A brush-like active polypeptide skin delivery system, its preparation method and application

Abstract

The application discloses a brush-like active polypeptide skin delivery system, a preparation method and application thereof. The system comprises a multi-functional polymer main chain and a plurality of active polypeptide side chains covalently grafted on the main chain; the main chain contains a plurality of reactive functional groups before the active polypeptide is grafted, and the active polypeptide is covalently coupled and grafted on the main chain to form a brush-like or bottle brush-like high molecular coupling structure. The main chain is selected from a polyamino acid main chain, a carbon-carbon polymer main chain or a polydisulfide main chain containing a sulfuric acid structure unit. The brush-like structure of the application can improve the delivery and stability of the active polypeptide in the skin and skin appendages, and is suitable for a variety of external use compositions.

Description

Technical Field

[0001] This invention belongs to the fields of polymer materials, biomaterials and skin delivery technology, specifically relating to a brush-like active polypeptide skin delivery system, its preparation method and application. Background Technology

[0002] Active peptides have significant application value in fields such as skin care, scalp care, barrier repair, anti-inflammation, antibacterial, tissue repair, and medical topical delivery due to their well-defined biological functions, strong structural designability, and ability to achieve different biological effects through sequence regulation.

[0003] For example, some peptides can participate in cell migration, extracellular matrix formation, angiogenesis-related signaling, barrier regulation, or intracellular function regulation, and therefore can be used as topical active ingredients or research models. In scalp and hair follicle care scenarios, some active peptides can also be used to regulate hair follicle-related signals, scalp microenvironment, sebum secretion, or oil-related states, thus having potential application value in topical products such as hair care, scalp care, oil control care, or post-cleansing care.

[0004] However, active peptides still face significant limitations in topical delivery. On the one hand, peptides typically have strong hydrophilicity, high molecular weight, or complex charge distribution, making it difficult to effectively penetrate barriers such as the stratum corneum, scalp, and hair follicles. On the other hand, peptides are susceptible to factors such as enzymatic degradation, conformational changes, aggregation, or adsorption, leading to decreased formulation stability, storage stability, and bioavailability.

[0005] Existing peptide delivery systems primarily focus on drug encapsulation or self-assembly into nanoparticles, but they still fall short in improving the delivery efficiency of active peptides to the skin and skin appendages, enzyme stability, and stability in complex topical environments. For topical products, delivery systems may also be affected by surfactants, ions, proteases, and other complex environmental factors.

[0006] For example, invention application CN122320815A discloses a polypeptide composition-hydrogel composite delivery system for regulating the stability of the skin's lipid membrane, its preparation method, and its application. The composite delivery system includes a polypeptide composition, a hydrogel matrix, a permeation enhancer, and deionized water. The polypeptide composition consists of an antimicrobial polypeptide, a prebiotic polypeptide, and an immunomodulatory polypeptide. The preparation method includes: dissolving the antimicrobial polypeptide, prebiotic polypeptide, and immunomodulatory polypeptide in phosphate buffer to obtain a polypeptide composition solution; dispersing the hydrogel matrix in deionized water, stirring to swell, adding the permeation enhancer, mixing, adding the polypeptide composition solution dropwise, adjusting the pH value, vacuum degassing, and freeze-drying to obtain the final product.

[0007] The invention application with publication number CN120267790A discloses an anti-aging topical compound polypeptide oil-encapsulated nanosuspension and its preparation method. The compound polypeptide oil-encapsulated nanosuspension includes a water-soluble polypeptide drug, a surfactant, and an oil phase matrix. The water-soluble polypeptide drug is encapsulated by the surfactant, freeze-dried, and then uniformly dispersed in the oil phase matrix to form the compound polypeptide oil-encapsulated nanosuspension.

[0008] Therefore, there is an urgent need to develop a polymer delivery platform that is structurally stable, can improve the delivery efficiency of active peptides in the skin and skin appendages, and is suitable for a variety of external application environments. Summary of the Invention

[0009] To address the problems of low skin delivery efficiency of active peptides, insufficient enzyme stability, and limited stability in complex topical environments, this invention provides a brush-like active peptide skin delivery system, its preparation method, and its application.

[0010] The present invention first provides a brush-like active peptide skin delivery system, comprising a multifunctional polymer backbone and a plurality of active peptide side chains covalently grafted onto the multifunctional polymer backbone; The multifunctional polymer backbone contains multiple reactive functional groups that can covalently couple with the active peptide or modified active peptide before grafting with the active peptide. The active peptide side chain is grafted onto the multifunctional polymer backbone through the covalent coupling reaction involving the reactive functional groups to form a brush-like or bottle-brush-like polymer coupling structure. The multifunctional polymer backbone is selected from one or more of the following: polyamino acid backbone, carbon-carbon polymer backbone, and polydisulfide backbone containing thiooctanoic acid structural units.

[0011] The reactive functional groups can be inherent in the multifunctional polymer backbone itself or introduced through chemical modification; the active polypeptide can also have complementary reactive groups that can covalently couple with the reactive functional groups introduced at the N-terminus, C-terminus, amino acid side chains, non-natural amino acid residues or linkers.

