Polypeptides and nanoassemblies and their preparation methods

Abstract

This disclosure provides a polypeptide and a nanoassembly of formula (I) and a method for preparing the same. In formula (I), R1 is selected from any of the following groups: aliphatic primary amine residues, aromatic primary amine residues, and residues based on modified polyethylene glycol; R2 is selected from any of the following groups: H, amino protecting group; wherein, when R2 represents an amino protecting group, R3 represents H, and when R2 represents H, R3 represents H or is absent; n includes 1 to 1000, representing the degree of polymerization of the amino acid monomers on the polypeptide backbone; x includes 1 to 3; wherein, when x = 1, the polypeptide backbone corresponds to 2,4-diaminobutyric acid, when x = 2, the polypeptide backbone corresponds to ornithine, and when x = 3, the polypeptide backbone corresponds to lysine amino acid residues. The polypeptide provided in this disclosure can, after ultraviolet light irradiation (or other types of side-chain protecting groups and their corresponding deprotection), expose amino groups on the side chains of the polypeptide backbone, and undergo amino cyclization attacking the amide bond, leading to self-degradation of the backbone.

Description

Technical Field

[0001] This disclosure relates to the field of polypeptide materials technology, and in particular to a polypeptide and nanoassembly and a method for preparing the same. Background Technology

[0002] Polypeptides, as protein mimics, possess biocompatibility, degradability, and stimulus-responsiveness, and have wide applications in drug targeting, gene delivery, and antibacterial agents. Polypeptides with various topologies can self-assemble into different nanostructures, thereby achieving diverse applications, which has attracted widespread attention in the field of biochemistry. Summary of the Invention

[0003] In view of this, the main objective of this disclosure is to provide a polypeptide and a method for preparing the same, as well as a method for preparing nanoassemblies, in order to at least partially solve at least one of the aforementioned technical problems.

[0004] To achieve the above objectives, as an embodiment of one aspect of this disclosure, a polypeptide of formula (I) is provided.

[0005]

[0006] R1 is selected from any of the following groups: aliphatic primary amine residues, aromatic primary amine residues, residues based on modified polyethylene glycol; R2 is selected from any of the following groups: H, amino protecting group; wherein, when R2 represents an amino protecting group, R3 represents H, and when R2 represents H, R3 represents H or is absent; n includes 1 to 1000, representing the degree of polymerization of amino acid monomers on the polypeptide backbone; x includes 1 to 3; wherein, when x = 1, the polypeptide backbone corresponds to 2,4-diaminobutyric acid (Dab), when x = 2, the polypeptide backbone corresponds to ornithine (Orn), and when x = 3, the polypeptide backbone corresponds to the amino acid residue of lysine (Lys).

[0007] As another embodiment of this disclosure, a method for preparing the polypeptide as described above is provided, comprising: mixing a monomer and an initiator, then dissolving them in a solvent; initiating monomer polymerization under heating conditions; wherein the molar ratio of the monomer to the initiator is from 1:1 to 1000:1, the concentration of the monomer is from 0.05 to 2M, and the initiator comprises protonated or deprotonated PEG. m -Amin initiator, protonated or deprotonated primary or secondary amine; the solvent includes any one of N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), and the monomer includes the compound shown in formula (V) or the compound shown in formula (VI).

[0008]

[0009] As another embodiment of this disclosure, a nano-assembly obtained by the self-assembly of the above-described polypeptide is provided. The polypeptide represented by formula (III) or formula (IV) is dissolved in an organic solvent to obtain an organic mixed solution. Then, it is added to stirred water by slow addition of water, direct dialysis, or rapid addition, respectively. Finally, the organic solvent is removed by dialysis to obtain the nano-assembly.

[0010] The polypeptides provided in the above embodiments of this disclosure can undergo degradation based on the polypeptide backbone. The specific process is as follows: after ultraviolet light irradiation (or other types of side-group protecting groups and their corresponding deprotection), the side groups of the polypeptide backbone expose amino groups, and amino cyclization attacks the amide bond, leading to backbone self-degradation. For example, the polypeptide prepared by formula (V) in the preparation method of the polypeptide can undergo hydrolysis-independent triggered active degradation. The specific process is as follows: under ultraviolet light (e.g., 365 nm) irradiation, the oNB group breaks, exposing the amino side groups of the polypeptide backbone, followed by amino cyclization degradation attacking the amide bond. In addition, the above-mentioned polypeptide self-assembly can yield nano-assemblies. The embodiments of this disclosure designed and synthesized a polypeptide with a multi-level self-assembly structure and cyclization-induced backbone self-degradation. Attached Figure Description

[0011] Figure 1A Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBDab 80 Gel permeation chromatogram;

[0012] Figure 1B Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBDab 80 The proton NMR spectrum;

[0013] Figure 1C Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBDab 80 Infrared spectrum;

[0014] Figure 2A Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76 Gel permeation chromatogram;

[0015] Figure 2B Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76The proton NMR spectrum;

[0016] Figure 3A Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PBocDab 10 Gel permeation chromatogram;

[0017] Figure 3B Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PBocDab 10 The proton NMR spectrum;

[0018] Figure 4A Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PBocO 10 Gel permeation chromatogram;

[0019] Figure 4B Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PBocO 10 The proton NMR spectrum;

[0020] Figure 5 Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PDab 10 The proton NMR spectrum;

[0021] Figure 6 Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-POrn 10 The proton NMR spectrum;

[0022] Figure 7A Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBDab 80 Transmission electron microscopy characterization of the self-assembled nanoassemblies;

[0023] Figure 7B Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBDab 80 Scanning electron microscope characterization of the self-assembled nanoassemblies;

[0024] Figure 7C Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBDab 80 Atomic force microscopy characterization of the self-assembled nanoassemblies;

[0025] Figure 8A Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76 Transmission electron microscopy characterization of the self-assembled nanoassemblies;

[0026] Figure 8B Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76 Scanning electron microscope characterization of the self-assembled nanoassemblies;

[0027] Figure 8C Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76 Characterization images of self-assembled nanoassemblies obtained by atomic force microscopy;

[0028] Figure 8D Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76 Characterization image of the self-assembled nanoassemblies by confocal electron microscopy;

[0029] Figure 9A Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76 Transmission electron microscopy characterization of the self-assembled ellipsoidal vesicle structure nanoassembly;

[0030] Figure 9B Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76 Atomic force microscopy characterization of the self-assembled ellipsoidal vesicle structure nanoassembly;

[0031] Figure 9C Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBO 76 Characterization image of the self-assembled ellipsoidal vesicle structure nanoassembly by confocal electron microscopy;

[0032] Figure 10 Polypeptide PEG according to an exemplary embodiment of the present disclosure 45 -b-PNBDab 80 Transmission electron microscopy characterization of the self-assembled vesicle structure nanoassemblies for UV-triggered responsive self-degradation.

