhydride monomers for ring-opening copolymerization. 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 monomers can be benzyl glutamate N-carboxyl ring anhydride, N6-Cbz-L-lysine N-carboxyl ring anhydride, benzyl aspartate N-carboxyl ring anhydride, and sarcosine N-carboxyl ring anhydride.

[0017] In some embodiments of the present invention, the amino acid N-carboxyl ring intracyclic anhydride monomer may also include other hydrophobic amino acid N-carboxyl ring intracyclic anhydride monomers, such as leucine N-carboxyl ring intracyclic anhydride monomers, isoleucine N-carboxyl ring intracyclic anhydride monomers, valine N-carboxyl ring intracyclic anhydride monomers, etc.

[0018] 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.

[0019] In some embodiments of the present invention, the amino acid polymer comprises a copolymer formed of an N-carboxycyclic anhydride monomer of tryptophan or a derivative thereof and an N-carboxycyclic anhydride (NCA) monomer of lysine or a derivative thereof.

[0020] In some embodiments of the present invention, the molar ratio of N-carboxycyclic anhydride of tryptophan or its derivative and N-carboxycyclic anhydride (NCA) of lysine or its derivative in the amino acid polymer is 0.01~20:1, and may also be 0.01~0.1:1, 0.1~1:1, 1~5:1, 5~10:1, 10~15:1, or 15~20:1.

[0021] In some embodiments of the present invention, the number average molecular weight of the amino acid polymer is 1,000–200,000 Da; it can also be 1,000–5,000 Da, 5,000–15,000 Da, 15,000–35,000 Da, 35,000–70,000 Da, 70,000–100,000 Da, 100,000–130,000 Da, 130,000–160,000 Da, or 160,000–200,000 Da.

[0022] 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.

[0023] In some embodiments of the present invention, the copolymer is a random copolymer or a block copolymer.

[0024] In some embodiments of the present invention, the block copolymer is a block copolymer with 2 to 10 blocks, and may also be a copolymer with 2, 3, 4, 5, 6, 7, 8, 9 or 10 blocks.

[0025] In some embodiments of the present invention, the length of each block is 10-100 amino acids, and may also be 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 amino acids.

[0026] In some embodiments of the present invention, the amino acid polymer comprises a homopolymer or copolymer having a structure of formula II;

[0027]

[0028] R1, R3, R4, R5, and R6 are each independently one of hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, or acetonitrile groups; R2 is one of hydrogen, trifluoromethyl, methyl, isopropyl, tert-butyl, or tert-butyloxycarbonyl; R is one of methyl, ethyl, propyl, butyl, tert-butyl, hexyl, pentyl, benzyl, or propargyl; n is the degree of polymerization, which is 5-1000.

[0029] A second aspect of the present invention provides a method for preparing the above-mentioned amino acid polymer, comprising performing a ring-opening polymerization reaction on an N-carboxyl ring anhydride monomer of one or more amino acids or their derivatives to form an amino acid polymer.

[0030] In a preferred embodiment, at least one amino acid N-carboxyl ring anhydride is an N-carboxyl ring anhydride of tryptophan or a derivative thereof.

[0031] In some preferred embodiments of the present invention, the plurality includes 2 to 10 types.

[0032] In some embodiments of the present invention, when preparing random copolymers, one or more N-carboxyl 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 anhydride of an amino acid or its derivative is tryptophan or its derivative.

[0033] In some embodiments of the present invention, when preparing block copolymers, one or more amino acids or their derivatives are subjected to stepwise polymerization of N-carboxyl ring anhydrides to form an amino acid polymer, wherein at least one amino acid or its derivative has N-carboxyl ring anhydride that is tryptophan or its derivative.

[0034] For example, when there are two N-carboxyl anhydrides in the amino acid ring: in the presence of an initiator, the N-carboxyl 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 anhydride of another amino acid or its derivative is added and subjected to a polymerization reaction to form a block amino acid copolymer; wherein at least one N-carboxyl anhydride of the amino acid or its derivative is tryptophan or its derivative.

[0035] When there are three N-carboxyl ring anhydride monomers of the amino acid or its derivative: 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, another N-carboxyl ring anhydride of the amino acid or its derivative is added and subjected to a polymerization reaction to form a block amino acid copolymer; wherein at least one N-carboxyl ring anhydride of the amino acid or its derivative is tryptophan or its derivative.

[0036] In some embodiments of the present invention, the method for preparing the N-carboxyl anhydride of the amino acid or its derivative is as follows: dissolving the amino acid or its derivative in a first organic solvent, adding triphosgene and an acid-binding agent to carry out a ring-closing reaction, and purifying to obtain the NCA monomer.

[0037] 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.

[0038] In some embodiments of the present invention, the derivative is a derivative derived from an amino acid; the derivative includes the introduction or substitution of functional groups in the main chain or side chain of the amino acid.

[0039] In some embodiments of the present invention, the functional groups include, but are not limited to, hydroxyl, alkyl, amino, carboxyl, and protecting groups; in some embodiments of the present invention, the protecting groups include, but are not limited to, benzoyl (Cbz), 9-fluorenyloxycarbonyl (Fmoc), etc.

[0040] In some embodiments of the present invention, the alkyl group includes, but is not limited to, methyl, ethyl, etc.