[0012] Preferably, the polyamino acid backbone is selected from one or more of polyglutamic acid, polyaspartic acid, polylysine, polyornithine, glutamic acid-lysine copolymer, glutamic acid-aspartic acid copolymer or their derivatives.

[0013] The polyamino acid backbone contains repeating units as shown in Formula I before grafting with the active polypeptide: Formula I, In formula I, m is a natural number between 2 and 10000. R may be the same or different in each repeating unit, and R is selected from one or more amino acid side chains that are not modified or contain reactive functional groups.

[0014] The carbon-carbon polymer backbone, before grafting with the active polypeptide, comprises a carbon-carbon backbone formed by polymerization of one or more vinyl monomers and multiple side chain functional groups, at least some of which are reactive functional groups capable of covalently coupling with the active polypeptide or modified active polypeptide.

[0015] Preferably, the carbon-carbon polymer backbone is selected from copolymers of polyacrylic acid, polymethacrylic acid, acrylic acid and / or methacrylic acid with one or more vinyl monomers, or one or more derivatives thereof.

[0016] The vinyl monomer is selected from one or more of acrylate monomers, methacrylate monomers, acrylamide monomers, methacrylamide monomers, styrene monomers, vinylpyrrolidone monomers, vinyl ether monomers, vinyl ester monomers, or their derivatives.

[0017] More preferably, the vinyl monomer is selected from one or more of the following: benzyl acrylate, benzyl methacrylate, alkyl acrylate, alkyl methacrylate, hydroxyalkyl acrylate, hydroxyalkyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, isobornyl acrylate, isobornyl methacrylate, 2,2,2-trifluoroethyl acrylate, 2,2,2-trifluoroethyl methacrylate, N-isopropylacrylamide, N,N-dimethylacrylamide, N-hydroxyethylacrylamide, vinylpyrrolidone, styrene, hydroxystyrene, sulfonated styrene, carboxystyrene, or their derivatives.

[0018] The polydisulfide backbone of the thioctic acid structural unit includes a polydisulfide backbone formed from thioctic acid or its derivatives, or the polydisulfide backbone of the thioctic acid structural unit includes a polydisulfide copolymer backbone formed by copolymerization of thioctic acid or its derivatives with vinyl monomers.

[0019] Preferably, the polydisulfide backbone of the thioctic acid-containing structural unit contains the structural unit shown in Formula II before grafting with the active polypeptide: Formula II, In formula II, a is a natural number between 1 and 10000. b is 0 or a natural number between 1 and 10000. R5 is hydrogen or methyl. R6 is a side group in the lipoic acid structural unit, selected from one or more reactive functional groups containing hydroxyl, alkoxy, amino, substituted amino, carboxyl, active ester, azide, alkynyl, cycloalkynyl, maleimide, mercapto, tetrazinyl, or transcyclooctenyl. Q is a side group retained in the repeating structural unit of the polymer after the polymerization of vinyl monomers, selected from one or more of alkyl, aryl, carboxyl, ester, amide, hydroxyl, amino, or polyether groups.

[0020] When b=0, the polydisulfide backbone of the thioctic acid structural unit is a polythioctic acid backbone, a polydisulfide backbone, or a derivative thereof formed from thioctic acid or its derivatives. When b>0, the polydisulfide backbone of the thioctic acid structural unit is a polydisulfide copolymer backbone formed by copolymerizing thioctic acid or its derivatives with vinyl monomers.

[0021] Preferably, the polydisulfide backbone of the thiooctanoic acid structural unit has free radical scavenging and / or antioxidant properties.

[0022] In each polymer backbone, the number of repeating polymer units can be selected according to the actual needs of the appropriate degree of polymerization, such as m in Formula I and a in Formula II. Generally, the degree of polymerization can be selected not to exceed 10,000, preferably 2-200, and more preferably 70-120.

[0023] In this invention, the chain-end structure of the multifunctional polymer backbone does not constitute a necessary limitation for forming the brush-like or bottle-brush-like polymer coupling structure. One or both chain ends of the multifunctional polymer backbone may have corresponding end groups or residues depending on the polymerization method and the initiator, chain transfer agent, capping agent, terminator, or end-group modifier used. The end groups or residues may include one or more of alkyl, alkoxy, acyl, hydroxyl, carboxyl, amino, mercapto, polyether, thiocarbonyl, trithiocarbonate, azide, or derivatives thereof, and may also include other residues formed by the initiator, chain transfer agent, capping agent, terminator, or end-group modifier. Unless otherwise stated, the specific chain-end structures shown in the structural formulas or drawings of this specification are exemplary chain-end structures in the corresponding embodiments and do not constitute a necessary limitation on the chain-end structure of the multifunctional polymer backbone.

[0024] Preferably, the reactive functional groups of the multifunctional polymer backbone before grafting with the active polypeptide are selected from one or more of the following: azide group, alkynyl group, cycloalkynyl group, dibenzocyclooctyne group, maleimide group, mercapto group, amino group, carboxyl group, hydroxyl group, aldehyde group, ketone group, hydrazine group, hydroxylamine group, tetraazine group, transcyclooctenyl group, norbornene group, active ester group, N-hydroxysuccinimide ester group, isocyanate group, isothiocyanate group, or anhydride group.