[0033] Figure 11A A self-assembled PEG according to an exemplary embodiment of the present disclosure 45 -b-POrn 10 Gel permeation chromatogram of self-assembled components at the molecular chain level; and

[0034] Figure 11B A self-assembled PEG according to an exemplary embodiment of the present disclosure 45 -b-POrn 10 Electrospray mass spectra of self-assembled organisms at the molecular chain level. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0036] In recent years, stimulus-responsive peptides have attracted widespread attention in the fields of biomaterials, nanomedicine, and controlled drug release. Among them, photosensitive polymers, due to their high selectivity and efficiency, show great potential for applications in drug delivery, polymer therapy, and smart surfaces. Biodegradable peptides have become an important class of biomedical polymer materials due to their good biocompatibility and biodegradability, ease of modifying functional groups on the main chain, and unique responsive chain conformational transitions and multi-level self-assembly properties. Currently, there are numerous reports on the research of pH-, heat-, light-, redox-responsive peptides and their nanomedicine applications, and several polyethylene glycol-peptide copolymer nanomedicine systems have been approved for clinical research internationally. Of particular note is the stimulus-responsive motif o-nitrobenzyl (oNB), which exhibits good stability under acidic and alkaline conditions and highly controllable photochemical properties, providing a spatiotemporally controllable UV-triggered responsive motif.

[0037] Since Hermann Leuchs invented α-amino acid-N-carboxylic anhydride (NCA) in 1906, NCA ring-opening polymerization has been a common method for preparing polypeptides. To improve the controllability of NCA polymerization, many new polymerization techniques have emerged in recent years. However, due to the high sensitivity of NCA and the numerous side reactions during polymerization, methods with precise terminal functional groups and narrow polydispersity have become less effective. The controlled synthesis of Mw / Mn) polypeptides remains a significant challenge. Previous reports on the characterization of NCA polymerization using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) show that impurity peaks related to side reactions are still clearly present. To address these issues, the applicant has previously developed a novel strategy using NPCA as the monomer, instead of unstable NCA, and primary amine hydrochloride as the initiator. This strategy initiates the living polymerization of NPCA under NCA monomer-starved conditions to prepare well-defined polypeptides, yielding products with predetermined molecular weights and narrow molecular weight distributions. It contains precise terminal functional groups and has no obvious byproducts. Therefore, this methodology establishes an effective strategy for the synthesis and functional application of polypeptide materials.

[0038] Inspired by the 21 amino acids that make up organisms, lysine is included among them. However, ornithine and 2,4-diaminobutyric acid (2,4-DABA), two homologues of lysine, are not components of organisms. This is likely because ornithine and 2,4-DABA can attack the amide bond of the main chain through side-group amino cyclization, leading to peptide chain instability. Of course, their cyclization degradation products are five-membered and six-membered ring structures, a thermodynamically supported process. Furthermore, given that natural proteins can be specifically degraded by enzymes, while synthetically produced peptides, due to their non-specificity, often require either vigorous hydrolysis conditions or very slow hydrolysis under mild conditions, this study highlights the importance of designing self-degradable peptide materials, despite the current popularity of self-degradable polymers (SIPs). This research aims to significantly expand the application areas of self-degradable peptide materials. Based on previously established polymerization methodologies, this invention prepares this class of photoresponsive amphiphilic block polypeptide materials and investigates their controllable self-assembly and responsive self-degradation behavior.

[0039] Based on the general inventive concept of this disclosure, a polypeptide of formula (I) is provided.

[0040]

[0041] R1 is selected from any of the following groups: aliphatic primary amine residues, aromatic primary amine residues, residues based on modified polyethylene glycol; R2 is selected from any of the following groups: H, amino protecting group; wherein, when R2 represents an amino protecting group, R3 represents H, and when R2 represents H, R3 represents H or is absent; n includes 1 to 1000, representing the degree of polymerization of amino acid monomers on the polypeptide backbone; x includes 1 to 3; wherein, when x = 1, the polypeptide backbone corresponds to 2,4-diaminobutyric acid (Dab), when x = 2, the polypeptide backbone corresponds to ornithine (Orn), and when x = 3, the polypeptide backbone corresponds to the amino acid residue of lysine (Lys).

[0042] The polypeptides provided in this embodiment can undergo degradation based on the polypeptide backbone. The specific process is as follows: after ultraviolet light irradiation (or other types of side-chain protecting groups and their corresponding deprotection), the side chains of the polypeptide backbone expose amino groups, and amino cyclization attacks the amide bonds, leading to the self-degradation of the backbone.

[0043] In some embodiments of this disclosure, after the protecting group of the polypeptide is triggered to be removed, its side amino groups can cyclize and attack the amide bonds of the polypeptide backbone, leading to the self-degradation of the backbone and the generation of cyclization products, namely a five-membered ring with a molecular weight of 100.06 generated in the 2,4-diaminobutyric acid (Dab) system; and a six-membered ring with a molecular weight of 114.08 generated in the ornithine (Orn) system.

[0044] In some embodiments of this disclosure, the side chain amino groups of the polypeptide block are in either a primary amine state or a protonated state. The counterion of the corresponding protonated primary amine includes Cl... - BF4 - ClO4 - PF6 - CF3COO - HCOO - , or CH3COO - wait.

[0045] In some embodiments of this disclosure, the side chain amino groups of the polypeptide block can be protected by amino protecting groups. According to embodiments of this disclosure, amino protecting groups include o-nitrobenzyloxycarbonyl (oNB), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenemethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl (or ethoxycarbonyl), etc.