[0041] In some embodiments of the present invention, the amino acid includes tryptophan or a derivative thereof; it may also include one or more of the following: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, cysteine, proline, histidine, arginine or a derivative thereof.

[0042] 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.

[0043] In some embodiments of the present invention, the polymerization reaction of the N-carboxyl anhydride of the amino acid or its derivative specifically includes the following steps:

[0044] An amino acid or its derivative containing an N-carboxyl ring anhydride, an initiator, a catalyst, and a second organic solvent is mixed to obtain a mixture, which is then subjected to ring-opening polymerization to obtain an amino acid polymer.

[0045] In some embodiments of the present invention, the N-carboxylated intracyclic anhydride monomer of tryptophan or its derivatives includes one or more of tryptophan-N-carboxylic acid intracyclic anhydride, 5-hydroxy-tryptophan-N-cyclic anhydride, methyltryptophan-N-cyclic anhydride, and 7-bromotryptophan-N-cyclic anhydride.

[0046] In some embodiments of the present invention, the acid-binding agent includes one or more of propylene oxide, epichlorohydrin, or pinene.

[0047] 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.

[0048] 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 to 30; it can also be 1:5 to 10, 1:10 to 20, or 1:10 to 30.

[0049] 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, or 1g:40~50mL.

[0050] In some embodiments of the present invention, the temperature of the ring-closing reaction is room temperature; preferably 10~35℃; and may also be 10~15℃, 15~25℃, or 25~35℃.

[0051] In some embodiments of the present invention, the ring-closing reaction takes 1-3 hours.

[0052] In some embodiments of the present invention, the purification includes extraction, washing, drying, rotary evaporation, and recrystallization.

[0053] In some embodiments of the present invention, the extractant used during extraction includes ethyl acetate, dichloromethane, toluene, diethyl ether, n-hexane, etc.

[0054] In some embodiments of the present invention, the washing includes 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.

[0055] In some embodiments of the present invention, the drying includes drying with anhydrous sodium sulfate, anhydrous calcium chloride, and anhydrous magnesium sulfate.

[0056] In some embodiments of the present invention, the recrystallization includes recrystallization using n-hexane, tetrahydrofuran, ethyl acetate, or petroleum ether.

[0057] In some embodiments of the present invention, the second organic solvent is one or more of toluene, benzene, tetrahydrofuran, dimethylformamide, dichloromethane, and ethyl acetate.

[0058] In some embodiments of the present invention, the second organic solvent is preferably dimethylformamide or dichloromethane.

[0059] In some embodiments of the present invention, the initiator includes amine compounds; preferably, mono-primary amines, di-primary amines, ter-primary amines, or poly-primary amines with C1–C10 straight-chain, branched, or cyclic structures.

[0060] In some embodiments of the present invention, the primary amine includes one or more of hexylamine, benzylamine, propylamine, butylamine, and tert-butylamine.

[0061] In some embodiments of the present invention, the diamine includes one or more of p-phenylenediamine, 1,6-hexanediamine, 1,3-propanediamine, 1,12-dodecanediamine, triethylenetetramine, and diethylenetriamine.

[0062] In some embodiments of the present invention, the catalyst comprises an acid and / or a base; the acid comprises Brønsted or Lewis acids, such as acetic acid, propionic acid, valeric acid, butyric acid, benzoic acid, methanesulfonic acid, triethylaluminum, diphenylzinc, pentafluorophenylboron; the base comprises 1,5,7-triazabicyclo(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, stigmine, tetramethylguanidine, triethylamine, N,N-diisopropylethylamine, tetramethylguanidine, potassium alkoxide, or sodium alkoxide. More preferably, the catalyst in this application is a mixture of acid and base. Acid-base mixtures have a synergistic catalytic effect. In some cases, using only acid can lead to side reactions and an increase in byproducts in ring-opening polymerization; in other cases, using only alkali can leave the molecular weight of the final product uncontrollable.

[0063] 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.

[0064] In some embodiments of the present invention, the molar ratio of the catalyst to the N-carboxyl 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, or 1:700~1000.

[0065] 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, or 1:40-50.

[0066] In some embodiments of the present invention, the molar ratio of the initiator to the N-carboxyl anhydride of the amino acid or its derivative is 1:20~500; it can also be 1:20~50, 1:50~100, 1:10~200, 1:200~300, 1:300~400, or 400~500.

[0067] In some embodiments of the present invention, in the mixed system for ring-opening polymerization, the volume percentage 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%, or 80%-90%.

[0068] In some embodiments of the present invention, the ring-opening polymerization temperature is 0-100℃; it can also be 0-10℃, 10-20℃, 20-30℃, 30-40℃, 40-50℃, 50-60℃, 60-80℃, or 80-100℃.

[0069] In some embodiments of the present invention, the ring-opening polymerization time 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.

[0070] In some embodiments of the invention, if the N-carboxyl anhydride of the amino acid or its derivative includes a protecting group, the method for preparing the polyamino acid further includes a deprotection step after polymerization; the deprotection step effectively removes the protecting group, restores the functional groups on the side chain of the amino acid, and enables it to participate in further chemical reactions or exhibit the desired biological activity.

[0071] In some embodiments of the present invention, the deprotection method preferably uses an acetic acid solution of TFA / HBr or Pd / C with NaBH4, and the amount of 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.