[0025] Preferably, the active polypeptide side chain is grafted onto the multifunctional polymer backbone via one or more of the following reactions: click reaction, cycloaddition reaction, amidation reaction, active ester-amine reaction, carboxyl-amine condensation reaction, mercapto-maleimide reaction, mercapto-ene reaction, oxime bond formation reaction, hydrazone bond formation reaction, esterification reaction, carbonate reaction, disulfide bond exchange reaction, or isocyanate-amine reaction.

[0026] More preferably, the active polypeptide side chain is grafted onto the multifunctional polymer backbone via an azide-alkynyl click reaction, a copper-free azide-cycloalkynyl click reaction, a BCN-azide click reaction, a DBCO-azide click reaction, a tetrazine-trans-cyclooctene reaction, a CuAAC reaction, a mercapto-maleimide reaction, or an active ester-amine reaction.

[0027] The grafting ratio of the active polypeptide to the reactive sites on the multifunctional polymer backbone before grafting can be 0.1%-100%, for example, 1%-99%, 5%-95%, 5%-90%, 5%-80%, 10%-80%, etc. The grafting ratio refers to the proportion of the number of sites on the multifunctional polymer backbone that have been grafted with the active polypeptide to the total number of reactive sites available for grafting the active polypeptide before grafting.

[0028] The grafting ratio refers to the proportion of the number of sites in the multifunctional polymer backbone that have been grafted with active peptides to the total number of reactive sites that can be used for grafting active peptides before grafting.

[0029] Preferably, the active polypeptide is a polypeptide with biological activities related to the regulation of skin, scalp, hair follicles, skin appendages or local tissues. The biological activities of the active polypeptide include one or more of the following: skin care, scalp care, hair follicle regulation, tissue repair, anti-inflammation, antibacterial, antioxidant or cell regulation.

[0030] More preferably, the active polypeptide includes one or more of QK, KLAK, or LTX-315.

[0031] Further preferred, the amino acid sequence of the active peptide KLAK is CKALKLAKKLAKLAK; the amino acid sequence of the active peptide QK is CKLTWQELYQLKYKGI; and the amino acid sequence of the active peptide LTX-315 is CKKWWKKWDipK-NH2, wherein Dip represents the non-natural amino acid residue 3,3-diphenylalanine.

[0032] The present invention also provides a method for preparing the brush-like active peptide skin delivery system, comprising the following steps: reacting the multifunctional polymer backbone with an active peptide or a modified active peptide in a solvent system, wherein the multifunctional polymer backbone contains a plurality of reactive functional groups that can covalently couple with the active peptide or the modified active peptide before grafting with the active peptide, and the active peptide side chain is grafted onto the multifunctional polymer backbone through the covalent coupling reaction involving the reactive functional groups to form a brush-like or bottle-brush-like polymer coupling structure.

[0033] In some embodiments, when the covalent coupling reaction is a click reaction or a cycloaddition reaction, the reaction temperature is 0-100 °C and the reaction time is 0.5-96 h.

[0034] In some embodiments, when the covalent coupling reaction is an amidation reaction or an active ester-amine reaction, the reaction temperature is 0-60 °C and the reaction time is 0.5-72 h.

[0035] The solvent system may include one or more of water, buffer solution, dimethyl sulfoxide, N,N-dimethylformamide, ethanol, methanol, acetonitrile, or tetrahydrofuran.

[0036] In some embodiments, the active polypeptide is first chemically modified to introduce reactive groups that match the reactive functional groups on the multifunctional polymer backbone, and then covalently coupled to the multifunctional polymer backbone.

[0037] More preferably, when the multifunctional polymer backbone contains an azide group, the active polypeptide is grafted onto the multifunctional polymer backbone via a strain-promoted azide-cycloalkyne click reaction after being modified with a cycloalkyne group. Further preferably, the cycloalkyne group is BCN (bicyclo[6.1.0]nonyne) or DBCO (dibenzocyclooctyne).

[0038] The present invention also provides a topical skin composition comprising the aforementioned brush-like active peptide skin delivery system and a topically acceptable matrix.

[0039] The topical acceptable matrix can be selected according to the specific application scenario of skin, scalp, hair or skin appendages, and may further include one or more of surfactants, moisturizers, thickeners, emulsifiers, solvents, preservatives, pH adjusters or other excipients acceptable in topical preparations.

[0040] In some embodiments, the topical skin composition further includes a surfactant.

[0041] The surfactant is selected from one or more of anionic surfactants, amphoteric surfactants, nonionic surfactants, cationic surfactants, or amino acid surfactants.

[0042] Preferably, the surfactant accounts for 0.5%-10% (w / v) of the total amount of the topical skin composition; more preferably, it accounts for 6%-7% (w / v). When multiple surfactants are used in combination, each surfactant may account for 1%-4% (w / v) of the total amount of the topical skin composition.

[0043] More preferably, the surfactant is selected from one or more of sodium dodecyl sulfate, sodium lauryl ether sulfate, cocamidopropyl betaine, sodium cocoyl glutamate, sodium lauroyl sarcosinate, sodium lauroyl glutamate, decyl glucoside, lauryl glucoside, polysorbate, or fatty alcohol polyether.

[0044] In some embodiments, the brush-like active peptide skin delivery system maintains at least one of its diffusion behavior, apparent hydration size, or structural integrity in a system containing the surfactant.