[0046] According to embodiments of this disclosure, in the R1 group, the aliphatic group of the aliphatic primary amine residue is selected from C groups that are substituted with or unsubstituted with halogen or phenyl groups. 1-30 Alkyl groups or C groups substituted with or unsubstituted with halogens or phenyl groups 2-30 Alkenyl; the aromatic group in the aromatic primary amine residue is selected from phenyl groups that are substituted with halogen or alkyl groups or are unsubstituted.

[0047] According to embodiments of this disclosure, the residues in R1 based on modified polyethylene glycol include groups represented by formula (II), wherein m includes 1 to 20000, and preferably, m is selected from any one of the following: 22, 44, 112.

[0048]

[0049] According to embodiments of this disclosure, the polypeptide includes a polypeptide of formula (III) or formula (IV), wherein m, n, and x are as defined above.

[0050]

[0051] In some embodiments of this disclosure, the structure shown in formula (III) is subjected to ultraviolet light irradiation to remove the side group oNB motif to obtain the structure shown in formula (VII); or the structure shown in formula (IV) is subjected to acidic conditions to remove the Boc motif to obtain the structure shown in formula (VII).

[0052]

[0053] According to embodiments of this disclosure, a method for preparing the polypeptide as described above includes: mixing a monomer and an initiator, then dissolving them in a solvent; initiating monomer polymerization under heating conditions; wherein the molar ratio of the monomer to the initiator is between 1:1 and 1000:1, the concentration of the monomer is between 0.05 and 2 M, and the initiator comprises protonated or deprotonated PEG. m - An amino initiator, a protonated or deprotonated primary or secondary amine; the solvent includes any one of N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), and dimethyl sulfoxide (DMSO); the monomer includes a compound of formula (V) or a compound of formula (VI).

[0054]

[0055] In some embodiments of this disclosure, the initiator has the structure shown in formula (VIII).

[0056]

[0057] In some embodiments of this disclosure, the reaction time ranges from 12 to 72 hours; the reaction is carried out under inert gas protection, vacuum, or open conditions.

[0058] According to embodiments of this disclosure, the method further includes: adding the reaction mixture obtained after monomer polymerization to cold diethyl ether for precipitation separation, or adding excess cold diethyl ether or petroleum ether to the concentrate of the reaction mixture for precipitation separation, and drying the obtained polymer under reduced pressure or vacuum.

[0059] In some embodiments of this disclosure, the chemical reaction for preparing the polypeptide of formula (III) from the monomer of formula (V) is shown in reaction formula (1).

[0060]

[0061] The specific preparation steps are as follows: In a fume hood with direct opening or a glove box filled with nitrogen, the monomer N-phenoxycarbonyl-protected α-amino acid (NPCA) shown in formula (V) is added to the polymerization tube. A polyethylene glycol-derived amino initiator is added according to a predetermined feed ratio. A solvent is added to dissolve the monomer. The reaction mixture is stirred in a glove box at a temperature of 50–80°C. 1 NPCA conversion was monitored by ¹H NMR. When the NPCA conversion was >99%, the polymer was precipitated from excess cold diethyl ether; after drying in a vacuum drying oven, PEG was obtained. m -b-polypeptide products. Number-average molecular weight (Mn) was determined by GPC analysis. n ), and molecular weight distribution coefficient (M w / M n ).pass 1 H NMR spectroscopy, PEG m Degree of polymerization (DP) of the polypeptide block in a β-polypeptide copolymer.

[0062] In some embodiments of this disclosure, the chemical reaction for preparing the polypeptide of formula (III) from the monomer of formula (V) is shown in reaction formula (1), and the specific preparation steps are as follows: polymerization is carried out in an open fume hood, and the initiator is PEG. m -NH3 + Cl - (m=45), the solvent is DMAc, the concentration of the monomer N-phenoxycarbonyl-protected α-amino acid (NPCA) is 0.25M, and the reaction temperature is 70℃.

[0063] In some embodiments of this disclosure, the polypeptide PEG of formula (III) is prepared from the monomer of formula (V). 45 -b-PNBDab 80 The chemical reaction is shown in reaction formula (2).

[0064]

[0065] The specific preparation steps are as follows: In a glove box filled with nitrogen, 100.0 mg (0.240 mmol, 80 eq.) of N... α -Phenoxycarbonyl-Nγ-o-nitrobenzyloxycarbonyl-2,4-diaminobutyric acid NPCA (NBDab NPCA) was added to the polymerization tube, along with 6.58 mg (0.003 mmol, 1.0 eq.) of the initiator PEG. 45 -NH3 + Cl -Then, 958 μL of N,N-dimethylacetamide (DMAc) solvent was added, and the reaction mixture was placed in a glove box at 70°C and stirred. 1 The conversion rate of NBDab NPCA was monitored by ¹H NMR. When the conversion rate of NBDab NPCA was >99%, the polymer was precipitated from excess cold diethyl ether. After drying in a vacuum drying oven, 96.8 mg of the product PEG was obtained. 45 -b-PNBDab. The obtained product was characterized by gel permeation chromatography, 1H NMR spectroscopy, and infrared spectroscopy. The test results show... Figure 1A , Figure 1B and Figure 1C middle.

[0066] like Figure 1A As shown, analysis by gel permeation chromatography (GPC) revealed that M n It is 23.8 kDa, M w / M n It is 1.13.

[0067] like Figure 1B As shown, through 1 ¹H NMR spectroscopy analysis showed that the degree of polymerization (DP) of the PNBDab block in the PEG-b-PNBDab copolymer was 80, therefore the copolymer was designated as PEG. 45 -b-PNBDab 80 .

[0068] like Figure 1C As shown, characterization by infrared spectroscopy indicates that the PEG-b-PNBDab copolymer represents the presence of peptide bonds in the main chain of the polypeptide.

[0069] In some embodiments of this disclosure, the polypeptide PEG of formula (III) is prepared from the monomer of formula (V). 45 -b-PNBO 76 The chemical reaction is shown in reaction formula (3).