[0072] A third aspect of the present invention provides the use of the above-described polyamino acid in any of the following:

[0073] (I) Preparation of drug carriers;

[0074] (II) Preparation of brain therapy drugs;

[0075] (III) Preparation of brain-targeting agents.

[0076] In some embodiments of the present invention, the drug includes nucleic acid, peptide, protein or small molecule drug.

[0077] In some embodiments of the present invention, the nucleic acid is selected from antisense oligonucleotides, siRNA, miRNA, mRNA, and gRNA.

[0078] In some embodiments of the present invention, the polypeptide or protein includes antibodies, complement, enzymes, transport proteins, cytokines, polypeptide hormones, etc.

[0079] In a fourth aspect, the present invention provides a targeting reagent comprising the polyamino acid polypeptide material described above.

[0080] In some embodiments of the present invention, the targeting agent further includes an imaging agent and / or a therapeutic agent.

[0081] In some embodiments of the present invention, the therapeutic agent includes, but is not limited to, nucleic acids, peptides, proteins, or small molecule drugs.

[0082] In some embodiments of the present invention, the nucleic acid is selected from antisense oligonucleotides, siRNA, miRNA, mRNA, and gRNA.

[0083] In some embodiments of the present invention, the polypeptide or protein includes antibodies, complement, enzymes, transport proteins, cytokines, polypeptide hormones, etc.

[0084] In some embodiments of the present invention, the active pharmaceutical ingredient is a pharmaceutically active ingredient that acts on the brain.

[0085] In some embodiments of the present invention, the imaging agent includes at least one of a radionuclide, biotin, fluorescent protein, antibody, horseradish peroxidase, and alkaline phosphatase.

[0086] In some embodiments of the present invention, the drug further includes excipients, including but not limited to physiologically, medically, pharmaceutically, and nutritionally acceptable excipients.

[0087] The term "excipients" as used herein refers to, but is not limited to, carriers, excipients, diluents, adjuvants, colorants, fragrances, preservatives, etc., that would be considered by those skilled in the art when formulating the compounds of the present invention to prepare pharmaceutical compositions.

[0088] In some embodiments of the present invention, the drug includes excipients for improving the water solubility or hydrophilicity of the drug.

[0089] In some embodiments of the present invention, the dosage form of the drug includes, but is not limited to, at least one of the following: solution, powder, tablet, granule, capsule, pill, drop, emulsion, injection, ointment, spray, gel, aerosol, suspension, transdermal patch, suppository, or implant.

[0090] In some embodiments of the present invention, the route of administration of the drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, oral administration, sublingual administration, nasal administration, nebulized administration, or transdermal administration.

[0091] The beneficial effects of the technical solution in this application are as follows:

[0092] This invention discloses a method for synthesizing polypeptide materials of polytryptophan or its derivatives with controllable molecular weight and narrow molecular weight distribution. Specifically, this invention introduces an acid and / or base as a catalyst to catalyze the ring-opening polymerization of intracyclic anhydrides of amino acids and their derivatives in the presence of a primary amine initiator, thereby obtaining polytryptophan with different number-average molecular weights. Furthermore, the polymer structure is tunable, the molecular weight is controllable, and the molecular weight distribution is narrow, wherein the number-average molecular weight of the polytryptophan is 1000–200000 Da; and the molecular weight distribution PDI < 1.3.

[0093] The method for synthesizing polypeptide materials using polytryptophan or its derivatives in this invention is highly efficient, requires small amounts, is simple to synthesize, operates under relatively mild reaction conditions, and produces no metal residue in the product. Its application in ring-opening polymerization offers advantages such as simple process, low cost, high reaction rate, controllable process, and narrow molecular weight distribution of the product. Furthermore, polymerization can be carried out under various solvent conditions.

[0094] This invention also provides the application of the above-mentioned polytryptophan polypeptide materials in the field of gene delivery. Furthermore, this invention has discovered that, compared with other amino acid polymers, polymers based on tryptophan cyclic anhydrides have brain-targeting properties, while polyamino acids prepared from other amino acid cyclic anhydrides do not. Polypeptide materials constructed based on tryptophan cyclic anhydrides can not only serve as carriers for efficient delivery of nucleic acid drugs, but also as brain-targeting agents, delivering drugs to the brain across the blood-brain barrier, which is beneficial for the treatment of brain diseases and has good application prospects. Attached Figure Description

[0095] Figure 1 The image shows the 1H NMR spectrum of the anhydride within the N-carboxyl ring of L-tryptophan and the infrared spectrum of polytryptophan.

[0096] Figure 2 The image shows the MALDI-TOF image of polytryptophan in Example 7.

[0097] Figure 3 (a) shows the circular dichroism chromatograms of L-type and D-type Trp-NCA. Figure 3 (b) shows the circular dichroism chromatograms of L-type and D-type polytryptophan.

[0098] Figure 4 (a) shows the GPC diagram of polytryptophan initiated by different monomer and initiator molar ratios, while (b) shows the relationship between the number-average molecular weight (Mn), theoretical molecular weight (Mn,th.), and polydispersity index (PDI) obtained from GPC analysis and the monomer and initiator molar ratios.

[0099] Figure 5This is the GPC diagram of multi-block poly(Cbz-L-lysine-b-polytryptophan).

[0100] Figure 6 This is a diagram showing the results of a cell experiment.