[0045] This invention also provides the application of the brush-like active peptide skin delivery system in the preparation of topical skin products. The topical skin products are cosmetics, personal care products, topical skin formulations, or locally administered formulations.

[0046] In some embodiments, the topical skin product is selected from shampoos, scalp care solutions, scalp serums, conditioners, hair masks, shower gels, facial cleansers, serums, or skin lotions.

[0047] In some embodiments, the dosage form of the topical skin product is selected from gels, creams, ointments, sprays, or patches.

[0048] This invention offers the following advantages: First, by covalently grafting multiple active polypeptide side chains onto a multifunctional polymer backbone to form a brush-like or bottle-brush-like polymer coupling structure, this invention can improve the delivery, accumulation, or local retention of active polypeptides in the skin, scalp, hair follicles, or other skin appendages. Second, the covalent brush structure can enhance the enzymatic stability and cellular uptake capacity of active polypeptides. Third, the covalent brush structure can improve the stability of the active polypeptide delivery system in surfactant-containing systems, thus facilitating its application in complex topical formulations such as shampoos and facial cleansers. Fourth, the chemical composition, degree of polymerization, type of active polypeptide, and grafting ratio of the multifunctional polymer backbone are all adjustable, allowing for structural design based on the delivery requirements of different skin, scalp, hair follicles, and other skin appendages. Fifth, when the multifunctional polymer backbone is a polydisulfide backbone containing thioctic acid structural units, it can also provide additional functions such as free radical scavenging and / or antioxidant effects. Attached Figure Description

[0049] Figure 1 The reaction process for preparing azide-modified polyglutamic acid backbone.

[0050] Figure 2 The reaction process for preparing the copolymer backbone of polythiooctanoic acid-vinyl monomer modified with azide.

[0051] Figure 3 The reaction process for preparing azide-modified polythiooctanoic acid backbone.

[0052] Figure 4 The reaction process for preparing azide-modified polyacrylic acid backbone.

[0053] Figure 5 The 1H NMR spectrum of the azide-modified polyglutamic acid backbone.

[0054] Figure 6 The 1H NMR spectrum of the azido-modified thioctic acid-benzyl acrylate copolymer backbone.

[0055] Figure 7 The 1H NMR spectrum of the azido-modified polythiooctanoic acid backbone.

[0056] Figure 8 The 1H NMR spectrum of the azide-modified polyacrylic acid backbone.

[0057] Figure 9 The results are shown in the gel permeation chromatography characterization of the backbones of four types of multifunctional polymers. Figure 9 In this context, A represents the azide-modified polyglutamic acid backbone. Figure 9 B in the figure represents the azido-modified thioctic acid-benzyl acrylate copolymer backbone; Figure 9 C in the figure represents the azide-modified polythiooctanoic acid backbone; Figure 9 D in the figure represents the azide-modified polyacrylic acid backbone.

[0058] Figure 10 The reaction process for the preparation of BCN-maleimide is described.

[0059] Figure 11 The 1H NMR spectrum is for BCN-maleimide.

[0060] Figure 12 The matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) characterization results of KLAK, LTX-315, and QK before and after BCN modification are presented. Figure 12 In this context, A represents unmodified KLAK; Figure 12 In this context, B stands for unmodified LTX-315; Figure 12 In this context, C represents unmodified QK; Figure 12 In this context, D represents BCN modifying KLAK; Figure 12In this context, E stands for BCN-modified LTX-315; Figure 12 In this context, F represents BCN modifying QK.

[0061] Figure 13 The results are shown in the polyacrylamide gel electrophoresis characterization of the brush-like active peptide skin delivery system.

[0062] Figure 14 The results show the particle size and zeta potential characterization of three brush-like active peptide skin delivery systems. Figure 14 In the figure, A represents the particle size distribution of the LTX-315 brush system based on the azido-modified polyglutamic acid backbone, the QK brush system based on the azido-modified thioctic acid-benzyl acrylate copolymer backbone, and the KLAK brush system based on the azido-modified polythioctic acid backbone. Figure 14 In the figure, B represents the zeta potential of the corresponding system. Data are expressed as mean ± standard deviation, n=3.

[0063] Figure 15 The results show the stability evaluation of three brush-like active peptide skin delivery systems in surfactant-containing systems. Figure 15 In this context, A represents the KLAK brush-like active peptide skin delivery system based on an azide-modified polythiooctanoic acid backbone; Figure 15 In this context, B represents the LTX-315 brush-like active peptide skin delivery system based on an azide-modified polyglutamic acid backbone. Figure 15 In this context, C represents the QK brush-like active peptide skin delivery system based on an azide-modified thioctic acid-benzyl acrylate copolymer backbone. Data are expressed as mean ± standard deviation, with n = 5 independent samples. One-way ANOVA was used for comparisons among multiple groups, followed by Tukey's post-hoc multiple comparison test; ns indicates no statistically significant difference.

[0064] Figure 16 The results show the delivery and hair follicle enrichment of the QK brush-like active peptide skin delivery system in the dorsal skin of live BALB / c mice.