[0070]

[0071] The specific preparation steps are as follows: In a glove box filled with nitrogen, 100.0 mg (0.232 mmol, 80 eq.) of N... α -Phenoxycarbonyl-N δ -o-nitrobenzyloxycarbonyl-ornithine NPCA (NBO NPCA) was added to the polymerization tube, along with 6.37 mg (0.029 mmol, 1.0 eq.) of the initiator PEG. 45 -NH3 + Cl -Then, 927 μL of N,N-dimethylacetamide (DMAc) solvent was added, and the reaction mixture was placed in a glove box at 70°C and stirred. 1 The conversion rate of NBONPCA was monitored by ¹H NMR. When the conversion rate of NBONPCA was >99%, the polymer was precipitated from excess cold diethyl ether. After drying in a vacuum drying oven, 95.3 mg of the product PEG was obtained. 45 -b-PNBO. The obtained product was characterized by gel permeation chromatography and 1H NMR spectroscopy. The test results showed... Figure 2A and Figure 2B middle.

[0072] like Figure 2A As shown, analysis by gel permeation chromatography (GPC) revealed that M n It is 24.1 kDa, M w / M n It is 1.10.

[0073] like Figure 2B As shown, through 1 ¹H NMR spectroscopy analysis showed that the degree of polymerization of the PNBO block in the PEG-b-PNBO copolymer was 76, therefore the copolymer was designated as PEG. 45 -b-PNBO 76 .

[0074] In some embodiments of this disclosure, the polypeptide PEG of formula (IV) is prepared from the monomer of formula (VI). 45 -b-PBocDab 10 The chemical reaction is shown in reaction formula (4).

[0075]

[0076] The specific preparation steps are as follows: In a nitrogen-filled glove box, 100.0 mg (0.296 mmol, 10 eq.) of BocDab NPCA was added to a polymerization tube, along with 64.93 mg (0.0296 mmol, 1.0 eq.) of the initiator PEG. 45 -NH3 + Cl - Then, 1.18 mL of N,N-dimethylacetamide (DMAc) solvent was added, and the reaction mixture was placed in a glove box at 70°C and stirred. 1 The conversion rate of BocDab NPCA was monitored by ¹H NMR. When the conversion rate of BocDab NPCA was >99%, the polymer was precipitated from excess cold diethyl ether. After drying in a vacuum drying oven, 153.6 mg of the product PEG was obtained. 45-b-PBocDab. The obtained product was characterized by gel permeation chromatography and 1H NMR spectroscopy. The test results show... Figure 3A and Figure 3B middle.

[0077] like Figure 3A As shown, analysis by gel permeation chromatography (GPC) revealed that M n It is 4.1 kDa, M w / M n It is 1.05.

[0078] like Figure 3B As shown, through 1 ¹H NMR spectroscopy analysis showed that the degree of polymerization of the PBocDab block in the PEG-b-PBocDab copolymer was 10; therefore, this copolymer was designated as PEG. 45 -b-PBocDab 10 .

[0079] In some embodiments of this disclosure, the polypeptide PEG of formula (IV) is prepared from the monomer of formula (VI). 45 -b-PBocO 10 The chemical reaction is shown in reaction formula (5).

[0080]

[0081] The specific preparation steps are as follows: In a nitrogen-filled glove box, 100.0 mg (0.283 mmol, 10 eq.) of BocO NPCA was added to the polymerization tube, along with 62.35 mg (0.0283 mmol, 1.0 eq.) of the initiator PEG. 45 -NH3 + Cl - Then, 1.14 mL of N,N-dimethylacetamide (DMAc) solvent was added, and the reaction mixture was placed in a glove box at 70°C and stirred. 1 The conversion rate of BocO NPCA was monitored by ¹H NMR. When the conversion rate of BocO NPCA was >99%, the polymer was precipitated from excess cold diethyl ether. After drying in a vacuum drying oven, 114.2 mg of the product PEG was obtained. 45 -b-PBocO. The obtained product was characterized by gel permeation chromatography and 1H NMR spectroscopy. The test results show... Figure 4A and Figure 4B middle.

[0082] like Figure 4A As shown, analysis by gel permeation chromatography (GPC) revealed that M n It is 4.2kDa, M w / M n It is 1.06.

[0083] like Figure 4B As shown, through 1 ¹H NMR spectroscopy analysis showed that the degree of polymerization of the PBocO block in the PEG-b-PBocO copolymer was 10; therefore, this copolymer was designated as PEG. 45 -b-PBocO 10 .

[0084] In some embodiments of this disclosure, the preparation method for preparing a biodegradable model block polypeptide molecular chain by deprotecting the side tert-butyloxycarbonyl (Boc) group of the polypeptide represented by formula (IV) is as shown in the chemical reaction shown in reaction formula (6).

[0085]

[0086] The specific preparation steps are as follows: (1) Dissolve the polypeptide shown in formula (IV) in an organic solvent, wherein the organic solvent includes tetrahydrofuran, dioxane, methanol, and ethanol; (2) After adding acid, react for 1 to 24 hours at a reaction temperature of -20℃ to 100℃, wherein the acid includes various organic acids and inorganic acids, such as hydrochloric acid, trifluoroacetic acid, hexafluorophosphate, tetrafluoroboric acid, perchloric acid, formic acid, acetic acid, etc., preferably hydrochloric acid; (3) After the reaction is completed, concentrate the reaction mixture and add excess cold diethyl ether or petroleum ether for precipitation separation; (4) Dry under reduced pressure in a vacuum drying oven to obtain the product.

[0087] In some embodiments of this disclosure, the polypeptide PEG represented by m=44, x=1, n=10 in formula (IV) 45 -b-PBocDab 10 Deprotection of the side group tert-butoxycarbonyl (Boc) was used to prepare a biodegradable model polypeptide chain PEG. 45 -b-PDab 10 The preparation method is as shown in the chemical reaction formula (7).