[0101] Figure 7 In vivo imaging of mice loaded with siRNA (time: 2h).

[0102] Figure 8 Brain distribution of KW-type materials and mice dissected 24 hours after tail vein injection of material-loaded siRNA.

[0103] Figure 9 for Figure 8 The statistical graph of average radiant efficiency in the imaging results.

[0104] Figure 10 It is a random copolymerized polypeptide K 80 W 40 Immunofluorescence staining image of brain slices from the material. Detailed Implementation

[0105] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0106] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0107] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0108] Example 1

[0109] In a 50 mL thick-walled pressure-resistant flask, add 1.0 g (4.9 mmol, 1.0 eq) of L-tryptophan and 15 mL of THF. Under magnetic stirring, add 3.4 mL (49 mmol, 10.0 eq) of propylene oxide and 749 mg (2.5 mmol, 0.5 eq) of triphosgene. Seal the container and stir at 25 °C for 1.5 h, then place in an ice bath at 4 °C. For safety, excess triphosgene is quenched by adding 15 mL of cold water at 4 °C. After stirring for 1–3 minutes, extract twice with ethyl acetate (EA, 20 mL). Combine the organic phases at room temperature, wash with saturated brine, and dry with anhydrous Na₂SO₄. After removing the solvent by vacuum rotary evaporation at 35 °C, the crude product was recrystallized from tetrahydrofuran / n-hexane (1:5 v / v) to obtain single crystals (1.0 g, 89% yield) of L-tryptophan-N-carboxylic acid intracyclic anhydride (Trp-NCA). The 1H NMR spectrum of Trp-NCA is shown below. Figure 1 NCA is stored in a glove box filled with argon gas at -15°C.

[0110] Example 2

[0111] 1.0 g (4.9 mmol, 1.0 eq) of D-tryptophan and 15 mL of THF were added to a 50 mL thick-walled pressure-resistant flask. Under magnetic stirring, 3.4 mL (49 mmol, 10.0 eq) of propylene oxide and 749 mg (2.5 mmol, 0.5 eq) of triphosgene were added sequentially. The container was sealed and stirred at 25 °C for 1.5 h, then placed in an ice bath at 4 °C. For safety, excess triphosgene was quenched by adding 15 mL of cold water at 4 °C. 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. 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 give a single crystal (0.99 g, 88% yield) of the intracyclic anhydride of D-tryptophan-N-carboxylic acid. NCA is stored in a glove box at -15°C under argon protection.

[0112] Example 3

[0113] In a 50 mL thick-walled pressure-resistant flask, add 1.0 g (4.5 mmol, 1.0 eq) of L-5-hydroxytryptophan and 15 mL of THF. 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. Seal the container and stir at 25 °C for 1.5 h, then place in an ice bath at 4 °C. For safety, excess triphosgene is quenched by adding 15 mL of cold water at 4 °C. After stirring for 1–3 minutes, extract twice with ethyl acetate (EA, 20 mL). At room temperature, combine the organic phases, wash with saturated brine, and dry with anhydrous Na₂SO₄. After removing the solvent by vacuum rotary evaporation at 35 °C, the crude product was recrystallized from tetrahydrofuran / n-hexane (1:5 v / v) to give a single crystal (1.0 g, 91% yield) of L-5-hydroxy-tryptophan-N-carboxylic acid anhydride. NCA was stored in an argon-filled glove box at -15 °C.

[0114] Example 4

[0115] Add 1.0 g (4.5 mmol, 1.0 eq) of DL-5-hydroxytryptophan and 15 mL of THF to a 50 mL thick-walled pressure-resistant flask. 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. Seal the container and stir at 25 °C for 1.5 h, then place in an ice bath at 4 °C. For safety, excess triphosgene was quenched by adding 15 mL of cold water at 4 °C. After stirring for 1–3 minutes, extract twice with ethyl acetate (EA, 20 mL). Combine the organic phases at room temperature, wash with saturated brine, and dry with anhydrous Na₂SO₄. After removing the solvent by vacuum rotary evaporation at 35 °C, the crude product was recrystallized from tetrahydrofuran / n-hexane (1:5 v / v) to give single crystals (0.85 g, 77% yield) of DL-5-hydroxy-tryptophan-N-carboxylic acid anhydride. NCA was stored in an argon-filled glove box at -15 °C.

[0116] Example 5

[0117] 0.5 g (0.23 mmol, 1.0 eq) of L-methyltryptophan and 15 mL of THF were added to a 50 mL thick-walled pressure-resistant flask. Under magnetic stirring, 1.6 mL (23 mmol, 10.0 eq) of propylene oxide and 340 mg (23 mmol, 0.5 eq) of triphosgene were added sequentially. The container was sealed and stirred at 25 °C for 1.5 h, then placed in an ice bath at 4 °C. After removing the solvent by rotary evaporation under vacuum 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 obtain a single crystal (0.85 g, 77% yield) of L-methyltryptophan-N-carboxylic acid intracyclic anhydride. NCA was stored in an argon-filled glove box at -15 °C.

[0118] The polymerization reaction in the technical solution will continue to be illustrated using specific embodiments, as follows.