[0065] Figure 17 Results of cellular uptake by the QK brush-like active peptide skin delivery system. Figure 17 In the image, A represents a representative confocal laser scanning microscope image of cells after treatment with Cy5-labeled free QK and a QK brush-like active peptide skin delivery system; Figure 17 B in the figure represents the representative flow cytometry histogram and the quantitative results of the average fluorescence intensity of Cy5 cells.

[0066] Figure 18 The results show the enzyme stability evaluation of the QK brush-skin delivery system. Free QK fell below the detection limit after 15 min, therefore the 240-min lane was not shown.

[0067] Figure 19Evaluation results of the ABTS radical cation scavenging ability of the azido-modified thioctic acid-benzyl acrylate copolymer backbone. Figure 19 In the figure, A represents the ABTS radical cation scavenging kinetics curves of the alpha-lipoic acid-benzyl acrylate copolymer backbone with different concentrations of azide modification within 0-240 min. Figure 19 B in the image shows representative well plate images at the start of the reaction and after 240 min of incubation. The final sample concentrations were 1, 2, 4, and 8 mg / mL, with three parallel wells in each group. Detailed Implementation

[0068] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Those skilled in the art, without departing from the concept of the present invention, may make equivalent substitutions or conventional adjustments to the main chain type, reactive groups, linkage methods, polypeptide sequences, surfactant types, and external matrices, all of which should fall within the scope of protection of the present invention.

[0069] Example 1: Preparation of a multifunctional polymer backbone This embodiment prepares an azide-modified multifunctional polymer backbone for constructing a brush-like active peptide skin delivery system. The backbone includes an azide-modified polyglutamic acid backbone, an azide-modified polythiooctanoic acid-vinyl monomer copolymer backbone, an azide-modified polythiooctanoic acid backbone, and an azide-modified polyacrylic acid backbone.

[0070] I. Preparation of azide-modified polyglutamic acid backbone Specific preparation methods are as follows: Figure 1 As shown. The following is a step-by-step description: Step 1: Preparation of polyglutamic acid benzyl ester macromolecular initiator γ-Benzyl-L-glutamic acid N-carboxycyclic anhydride (compounds 1-2, BLG-NCA, 600 mg, 2.28 mmol) was dissolved in anhydrous N,N-dimethylformamide (DMF, 10 mL), and amino-PEG12-methoxy (compound 1-1, 40 μL, 0.076 mmol) was added as an initiator. The reaction was stirred at 4 °C for 3 days. After the reaction was completed, the reaction solution was concentrated and precipitated in 40 mL of ice-cold diethyl ether, and the precipitation was repeated 3 times. The resulting white precipitate was dried under vacuum to obtain 308.5 mg of polyglutamic acid benzyl ester macromolecular initiator (compounds 1-3), with a yield of 58%. Based on the molar ratio of BLG-NCA to amino-terminated polyethylene glycol (amino-PEG12-methoxy), the target degree of polymerization of the polyglutamic acid benzyl ester segment in the obtained macromolecular initiator was approximately 30. When the degree of polymerization of the polyglutamic acid backbone to be used is less than 30, the target backbone can be obtained by directly controlling the anhydride within the amino-PEG12-methoxy and γ-benzyl-L-glutamic acid N-carboxyl rings, skipping step 2 and proceeding directly to step 3.

[0071] Step 2: Preparation of polyglutamic acid backbone 131.6 mg (0.5 mmol) of γ-benzyl-L-glutamic acid N-carboxyl ring anhydride (compounds 1-2) and 5.28 mg (0.02 mmol) of 18-crown ether-6 were dissolved in 9 mL of dichloromethane (DCM) to obtain a solution of γ-benzyl-L-glutamic acid N-carboxyl ring anhydride. Separately, 70 mg (approximately 0.01 mmol) of the aforementioned macromolecular initiator (compounds 1-3) was dissolved in 1 mL of dichloromethane and rapidly added to the γ-benzyl-L-glutamic acid N-carboxyl ring anhydride solution. The reaction system was stirred at room temperature for 2 h. Subsequently, 50 μL of pentanoic anhydride (compounds 1-4) and 10 μL of N,N-diisopropylethylamine (DIPEA) were added, and the mixture was stirred overnight at room temperature to block the terminal amino groups of the polymer. The reaction solution was precipitated with ice-cold diethyl ether and dried under vacuum to obtain end-blocked poly(γ-benzyl-L-glutamic acid) (compounds 1-5). The degree of polymerization of poly(γ-benzyl-L-glutamic acid) can be freely controlled by adjusting the ratio of polybenzyl glutamic acid ester to the anhydride within the N-carboxyl ring of γ-benzyl-L-glutamic acid in the macromolecular initiator.

[0072] 103.6 mg of poly(γ-benzyl-L-glutamic acid) (compounds 1-5) was dissolved in 1.24 mL of tetrahydrofuran to obtain a poly(γ-benzyl-L-glutamic acid) solution. 29.35 mg of lithium hydroxide was dissolved in 403 μL of ultrapure water and cooled to 0 °C, followed by the addition of the poly(γ-benzyl-L-glutamic acid) solution. The reaction system was stirred at 4 °C for 12 h to hydrolyze and remove the benzyl ester protecting group from the side chain. After the reaction was complete, the mixture was extracted three times with diethyl ether, retaining the aqueous phase, and dialyzed against ultrapure water overnight using a dialysis membrane with a molecular weight cutoff of 3 kDa. The dialysate was lyophilized to obtain a white powdery polyglutamic acid backbone (compounds 1-6).