[0088]

[0089] The specific preparation steps are as follows: (1) Take 100mg of the polypeptide PEG shown in formula (IV) when m=44, x=1, n=10. 45 -b-PBocDab 10(1) Dissolve in 1 mL of the organic solvent dioxane; (2) Add 2 mL of 4 M dioxane hydrochloride solution and react at room temperature (25°C) for 6 h; (3) Concentrate the mixture obtained from the reaction by half and add excess cold diethyl ether for precipitation separation; (4) Dry the obtained product under reduced pressure in a vacuum drying oven, and characterize the obtained product by 1H NMR spectrum. The test results show that... Figure 5 middle.

[0090] like Figure 5 As shown, through 1 H NMR spectroscopy confirmed the structure of the product to be PEG. 45 -b-PDab 10 .

[0091] In some embodiments of this disclosure, the polypeptide PEG represented by m=44, x=2, n=10 in formula (IV) 45 -b-PBocO 10 Deprotection of the side group tert-butoxycarbonyl (Boc) was used to prepare a biodegradable model polypeptide chain PEG. 45 -b-POrn 10 The preparation method is as shown in the chemical reaction formula (8).

[0092]

[0093] The specific preparation steps are as follows: (1) Take 100mg of the polypeptide PEG shown in formula (IV) when m=44, x=1, n=10. 45 -b-PBocO 10 (1) Dissolve in 1 mL of the organic solvent dioxane; (2) Add 2 mL of 4 M dioxane hydrochloride solution and react at room temperature (25°C) for 6 h; (3) Concentrate the mixture obtained from the reaction by half and add excess cold diethyl ether for precipitation separation; (4) Dry the obtained product under reduced pressure in a vacuum drying oven, and characterize the obtained product by 1H NMR spectrum. The test results show that... Figure 6 middle.

[0094] like Figure 6 As shown, through 1 H NMR spectroscopy confirmed the structure of the product to be PEG. 45 -b-POrn 10 .

[0095] According to embodiments of this disclosure, a nano-assembly obtained by the self-assembly of the above-mentioned polypeptide is prepared by dissolving the polypeptide represented by formula (III) or formula (IV) in an organic solvent to obtain an organic mixed solution; then adding water to stirred water by slow addition, direct dialysis or rapid addition, and finally dialysis to remove the organic solvent to obtain the nano-assembly.

[0096] According to embodiments of this disclosure, the nanoassemblies include: spherical vesicle structures, ellipsoidal vesicle structures, spherical micelle structures, and disk-shaped sheet assemblies.

[0097] In some embodiments of this disclosure, the nanostructures of the prepared PEG-b-PNDab and PEG-b-PNBO are regulated. Specifically, the obtained PEG-b-polypeptide is dissolved in an organic solvent, and then added to stirred water over a period of 1 to 10 seconds using direct dialysis, or by adding ultrapure water dropwise to the organic solution over a period of 0.5 to 5 hours using a flow injection pump. During this process, the solubility of the polypeptide blocks gradually decreases, causing them to collapse into a core and assemble. The hydrophilic PEG chain acts as a stabilizer on the periphery. By controlling the assembly conditions, particularly by changing the type of initial co-solvent, the method of water addition, and the rate of addition, various nanostructures with different morphologies can be obtained.

[0098] In some embodiments of this disclosure, the preparation steps of the nano-assemblies include: (1) dissolving the polypeptide in an organic solvent to obtain an organic mixed solution with a polypeptide concentration of 0.1 mg / mL to 1000 mg / mL, wherein the organic solvent includes at least one of tetrahydrofuran, DMSO, and DMF; (2) mixing the organic solvent and deionized water in two ways: the first way is to inject a first volume of the organic mixed solution into a second volume of water in a time of 1s to 10s, wherein the second volume is a multiple of the first volume of 0.5 to 1000 times; the second way is to add a fourth volume of deionized water into a third volume of the organic mixture through a syringe pump in a second time, wherein the second time is 0.5h to 5h, and the fourth volume is a multiple of the third volume of 0.5 to 1000 times; (3) dialysis to remove the organic solvent to obtain the nano-assemblies. When the organic solvent and deionized water are mixed in the first way, the polypeptide shown in formula (III) is used to prepare a nano-assembly with a diameter of 50-1000 nm spherical vesicle structure; the polypeptide shown in formula (IV) is used to prepare a nano-assembly with a diameter of 100-400 nm spherical micelle structure; when the organic solvent and deionized water are mixed in the second way, the polypeptide shown in formula (III) is used to prepare a nano-assembly with a diameter of 100-2000 nm disc-shaped sheet structure; and the polypeptide shown in formula (IV) is used to prepare a nano-assembly with a diameter of 1000-10000 nm ellipsoidal vesicle structure.

[0099] In some embodiments of this disclosure, PEG 45 -b-PNBDab 80The steps for preparing nanoassemblies from the polypeptide include: (1) dissolving 5 mg of polypeptide in 0.5 mL of organic solvent tetrahydrofuran to obtain an organic mixed solution with a polypeptide concentration of 10 mg / mL; (2) injecting the obtained organic mixed solution into 0.5 mL of water in 3 s at a time, and continuing to stir at 1500 rpm for 20 mins; (3) dialyzing the obtained polymer dispersion after stirring using a cellulose membrane with a molecular weight cutoff of MWCO = 3500 Da for 24 h, replacing the deionized water every 6 h; finally, adjusting the total volume of the dialysate to 5 mL with deionized water, and further diluting the dispersion to a specific concentration for later use; (4) characterizing the obtained product by transmission electron microscopy, scanning electron microscopy and atomic force microscopy, and the results are shown in the figure below. Figure 7A , Figure 7B and Figure 7C middle.

[0100] like Figure 7A part a in, such as Figure 7B part b in and such Figure 7C As shown in section c, the structure of the resulting assembly is a small vesicle structure.

[0101] In some embodiments of this disclosure, PEG 45 -b-PNBDab 80 The steps for preparing nanoassemblies from polypeptides include: (1) dissolving the polypeptide in the organic solvent tetrahydrofuran to obtain an organic mixed solution with a concentration of 0.1 mg / mL to 1000 mg / mL; (2) dialyzing the polymer solution directly using a cellulose membrane with a molecular weight cutoff of MWCO = 3500 Da for 24 h, replacing the deionized water every 6 h; finally, adjusting the total volume of the dialysate to 5 mL with deionized water, and further diluting the dispersion to a specific concentration for later use; (4) characterizing the obtained product by transmission electron microscopy, scanning electron microscopy and atomic force microscopy, and the results are shown in the figure below. Figure 7A , Figure 7B and Figure 7C middle.