[0119] Example 6

[0120] In a 4 mL vial, add 8.69 μL of catalyst DMAP (0.1 M, 12.2 mg / mL, solvent 50% DMF (where 50% DMF contains DMF and DCM in a 1:1 volume ratio), 11.48 μL of n-hexylamine (7.6 mg / 1 mL 50% DMF), then add 153.7 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and then add 43.5 μL of monomer L-tryptophan N-carboxycyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF). In the resulting reaction system, [M]0 = 0.2. mol / L, [M]0 / [I]0 / [DMAP]0=50 / 1 / 1, where M is the anhydride within the N-carboxyl ring of tryptophan monomer, and I is the initiator n-hexylamine. The reaction was carried out at 25°C for 4 hours, and then precipitated twice with cold anhydrous diethyl ether. The polymer was obtained by vacuum drying to constant weight.

[0121] Where Conv. = 99%, M n,GPC = 20.8 kg / mol, PDI = 1.18.

[0122] Conversion rates were calculated using NMR spectroscopy. The peak at 10.8 ppm is a characteristic peak of the indole ring NH in the monomer, and the peak at 10.4 ppm is a characteristic peak of the indole ring NH in the polymer. Conversion rates can be calculated using the TMS internal standard method.

[0123] Example 7

[0124] In a 4 mL vial, add 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. Then add 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and then add 43.5 μL of monomer L-tryptophan N-carboxycyclic 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). Control the temperature at 25°C and react for 3 h. Then precipitate twice with cold anhydrous diethyl ether and vacuum dry to constant weight to obtain polymer W. 50 Where Conv. = 99%, M n,GPC = 10.5 kg / mol, PDI = 1.21. For detailed mass spectrometry results, please refer to [link to relevant documentation]. Figure 2 ,Depend on Figure 2 The table shows that the structure and molecular weight of the final product conform to the theory.

[0125] The polymerization process of D-type Trp-NCA is similar, except that the raw material used is changed from the anhydride within the N-carboxyl ring of L-type tryptophan to D-type. The polymerization feed for methyltryptophan can also be implemented using this example. The detection process involves taking a 1 mm cuvette, adding 400 μL of a 0.1 mg / mL tetrahydrofuran solution of polytryptophan, monitoring the CD signal at 210 nm–260 nm, and converting the unit to molar value. The molar ellipticity [θ] (unit: deg / cm) is calculated by multiplying the measured mdeg value by 1000, dividing by the optical path length (mm), and then dividing by the concentration (mM). 2 .dmol -1 ).

[0126] For details of its circular dichroism (CD) spectrum results, please refer to Figure 3 The CD results showed that the L-type exhibited a significant negative peak at 218 nm and an upward Trp-Trp stacking peak at 229 nm, indicating an α-helical tendency in its secondary structure, while the D-type showed the opposite peak shape. The negative peak of methyltryptophan shifted to 220 nm, indicating a more stable α-helical tendency.

[0127] Example 8

[0128] In a 4 mL vial, add 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. Then add 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and add 43.5 μL of monomer L-tryptophan N-carboxycyclic 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). Control the temperature at 25°C and react for 3 h. Then precipitate twice with cold anhydrous diethyl ether and vacuum dry to constant weight to obtain the polymer; where Conv. = 99%, M n,GPC = 15.5 kg / mol, PDI = 1.21.

[0129] Example 9

[0130] In a 4 mL vial, add 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. Then add 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and add 43.5 μL of monomer L-tryptophan N-carboxycyclic 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). Control the temperature at 25°C and react for 4 h. Then precipitate twice with cold anhydrous diethyl ether and vacuum dry to constant weight to obtain the polymer, where Conv. = 99%, M n,GPC = 19.8 kg / mol, PDI = 1.03.

[0131] Example 10

[0132] In a 4 mL vial, add 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. Then add 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and add 43.5 μL of monomer L-tryptophan N-carboxycyclic 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). Maintain the reaction temperature at 25°C for 5 h. Then precipitate twice with cold anhydrous diethyl ether and vacuum dry to constant weight to obtain the polymer, where Conv. = 99%, M n,GPC = 28.6 kg / mol, PDI = 1.02.

[0133] Example 11

[0134] In a 4 mL vial, add 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. Then add 151.2 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and add 43.5 μL of monomer L-tryptophan N-carboxycyclic 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). Control the temperature at 25°C and react for 7 h. Then precipitate twice with cold anhydrous diethyl ether and vacuum dry to constant weight to obtain the polymer, where Conv. = 99%, M n,GPC = 32.2 kg / mol, PDI = 1.03; its infrared characterization results are detailed in [link to infrared characterization results]. Figure 1 .

[0135] In this invention, the GPC characterization method involves preparing a 5 mg / ml solution of the polymer (0.1 M LiBr in DMF solution) and characterizing its molecular weight using gel permeation chromatography. The molecular weight of the polymer is determined based on the dn / dc value of each polymer sample using an internal calibration system processed by ASTRA software (version 8.12, Wyatt Technology, Santa Barbara, California, USA). For polytryptophan, the dn / dc value is 0.1250.

[0136] Additionally, the GPC diagrams of the polytryptophan prepared in Examples 7-10 and 11 are shown below. 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 in line with the theoretical value.

[0137] Example 12

[0138] In a 4 mL vial, add 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. Then add 151.2 μL of a mixed solvent DMF / DCM (2:8, v / v), shake well, and add 43.5 μL of monomer L-tryptophan N-carboxycyclic 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). Control the temperature at 25°C and react for 7 h. Then precipitate twice with cold anhydrous diethyl ether and vacuum dry to constant weight to obtain the polymer, where Conv. = 99%, M n,GPC = 35.2 kg / mol, PDI = 1.05.