[0073] Step 3: Preparation of azide-modified polyglutamic acid backbone Weigh 30 mg of the polyglutamic acid backbone (compounds 1-6), approximately 0.231 mmol based on the repeating glutamic acid unit and its side chain carboxyl groups, and dissolve it together with 76 mg (0.346 mmol) of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethylamine (compounds 1-7) in 1 mL of methanol to obtain a mixed solution. Separately, dissolve 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine salt (compounds 1-8, 103 mg) in 0.5 mL of methanol and slowly add it to the above mixed solution. The reaction system is stirred overnight at room temperature to allow the pGA80 side chain carboxyl groups to undergo an amidation reaction with the azide-containing small molecule amine.

[0074] After the reaction was completed, a saturated sodium bicarbonate solution was added to quench the reaction, and the solution was dialyzed against ultrapure water for 2 days using a dialysis membrane with a molecular weight cutoff of 3 kDa. The dialysate was lyophilized to obtain the azide-modified polyglutamic acid backbone (compounds 1-9), which was then stored at -20 °C for later use.

[0075] Figure 1 Compounds 1-9 shown contain methoxy-terminated polyethylene glycol chain-end residues formed by an amino-PEG12-methoxy initiator at one end, and tervapotranilate chain-ends formed by tervapotranose anhydride at the other end. The above-described chain-end structures are specific chain-end structures formed by the polymerization and capping methods used in this embodiment, and do not constitute a necessary limitation on the chain-end structure of the polymer backbone in the brush-like active peptide skin delivery system.

[0076] The structure and molecular weight distribution of the obtained azide-modified polyglutamic acid backbone were characterized by 1H NMR spectroscopy and gel permeation chromatography, respectively. The results are shown in the figure. Figure 5 and Figure 9 As shown. The resulting backbone was further used to undergo a strain-promoted azido-cycloyne click reaction with a BCN (bicyclic [6.1.0]nonyne)-modified active polypeptide.

[0077] II. Preparation of azide-modified polythiooctanoic acid-vinyl monomer copolymer backbone Specific preparation methods are as follows: Figure 2 As shown. The following is a step-by-step description: Step 1: Preparation of the thioctic acid-benzyl acrylate copolymer backbone Under argon protection, lipoic acid (compound 2-1, 206.33 mg, 1.0 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 1 mL), and benzyl acrylate (compound 2-2, 450.56 μL, 3.0 mmol) was added. Separately, chain transfer agents 2-(dodecyltrithiocarbonyl)-2-methylpropionic acid (compound 2-3, 0.013 mmol) and azobisisobutyronitrile (AIBN, 0.002 mmol) were dissolved in 100 μL of anhydrous THF and added to the above mixture. The reaction system was stirred at 70 °C for 6 h to allow lipoic acid and benzyl acrylate to undergo free radical ring-opening copolymerization under reversible addition-fragmentation chain transfer conditions, yielding a random copolymer backbone of lipoic acid-benzyl acrylate. After the reaction was complete, the reaction solution was added to 40 mL of ice-cold anhydrous diethyl ether to precipitate, and the precipitation was repeated three times. The resulting precipitate was dried under vacuum to obtain a yellow, viscous thioctic acid-benzyl acrylate copolymer backbone (compounds 2-4). The degree of polymerization of the thioctic acid-benzyl acrylate copolymer backbone can be freely controlled by adjusting the ratio of 2-(dodecyltrithiocarbonate)-2-methylpropionic acid and thioctic acid to benzyl acrylate.

[0078] Step 2: Preparation of the azido-modified thioctic acid-benzyl acrylate copolymer backbone 0.4678 mmol of the above-mentioned thioctic acid-benzyl acrylate copolymer backbone, calculated based on the carboxyl groups of the side chains in the thioctic acid structural unit, was dissolved in 4 mL of THF. Azide-PEG9-amine (compound 2-5, 338.27 mg, 0.7 mmol, 1.5 equivalents based on carboxyl groups) was added. The reaction system was placed in an ice-water bath, and 4-dimethylaminopyridine (compound 2-6, DMAP, 57.15 mg, 0.4678 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (compound 2-7, EDCI, 179.3 mg, 0.9356 mmol) were added slowly in sequence. The reaction system was stirred at 0 °C for 10 min, then brought to room temperature and stirred overnight to allow the carboxyl groups in the thioctic acid structural unit to undergo an amidation reaction with the azide-PEG9-amine.

[0079] After the reaction was complete, the mixture was diluted with dichloromethane and washed successively with 1 mol / L hydrochloric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was precipitated in 40 mL of ice-cold anhydrous diethyl ether, and the precipitation was repeated three times. The product was dried under vacuum to obtain a light yellow viscous azido-modified thioctic acid-benzyl acrylate copolymer backbone (compounds 2-8). The structure and molecular weight distribution of the obtained azido-modified thioctic acid-benzyl acrylate copolymer backbone were characterized by 1H NMR spectroscopy and gel permeation chromatography, respectively. The results are shown in the figure. Figure 6 and Figure 9 As shown. The resulting backbone was further used to undergo a strain-promoted click reaction with BCN-modified QK.