[0102] like Figure 7A The d part, such as Figure 7B The e part and such Figure 7C As shown in part f, the structure of the resulting assembly is a large vesicle structure.

[0103] In some embodiments of this disclosure, PEG 45 -b-PNBDab 80The steps for preparing nanoassemblies from the polypeptide include: (1) dissolving 5 mg of polypeptide in 0.5 mL of organic solvent tetrahydrofuran to obtain an organic mixed solution with a polypeptide concentration of 10 mg / mL; (2) adding 0.5 mL of deionized water dropwise to 1 mL of organic mixed solution over 1 hour (1 mL / h) while stirring the organic mixed solution at 600 rpm; (3) dialyzing the polymer dispersion obtained after stirring with a cellulose membrane with a molecular weight cutoff of MWCO = 3500 Da for 24 hours, replacing the deionized water every 6 hours; finally, adjusting the total volume of the dialysate to 5 mL with deionized water, and further diluting the dispersion to a specific concentration for later use; (4) characterizing the obtained product by transmission electron microscopy, scanning electron microscopy and atomic force microscopy, and the results are shown in the figure below. Figure 7A , Figure 7B and Figure 7C middle.

[0104] like Figure 7A The g part, such as Figure 7B The h part and such Figure 7C As shown in part i, the structure of the resulting assembly is a disk-shaped sheet structure.

[0105] In some embodiments of this disclosure, PEG 45 -b-PNBO 76 The steps for preparing nanoassemblies from the polypeptide include: (1) dissolving 5 mg of polypeptide in 0.5 mL of organic solvent tetrahydrofuran to obtain an organic mixed solution with a polypeptide concentration of 10 mg / mL; (2) injecting the obtained organic mixed solution into 0.5 mL of water in 3 s at a time, and continuing to stir at 1500 rpm for 20 mins; (3) dialyzing the obtained polymer dispersion after stirring with a cellulose membrane with a molecular weight cutoff of MWCO = 3500 Da for 24 h, replacing the deionized water every 6 h; finally, adjusting the total volume of the dialysate to 5 mL with deionized water, and further diluting the dispersion to a specific concentration for later use; (4) characterizing the obtained product by transmission electron microscopy, scanning electron microscopy and atomic force microscopy, and the results are shown in the figure below. Figure 8A , Figure 8B and Figure 8C middle.

[0106] like Figure 8A part a in, such as Figure 8B The d part and such Figure 8C As shown in part g, the structure of the resulting assembly is a spherical micelle structure.

[0107] In some embodiments of this disclosure, PEG 45 -b-PNBO 76The steps for preparing nanoassemblies from the polypeptide include: (1) dissolving 5 mg of polypeptide in 0.5 mL of organic solvent dioxane to obtain an organic mixed solution with a polypeptide concentration of 10 mg / mL; (2) adding 0.5 mL of deionized water dropwise to 1 mL of the organic mixture over 1 hour (1 mL / h) while stirring the sample at 600 rpm; (3) dialyzing the polymer dispersion obtained after stirring with a cellulose membrane with a molecular weight cutoff of MWCO = 3500 Da for 24 hours, replacing the deionized water every 6 hours; finally, adjusting the total volume of the dialysate to 5 mL with deionized water, and further diluting the dispersion to a specific concentration for later use; (4) characterizing the obtained product by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy, and the results are shown in the figure below. Figure 8A , Figure 8B , Figure 8C middle.

[0108] like Figure 8A part b in Figure 8B part e in Figure 8C As shown in the h part, the structure of the resulting assembly is a spherical vesicle structure.

[0109] In some embodiments of this disclosure, PEG 45 -b-PNBO 76 The steps for preparing nanoassemblies from the polypeptide include: (1) dissolving 5 mg of polypeptide in 0.5 mL of organic solvent tetrahydrofuran to obtain an organic mixed solution with a polypeptide concentration of 10 mg / mL; (2) adding 0.5 mL of deionized water dropwise to 1 mL of the organic mixture over 1 hour (1 mL / h) while stirring the sample at 600 rpm; (3) dialyzing the polymer dispersion obtained after stirring with a cellulose membrane with a molecular weight cutoff of MWCO = 3500 Da for 24 hours, replacing the deionized water every 6 hours; finally, adjusting the total volume of the dialysate to 5 mL with deionized water, and further diluting the dispersion to a specific concentration for later use; (4) characterizing the obtained product by transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and confocal electron microscopy, and the results are shown in the figure below. Figure 8A , Figure 8B , Figure 8C and Figure 8D middle.

[0110] like Figure 8A part c in Figure 8B part f in Figure 8C The i and j parts and Figure 8D As shown, the structure of the resulting assembly is a disk-shaped layered structure.

[0111] According to embodiments of this disclosure, a method for preparing the nano-assemblies as described above includes: dissolving the above-described polypeptide in an organic solvent to obtain an organic mixed solution; and dialysis to remove the organic solvent to obtain the nano-assemblies.

[0112] In some embodiments of this disclosure, the preparation steps of the nanoassemblies include: (1) dissolving the polypeptide in the organic solvent tetrahydrofuran to obtain an organic mixed solution with a polypeptide concentration of 0.1 mg / mL to 1000 mg / mL; (2) dialyzing the polymer solution directly using a cellulose membrane with a molecular weight cutoff of MWCO = 3500 Da for 24 h, replacing the deionized water every 6 h; finally, adjusting the total volume of the dialysate to 5 mL with deionized water, and further diluting the dispersion to a specific concentration for later use; (4) characterizing the obtained product by transmission electron microscopy, scanning electron microscopy, atomic force microscopy and confocal electron microscopy, and the results are shown in Figure 9.

[0113] like Figure 9A , Figure 9B and Figure 9C As shown, the structure of the obtained assembly is a flattened spherical vesicle structure.