[0139] Example 13

[0140] 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. Then, 348 μL of a mixed solvent DMF / DCM (1:1, v / v) was added, and the mixture was shaken well. 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 the reaction was monitored by infrared spectroscopy at 1850 cm⁻¹. -1 and 1790 cm -1 The disappearance of the peak proved that the monomer was completely precipitated. Then, it was precipitated twice with cold anhydrous diethyl ether and dried under vacuum 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. Figure 5 .

[0141] Example 14

[0142] In a 4 mL vial, add 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. Then add 348 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and add 173.9 μL of pre-mixed monomers N6-benzyloxycarbonyl-L-lysine cyclic anhydride (1 M) and 173.9 μL of monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL). Control the temperature at 25°C and react for 4 h. The reaction can be monitored by infrared spectroscopy at 1850 cm⁻¹. -1 and 1790 cm -1 The disappearance of the peak proved that the monomer was completely formed. Then, it was precipitated twice with cold anhydrous diethyl ether and dried under vacuum 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.

[0143] In this invention, PZLL represents poly(N6-benzyloxycarbonyl-L-lysine), PLW represents poly(L-tryptophan N-carboxyl intracyclic anhydride), and ran represents a random copolymer. Other materials used in this example include PZLL. 20 -ran-PLW 10 PZLL 15 -ran-PLW 15 PZLL 30 -ran-PLW 30 PZLL 80 -ran-PLW 40 All of these can be synthesized using a method similar to this case. By changing the proportion of monomers added, the material can be obtained within 8 hours.

[0144] Example 15

[0145] In a 4 mL vial, add 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. Then add 348 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and add 278 μL of pre-mixed monomer N6-benzyloxycarbonyl-L-lysine cyclic anhydride (1 M) and 139 μL of monomer L-tryptophan N-carboxyl cyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL). Control the temperature at 25°C and react for 4 h. The reaction can be monitored by infrared spectroscopy at 1850 cm⁻¹. -1 and 1790 cm -1 The disappearance of the peak proved that the monomer was completely formed. Then, it was precipitated twice with cold anhydrous diethyl ether and dried under vacuum to constant weight to obtain the random polymer PZLL. 80 -ran-PLW 40 Where Conv. = 99%, M n,GPC = 25.0 kg / mol, PDI = 1.05.

[0146] Example 16

[0147] The synthesis of the block copolymer peptide was carried out 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 DMF / DCM (1:1, v / v) was added, and the mixture was shaken well. 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 disappearance of the 1850 and 1790 peaks was monitored by infrared spectroscopy, indicating complete monomer synthesis. Then, 173.9 μL of the second monomer was added. L-tryptophan N-carboxycyclic intracyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL) was reacted at 25°C for 4 hours, and the reaction was monitored at 1850 cm⁻¹ using infrared spectroscopy. -1 and 1790 cm -1 The disappearance of the peak proved that the monomer was completely precipitated. Then, it was precipitated twice with cold anhydrous diethyl ether and dried under vacuum to constant weight to obtain polymer PZLL. 50 -b-PLW 50 Where Conv. = 99%, M n,GPC= 23.8 kg / mol, PDI = 1.05, PZLL 50 -b-PLW 50 See GPC diagram Figure 5 .

[0148] PZLL represents poly(N6-benzyloxycarbonyl-L-lysine), PLW represents poly(L-tryptophan N-carboxycyclic intracyclic anhydride), b represents block, and 50 represents the degree of polymerization of each segment. Other materials used in this case, such as PZLL... 20 -b-PLW 10 PZLL 15 -b-PLW 15 PZLL 30 -b-PLW 30 PZLL 80 -b-PLW 40 All of these can be synthesized using a method similar to this case. By changing the proportion of monomers added, the material can be obtained within 8 hours.

[0149] Example 17 Synthesis of Block Copolymer Peptides

[0150] In a 4 mL vial, add 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. Then add 348 μL of a mixed solvent DMF / DCM (1:1, v / v), shake well, and add 173.9 μL of monomer N6-benzyloxycarbonyl-L-lysine cyclic anhydride (ZLL-NCA, 1 M, 306.3 mg / 1 mL 50% DMF), ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 50 / 1 / 1). Control the temperature at 25°C and react for 1 h. The reaction can be monitored by infrared spectroscopy at 1850 cm⁻¹. -1 and 1790 cm -1 The disappearance of the peak confirmed complete monomerization. Then, 173.9 μL of the second monomer, L-tryptophan N-carboxycyclic anhydride (Trp-NCA, 1 M, 230 mg / 1 mL 50% DMF), was added, and the reaction was carried out at 25°C for 4 hours. Infrared spectroscopy was used to monitor the reaction at 1850 cm⁻¹. -1 and 1790 cm -1The disappearance of the peak confirmed complete monomerization. Then, 173.9 μL of the third monomer, N6-benzyloxycarbonyl-L-lysine cyclic anhydride (ZLL-NCA, 1 M, 306.3 mg / 1 mL), was added ([M]0 = [AcOH]0 = 0.2 mol / L, [M]0 / [I]0 / [DMAP]0 = 50 / 1 / 1). The reaction was carried out at 25°C for 1 h. The product was precipitated twice with cold anhydrous diethyl ether and dried under vacuum to constant weight to obtain the block copolymer peptide 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 See GPC diagram Figure 5 .