[0080] III. Preparation of Azide-Modified Polythiooctanoic Acid Backbone Specific preparation methods are as follows: Figure 3 As shown. The following is a step-by-step description: Step 1: Preparation of 3-azidopropylthioclate monomer Lipoic acid (compound 3-1, 30.0 g, 0.145 mol) was dissolved in anhydrous dichloromethane (300 mL) and cooled to 0 °C in an ice-water bath. 1,1′-carbonyldiimidazole (compound 3-3, CDI, 25.9 g, 0.160 mol) was added in portions with stirring, followed by a reaction at room temperature for 30 min. Then, 3-azido-1-propanol (compound 3-2, 13.2 g, 0.131 mol) and 4-dimethylaminopyridine (compound 3-4, DMAP, 1.8 g, 14.5 mmol) were added sequentially, and the mixture was stirred overnight at room temperature.

[0081] After the reaction was complete, the mixture was washed three times with 100 mL of saturated sodium chloride aqueous solution each time. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 20:1 to give 31.1 g of a pale yellow oily 3-azidopropylthioclate monomer (compounds 3-5), with a yield of 82%.

[0082] Step 2: Preparation of azide-modified polythiooctanoic acid backbone Under nitrogen protection, azide-polyethylene glycol-mercapto-hydrazine (compound 3-6, N3-PEG-SH, number average molecular weight approximately 2 kDa, 86.9 mg, 0.04 mmol) was dissolved in anhydrous tetrahydrofuran (400 μL), and tetramethylguanidine (compound 3-7, TMG, 5.5 μL, 0.04 mmol) was added and mixed for 1 min to obtain an initiator solution. Separately, the above-mentioned 3-azidopropyl thiooctanoate monomer (878.6 mg, 3.04 mmol) was dissolved in anhydrous tetrahydrofuran (400 μL), and the initiator solution was rapidly added under vigorous stirring at room temperature. After reacting for 2 h, 2,2,2-trifluoroethyl acrylate (compound 3-8, 101 μL, 0.80 mmol) was added to terminate the polymerization and adjust the polymer end groups.

[0083] After the reaction was complete, the reaction solution was added to 30 mL of ice-cold diethyl ether to precipitate, and the precipitation was repeated 5 times. The obtained product was dried under vacuum to obtain 715 mg of a colorless, viscous azido-modified polythiooctanoic acid backbone (compounds 3-9), with a yield of 74%. The degree of polymerization of the azido-modified polythiooctanoic acid backbone can be freely controlled by adjusting the ratio of azido-polyethylene glycol-mercapto and 3-azidopropylthiooctanoate. The structure and molecular weight distribution of the obtained azido-modified polythiooctanoic acid backbone were characterized by 1H NMR spectroscopy and gel permeation chromatography, respectively. The results are shown in the figure. Figure 7 and Figure 9 As shown. The resulting backbone was further used to undergo a strain-promoted click reaction with BCN-modified KLAK.

[0084] IV. Preparation of Azide-Modified Polyacrylic Acid Backbone Specific preparation methods are as follows: Figure 4 As shown. The following is a step-by-step description: Step 1: Preparation of polyacrylic acid backbone Under nitrogen protection, acrylic acid (compound 4-1, 343.5 μL, 5 mmol) was dissolved in anhydrous tetrahydrofuran (400 μL) to obtain an acrylic acid solution. Separately, chain transfer agents 2-(dodecyltrithiocarbonyl)-2-methylpropionic acid (compound 4-2, 0.042 mmol) and azobisisobutyronitrile (AIBN, 0.008 mmol) were dissolved in anhydrous THF 100 μL and added to the acrylic acid solution. The reaction system was stirred at 70 °C for 20 min. After the reaction was complete, the reaction solution was added to 40 mL of ice-cold diethyl ether to precipitate, and the precipitation was repeated three times. The resulting pale yellow flocculent polymer was collected and vacuum dried to obtain the polyacrylic acid backbone (compound 4-3). The degree of polymerization of the polyacrylic acid can be freely controlled by adjusting the ratio of 2-(dodecyltrithiocarbonyl)-2-methylpropionic acid and acrylic acid.

[0085] Step 2: Preparation of azide-modified polyacrylic acid backbone 0.5 mmol of the polyacrylic acid backbone (based on side chain carboxyl groups) was dissolved in 4 mL of THF, and azido-PEG2-amine (compound 4-4, 130.7 mg, 0.75 mmol, 1.5 equivalents based on carboxyl groups) was added. The reaction system was placed in an ice-water bath, and 4-dimethylaminopyridine (compound 4-5, 61.1 mg, 0.5 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (compound 4-6, 191.7 mg, 1.0 mmol) were added slowly in sequence. The mixture was stirred at 0 °C for 10 minutes, then brought to room temperature and stirred overnight to allow the side chain carboxyl groups of the polyacrylic acid backbone to undergo an amidation reaction with azido-PEG2-amine.