[0114] In some embodiments of this disclosure, the mechanism of cyclization degradation of the polypeptide backbone is as follows:

[0115]

[0116] The specific process is explained as follows: The side amino groups of the polypeptide backbone are first protected by o-nitrobenzyloxycarbonyl (oNB). After UV irradiation, the oNB groups break, exposing the naked amino groups, which then undergo cyclization and attack the amide bonds of the polypeptide backbone. Further, this process leads to the self-degradation of the polypeptide backbone. This is manifested in the gradual destruction and degradation of assemblies prepared from the amphiphilic polypeptides through self-assembly under neutral or alkaline conditions after light irradiation. With prolonged time, the vesicle surface rupture process can be directly observed, and its degree of degradation increases over time. The number of vesicles in the system gradually decreases, while the number of fragments increases, eventually leading to near-complete degradation. Therefore, the carriers constructed from polyethylene glycol-b-poly(o-nitrobenzyloxycarbonyl) protected by non-natural amino acids can undergo responsive self-degradation under external stimuli, and ultimately, complete degradation can occur. Therefore, this peptide material is expected to be used in many biological applications in the future, such as stimulus-responsive drug carriers and targeted delivery, and its self-degradation helps it to be self-cleared after it has performed its function.

[0117] In some embodiments of this disclosure, the cyclization degradation of polypeptide assemblies was studied. The experiments investigating the cyclization degradation of polypeptide assemblies used PEG. 45 -b-PNBDab 80 The resulting vesicle assembly (hydrodynamic radius, <R h Taking the degradation of PEG (~107nm) under pH=10.0 conditions as an example, the specific experimental operation steps are as follows: (1) Degradation of PEG (~107nm) under pH=10.0 conditions ... 45 -b-PNBDab 80 The assembled vesicle assembly was dispersed in 5 mL of 10 mM borax-sodium hydroxide buffer solution to obtain a dispersion of the assembly; wherein, the buffer solution contained 25 μL of 10 mg / mL aminourea hydrochloride stock solution as a scavenging agent for nitrosobenzaldehyde; (2) the above assembly dispersion was irradiated with 365 nm ultraviolet light at room temperature of 25 °C for 40 min to obtain a treated assembly dispersion; (2) the above treated assembly dispersion was cultured under light-protected conditions at 25 °C and samples were taken at specific time points for continuous tracking tests. The specific experimental operation steps include: First, TEM electron microscope samples are prepared for observation. After irradiation with 365 nm ultraviolet light for 40 min, 10 μL of the treated assembly dispersion is dropped onto a copper grid coated with Formvar and carbon support film to prepare a sample for TEM observation. Secondly, during the subsequent continuous light-protected culture at room temperature for different number of days, samples for TEM observation were prepared daily at predetermined time points (every 24 hours apart) by dropping 10 μL of the assembly dispersion after UV irradiation onto a copper grid coated with Formvar and a carbon support film. The transmission electron microscopy characterization results of the self-degradation of the vesicle assembly are shown in... Figure 10 Regarding the degradation study of the assembly under neutral conditions (pH=7.4), except for the "buffer solution being 10mM PB buffer", the experimental procedures were the same as those under "alkaline conditions".

[0118] like Figure 10 As shown, the assemblies prepared by the self-assembly of polypeptides, after being exposed to light and further cultured under alkaline conditions, gradually break down and degrade. Furthermore, with prolonged exposure, the vesicle surface rupture process and its increasing severity can be directly observed, with the number of vesicles gradually decreasing and the number of fragments increasing, eventually leading to near-complete degradation.

[0119] In some embodiments of this disclosure, the mechanism of cyclization degradation of polypeptide cyclic molecular chains was investigated. The degradation was tracked using gel permeation chromatography (GPC) with PEG as an example. 45 -b-POrn 10Taking the degradation of PEG under pH=7.4 as an example, the specific experimental steps are as follows: (1) Take 20 mg of PEG as an example. 45 -b-POrn 10 (0.00588 mmol) was dissolved in 4 mL of 10 mM PB buffer and incubated in an incubator at 37 °C; (2) 0.4 mL of sample was taken at different time points and hydrochloric acid was added to adjust the pH to 3; (3) 5 mL of toluene solution was added and the mixture was azeotropically dried under reduced pressure several times to ensure complete dehydration; (4) The degraded mixture was sampled and dissolved in 300 μL of DMF, and 1.92 mg of Et3N (0.0188 mmol, 4 eq.) and 1.612 mg of benzyl chloroformate (0.0096 mmol, 2 eq.) were added; (5) After stirring at room temperature for 5 h, the reaction mixture was azeotropically dried and the DMF GPC was tested to monitor the change in molecular weight of the main peak of the molecular chain. For the initial control group that has not yet been degraded, the experimental steps are as follows: (1) 5 mg of the initial de-Boc polymer PEG was added. 45 -b-POrn 10 Dissolved in 400 μL DMF, 1.92 mg of Et3N (0.0188 mmol, 4 eq.) and 1.612 mg of benzyl chloroformate (0.0096 mmol, 2 eq.) were added, and the mixture was reacted at room temperature for 5 h to obtain the reaction mixture; (2) After the reaction mixture was azeotropically dried, GPC was tested, and the molecular weight change of the main peak of the molecular chain was monitored. The test results are shown in Figure 11A Before and after degradation, 100 μL of the aqueous phase sample of the above model molecular chain was taken and directly used for HRESI MS analysis to determine the presence or absence of cyclized products. The characterization spectrum of the polypeptide's cyclization and self-degradation at the molecular chain level is shown in... Figure 11B Similarly, studies on degradation under alkaline conditions (pH = 10.0) were conducted using a similar procedure in a 10 mM borax-sodium hydroxide buffer solution.