[0151] Example 18 Deprotection step of polypeptide

[0152] 3.0 g of the copolymer was dissolved in 18.0 mL of trifluoroacetic acid, and 9.0 mL of a hydrogen bromide / acetic acid mixture was slowly added. 9 mL of diethyl phosphite was added to protect the tryptophan side group indole. After stirring at 30 °C for 2 h, the mixture was precipitated in excess diethyl ether, washed, filtered, and vacuum dried. The resulting solid was dissolved in a small amount of water and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The solid was purified by dialyzing in distilled water and then freeze-dried to obtain a white solid (1.5 g, Yield: 50%).

[0153] 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.

[0154] The cytotoxicity experiments in this application are as follows:

[0155] The cytotoxicity of different formulations was detected using the CCK-8 assay. The simplified procedure is as follows: 293T-luc cells were seeded at a density of 5000 cells per well in 96-well plates and incubated overnight to allow cell adhesion. Then, blank formulations and siLUC vector formulations with different N / P ratios (10, 15, 20, 25, 30) were added, and the cells were cultured for another 48 hours. Next, CCK-8 reagent was added to each well, and the cells were incubated for another hour. Finally, the absorbance was measured at 450 nm using a microplate reader. The cytotoxicity results are shown below. Figure 6As shown, the results indicate that pure polylysine has high toxicity, while the random and block copolymers formed with polytryptophan are almost non-toxic, indicating that the material is safe and reliable. The transfection experiment in this application is as follows: 5000 293T-luc cells were seeded in 96-well plates 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 ng LUC siRNA and complexes formed with different formulations at N / P ratios (10, 15, 20, 25, 30). The cells were cultured until the detection time point, and the fluorescence intensity was measured using a luciferase assay kit and a microplate reader. The Lipofectamine 2000 transfection group was used as a positive control. Data are expressed as mean ± standard deviation (n=3). The transfection experiment results are shown below. Figure 6 As shown, the results indicate that the transfection efficiency of KW series materials loaded with siRNA is comparable to that of Lipofectamine 2000, demonstrating their effectiveness as vectors.

[0156] Example 19 Polypeptide fluorescent labeling (K 50 -W 50 -Cy5)

[0157] Dissolve 9 μL of 1 mg / mL Cy5-NHS ester in 100 μL of DMSO, then add it to a container containing peptide K. 50 -ran-W 50 In a 400 μL DMSO solution containing 10 mg of the dye Cy5-NHS ester, the ratio of dye to peptide was 1:50. Then, 0.5 μL of triethylamine (10 μL / 1 ml) was added to the mixture, and the reaction mixture was stirred overnight at room temperature in the dark. Water was then added and the mixture was placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The solution was purified by dialyzing in distilled water and freeze-dried to obtain a blue solid (7.1 mg, Yield: 71%).

[0158] Other peptide markers, such as Cy5, can also be added in the same proportion as in this implementation example.

[0159] Example 20: Preparation method of gene delivery system (N / P = 10)

[0160] (1) Preparation of polylysine-polytryptophan (K) 80 -ran-W 40 A nuclease-free DEPC aqueous solution (1 mg / mL) of random polypeptide (KW).

[0161] (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 thoroughly for 30 s, mechanically mix 10 times at 25℃; then incubate at 37℃ for 15 min; mix thoroughly, the mass ratio of the polypeptide to the target gene in the mixture is 6.14 μg: 1.4 μg. (siRNA sequence S: GCGACGACUGCCUAAGAUdTdT, AS: AUCUUAGGCAGAUCGUCGCdTdT)

[0162] Example 21 Preparation method of gene delivery system (N / P = 15)

[0163] (1) Preparation of polylysine-polytryptophan (K) 80 -ran-W 40 A nuclease-free DEPC aqueous solution (1 mg / mL) of random polypeptide (KW).

[0164] (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 thoroughly for 30 s, mechanically mix 10 times at 25℃; then incubate at 37℃ for 15 min; mix thoroughly, 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)

[0165] Example 22: Preparation method of the delivery system (N / P = 20)

[0166] (1) Preparation of polylysine-polytryptophan (K) 80 -ran-W 40 A nuclease-free DEPC aqueous solution (1 mg / mL) of random polypeptide (KW).

[0167] (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 thoroughly for 30 s, mechanically mix 10 times at 25℃; then incubate at 37℃ for 15 min; mix thoroughly, the mass ratio of the polypeptide to the target gene in the mixture is 12.3 μg: 1.4 μg. (siRNA sequence S: GCGACGACUGCCUAAGAUdTdT, AS: AUCUUAGGCAGAUCGUCGCdTdT)

[0168] Similar to the compounding method for block copolymer peptides and genes, the amount of siRNA added is adjusted based on the actual degree of polymerization and the N / P ratio.