[0120] like Figure 11BAs shown, a typical characteristic of PEG in HRESI MS is an adjacent mass spectrum peak (m / z) spacing of 11, corresponding to signal peaks with 4 charges (z). No residual high-molecular-weight peptides or other high-molecular-weight signals are visible in the HRESI MS spectrum, indicating that the peptides have undergone significant degradation. Under both neutral and alkaline conditions, the highest abundance of cyclic small molecule signals can be clearly found in the low molecular weight region: a five-membered ring with m / z = 100.06, originating from the 2,4-diaminobutyric acid (Dab) system, and a six-membered ring with m / z = 114.08, originating from the ornithine system. Whether in the assembled or degraded model molecular chain HRESI MS spectra, cyclic small molecule signals are the most abundant substances, while only residual PEG signals (m / z = 11, z = 4) are present in the high molecular weight region. The polypeptide backbone is ultimately completely converted into the remaining polyethylene glycol (PEG) and two highly abundant small cyclic products (five-membered and six-membered rings).

[0121] pass Figure 11A Gel permeation chromatography (GPC) and Figure 11B High-resolution electrospray ionization mass spectrometry (HRESI MS) characterization analysis can fully verify that the polypeptide molecular chain with exposed amino groups after deprotection of the side groups of the polypeptide has a clean and complete degradation process.

[0122] The photoresponsive polypeptides prepared in this disclosure undergo oNB group cleavage under ultraviolet light (e.g., 365 nm) irradiation, exposing amino side groups on the polypeptide backbone. This is followed by cyclization degradation through amino-amide bond attack, and the assemblies based on these polypeptides also degrade simultaneously. Furthermore, mass spectrometry verification based on molecular chain model degradation experiments further confirms this cyclization degradation process. Following further photolysis, the backbone can spontaneously degrade into small cyclic products, thus representing a hydrolysis-independent triggered active degradation process. This disclosure studies responsive degradation at the assembly level and provides evidence for cyclization degradation at the molecular chain level.

[0123] This disclosure proposes a novel theory based on the cyclization degradation of polypeptide backbones. Upon exposure to ultraviolet light (or other types of side-chain protecting groups and their corresponding deprotection), the side chains of the polypeptide backbone expose amino groups, leading to amino cyclization and attack on the amide bonds, resulting in the self-degradation of the backbone. This process was fully validated using high-resolution electrospray ionization mass spectrometry (HRESI MS).

[0124] This disclosure utilizes a solution-based molecular self-assembly control technique to prepare various uniform and stable hierarchical nanostructures of amphiphilic block polypeptides without the presence of any additives or templates. Considering the significant influence of the nanostructure on the function of polypeptide materials, the self-assembly of responsive polypeptides with various morphologies was further achieved. Amphiphilic photoresponsive polypeptides can self-assemble into different morphologies in aqueous solution depending on the assembly method. Typical morphologies in this invention mainly include: spherical micelles, disc-shaped sheets, spherical vesicles, and ellipsoidal vesicles. These assemblies possess very interesting nanostructures, showing excellent prospects for biological applications. These nanostructures can all serve as potential delivery carriers for drugs, nucleic acids, and various reagents for biological or chemical applications. The structures of the above-mentioned assemblies are very stable and can remain unchanged for several years. Furthermore, the prepared disc-shaped sheet structures have uniform and regular morphological dimensions, and are expected to become two-dimensional materials.

[0125] This disclosure constructs a phototriggered responsive peptide material system, based on the previously established NPCA polymerization methodology, through the macromolecular PEG-NH3... + Cl - The active-controlled polymerization of NBO NPCA and NBDab NPCA was initiated, introducing the oNB motif onto the side chain of the polypeptide, synthesizing a polypeptide based on ornithine and 2,4-diaminobutyric acid with the photoresponsive oNB group protected on the side chain. Then, considering the significant influence of the nanostructure of the polypeptide material on its function, this disclosure further achieves the controlled self-assembly of photoresponsive amphiphilic block polypeptides with various morphologies. The amphiphilic block polypeptides can form different morphologies of nanostructures depending on the assembly conditions, including spherical micelles, disc-shaped sheets, large / small spherical vesicles, and ellipsoidal vesicles. The controlled self-assembly process of the ellipsoidal vesicles was monitored in situ using confocal laser scanning microscopy (CLSM). This disclosure designs and synthesizes a polypeptide with a multi-level self-assembly structure and cyclization-induced main chain self-degradation.

[0126] The specific embodiments described above further illustrate the purpose, technical solutions, and beneficial effects of this disclosure. It should be understood that the above descriptions are merely specific embodiments of this disclosure and are not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A poly-polypeptide of formula (III) or (IV) : wherein m is 1-20000; n is 1-1000, representing the degree of polymerization of the amino acid monomers in the poly-polypeptide backbone; x is 1-2; wherein when x = 1, the poly-polypeptide backbone corresponds to 2, 4-diaminobutyric acid, and when x = 2, the poly-polypeptide backbone corresponds to ornithine. Equation (III); Formula (IV); wherein 2.The poly-polypeptide of claim 1, wherein m is selected from any one of 22, 44, and 112. 3.A method for preparing the poly-polypeptide of claim 1, comprising: mixing a monomer and an initiator, and then adding a solvent to dissolve; initiating polymerization of the monomer under heating; wherein the molar ratio of the monomer to the initiator is 1:1-1000:1, the concentration of the monomer is 0.05-2M, the initiator is a compound of formula (VIII), the solvent is any one of N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, and dimethyl sulfoxide, and the monomer is a compound of formula (V) or (VI) ; precipitating and separating the reaction mixture obtained after polymerization of the monomer by adding cold diethyl ether, or adding excess cold diethyl ether or petroleum ether to the concentrated solution of the reaction mixture, and then precipitating and separating, and drying the obtained polymer under reduced pressure or vacuum. The nano-assembly is self-assembled from the poly-polypeptide of claim 1. The nano-assembly comprises a spherical vesicular structure, an ellipsoidal vesicular structure, a spherical micellar structure, and a discoidal lamellar assembly. 7.A method for preparing the nano-assembly of claim 5, comprising: dissolving the poly-polypeptide of claim 1 in an organic solvent to obtain an organic mixed solution; and removing the organic solvent by dialysis to obtain the nano-assembly. ​ ​ ​ ​ ​ Formula (V); Formula (VI); Formula (VIII).

4. The method of claim 3, further comprising: ​ 5. A nanoassembly, characterized in that, ​ 6. The nanoassembly of claim 5, wherein, ​ ​ ​ ​

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