[0169] Example 23 In vivo imaging of the brain-targeted delivery platform KW and KW@siRNA

[0170] 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 and random copolymer K of siRNA-loaded labeled 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 at specific time intervals to detect their targeting ability. Simultaneously, 24 hours later, the mice were anesthetized, the thoracic cavity was cut along the manubrium of the sternum, the pericardium was cut open to expose the heart, and the left ventricle was cut at a 30-45° angle to the apex. The perfusion needle was inserted into the aorta through the left ventricle, the needle was fixed, and the right atrial appendage was cut open. The blood was then rapidly flushed with physiological saline (20-40 ml for mice) until the outflowing fluid was light in color and basically clear (rapid whitening of the liver, eyes, and paws is an effective observation sign of blood removal). The mice exhibited twitching of the limbs and tail. Perfusion was considered successful when the liver turned white and the internal organs swelled. The brain and internal organs were then dissected and imaged. The results showed that the brain-targeting delivery platform obtained in this embodiment could rapidly and accurately target the brain tissue in mice.

[0171] Figures 7-9 The results showed that the mouse brain exhibited significant Cy5 fluorescence, which was significantly stronger than that of the Cy5 group and the positive control Angiopep-2 commercial brain-targeting peptide group; indicating that the KW series polypeptides obtained in this embodiment can rapidly and accurately target to the surrounding brain tissue in mice. Furthermore, the degree of enrichment in the brain showed a positive correlation with the increase of polymerization degree and tryptophan content, highlighting the necessity of long-chain polytryptophan with a high tryptophan content. Figure 9 It can also be seen that the enrichment degree of brain material is positively correlated with tryptophan content, and the higher the degree of polymerization, the better.

[0172] Example 24

[0173] Based on Example 23, the distribution of the brain-targeted delivery platform KW in brain tissue was further investigated. The frozen section and immunofluorescence staining steps are as follows:

[0174] (1) Fixation of frozen sections: Bake frozen sections in an oven at 37°C for 10-20 min and drain off the water. Fix in fixative for 30 min, and wash three times in PBS (pH 7.4) on a decolorizing shaker for 5 min each time.

[0175] (2) Antigen retrieval: During the retrieval process, excessive evaporation of the buffer solution should be prevented, and the slide should not be dried out. After retrieval, allow it to cool naturally. Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time.

[0176] (3) Circle blocking with serum: After the slide is slightly dried, draw a circle around the tissue with a histochemical pen, 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.

[0177] (4) Add primary antibody: Add the prepared primary antibody, place the slice flat in a humidified box and incubate overnight at 4°C.

[0178] (5) Add secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. Add the corresponding secondary antibody and incubate at room temperature in the dark for 50 minutes.

[0179] (6) Counterstaining cell nuclei with DAPI: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 min each time. Add DAPI staining solution and incubate at room temperature in the dark for 10 min.

[0180] (7) Quenching tissue autofluorescence: Place the slide in PBS (pH 7.4) and wash it 3 times on a decolorizing shaker for 5 min each time. Add autofluorescence quencher B solution for 5 min, and rinse with running water for 10 min.

[0181] (8) Mounting: Mount the slide with anti-fluorescence quenching mounting medium.

[0182] (9) Image acquisition: DAPI excitation wavelength 330-380nm, emission wavelength 420nm; 488 excitation wavelength 465-495nm, emission wavelength 515-555nm; CY5 excitation wavelength 608-648nm, emission wavelength 672-712nm.

[0183] Figure 10 The results showed that the random copolymer peptide K 80 W 40 It is distributed in various brain regions, including the cerebral cortex, hippocampus, prefrontal cortex, and cerebellum, with the cerebellum having the most distribution.

[0184] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered 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 tryptophan or its derivatives undergoes ring-opening polymerization to form an amino acid polymer. The ring-opening polymerization uses a primary amine initiator and is catalyzed by both acid and base. The polymerization is carried out in a second organic solvent, which is a combination of dimethylformamide and one or more selected from toluene, tetrahydrofuran, dichloromethane, and ethyl acetate. The molecular weight distribution of the amino acid polymer is described below. PDI < 1.3; the acid catalyst is acetic acid, and the base catalyst is 4-dimethylaminopyridine.

2. The preparation method according to claim 1, characterized in that, The primary amine initiator is selected from one or more mono- or poly-primary amines with C1–C10 straight-chain, branched, or cyclic structures.

3. The preparation method according to claim 2, characterized in that, The monobasic primary amine includes one or more of hexylamine, benzylamine, propylamine, butylamine, and tert-butylamine, and the polybasic primary amine includes one or more of p-phenylenediamine, 1,6-hexanediamine, 1,3-propanediamine, 1,12-dodecanediamine, triethylenetetramine, and diethylenetriamine.

4. The preparation method according to claim 1, characterized in that, The method for preparing the N-carboxyl ring anhydride of tryptophan or its derivatives is as follows: tryptophan or its derivatives are dissolved in a first organic solvent, and then subjected to a ring-closure reaction with triphosgene and an acid-binding agent, and then purified to obtain the anhydride.

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. An amino acid polymer obtained by the preparation method according to any one of claims 1 to 5.

7. The amino acid polymer according to claim 6, characterized in that, The number-average molecular weight of the amino acid polymer is 1,000–200,000 Da.

8. The use of the amino acid polymer as described in claim 6 or 7 in any of the following; 1) Preparation of drug carriers; 2) Preparation of brain therapy drugs; 3) Preparation of brain-targeting agents.

9. A targeting agent comprising the amino acid polymer of claim 6 or 7.

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

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