[0086] After the reaction was complete, the mixture was diluted with dichloromethane and washed successively with 1 mol / L hydrochloric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was precipitated in 40 mL of ice-cold anhydrous diethyl ether, and the precipitation was repeated three times to obtain the azide-modified polyacrylic acid backbone (compounds 4-7). The structure and molecular weight distribution of the obtained azide-modified polyacrylic acid backbone were characterized by 1H NMR spectroscopy and gel permeation chromatography, respectively. The results are shown in the figure. Figure 8 and Figure 9 As shown. The resulting backbone was further used to undergo a strain-promoted azide-cycloalkyne click reaction with BCN-modified active peptides to construct a carbon-chain polymer backbone-type brush-like active peptide skin delivery system.

[0087] V. Subsequent applications of azide-modified main chains The backbones of the four types of azide-modified multifunctional polymers mentioned above all contain multiple azide groups, which can undergo click reactions with active peptides modified with DBCO, alkynyl, cycloalkynyl or other complementary reactive functional groups, so that the active peptides are covalently grafted onto the polymer backbone to form a brush-like or bottle-brush-like active peptide skin delivery system.

[0088] Example 2: Preparation of a brush-shaped active peptide skin delivery system In this embodiment, the azido-modified multifunctional polymer backbone prepared in Example 1 was subjected to a strain-promoted azido-cycloalkyne click reaction with BCN-modified active peptides to prepare KLAK, LTX-315 and QK brush-like active peptide skin delivery systems, respectively.

[0089] Active peptides can have reactive functional groups introduced at the N-terminus, C-terminus, amino acid side chains, non-natural amino acid residues, or linkers, which can covalently couple with the multifunctional polymer backbone. In this example, KLAK, LTX-315, and QK are used as representative active peptides, and a BCN group is introduced by using an N-terminal cysteine ​​residue. Other active peptides selected according to the target topical function can also be grafted onto the multifunctional polymer backbone by introducing corresponding reactive functional groups in the same or similar way.

[0090] The KLAK, LTX-315, and QK used in this embodiment were all purchased from Shanghai Jier Biochemical Co., Ltd., and cysteine ​​residues were introduced at the N-terminus of the corresponding parent peptides for subsequent chemical coupling. For simplicity, unless otherwise specified, they will be referred to as KLAK, LTX-315, and QK respectively, and their specific amino acid sequences are shown in Table 1.

[0091] Table 1. Amino acid sequences of active peptides

[0092] Dip represents non-natural amino acid residues.

[0093] I. Preparation of BCN-modified active peptides 1. Preparation of BCN-maleimide Specific preparation methods are as follows: Figure 10 As shown.

[0094] Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimide carbonate (compound 5-1, 50 mg, 0.172 mmol), 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (compound 5-2, 36 mg, 0.2064 mmol), and N,N-diisopropylethylamine (DIPEA, 100 μL) were added to a round-bottom flask and dissolved in anhydrous dichloromethane (DCM, 5 mL). The reaction was carried out at room temperature for 1 h. After the reaction was completed, the reaction solution was washed three times with 10 mL of brine each time. The organic layer was separated and dried over anhydrous sodium sulfate. After filtering to remove the anhydrous sodium sulfate, the organic phase was concentrated under reduced pressure.

[0095] The residue was purified by silica gel rapid column chromatography using a petroleum ether / ethyl acetate mixture (20:1 v / v) as eluent to give a white solid compound, BCN-maleimide (compound 5-3, 46.38 mg, 85% yield). The structure of the obtained BCN-maleimide was confirmed by 1H NMR spectroscopy; its representative NMR spectrum is shown below. Figure 11 .

[0096] 2. Preparation of BCN-modified active peptides The specific dosages of the three active peptides and BCN-maleimide are shown in Table 2.

[0097] Table 2 Reactant Feed Amounts for BCN-Modified Active Peptides

[0098] Dissolve the KLAK, QK, or LTX-315 samples (as shown in Table 2) in N,N-dimethylformamide (DMF, 2 mL). Add 1.8 times the amount of the active peptide prepared in the previous step, as shown in Table 2. Stir the reaction mixture overnight at room temperature to allow the maleimide groups in the BCN-maleimide to undergo an addition reaction with the thiol groups of the N-terminal cysteine ​​residues of the active peptide, yielding BCN-modified KLAK, BCN-modified QK, and BCN-modified LTX-315, respectively.

[0099] After the reaction was complete, 10 mL of diethyl ether was added to the reaction solution to precipitate the product. The precipitate was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected. The precipitate was redissolved in a small amount of DMF, and another 10 mL of diethyl ether was added to redissolve the product. The precipitate was then collected by centrifugation at 5000 rpm for 5 min. This redissolution and diethyl ether precipitation process was repeated once, for a total of three diethyl ether precipitations for each product. The resulting product was then lyophilized after removing residual diethyl ether.

[0100] The molecular weights of three bioactive peptides before and after BCN modification were characterized using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF mass spectrometry). Representative MALDI-TOF mass spectrometry results are shown below. Figure 12 Unmodified KLAK has a theoretical molecular weight of 1642 Da, with a major mass spectrum peak at m / z 1640.8; unmodified LTX-315 has a theoretical molecular weight of 1542 Da, with a major mass spectrum peak at m / z 1544.0; unmodified QK has a theoretical molecular weight of 2014 Da, with a major mass spectrum peak at m / z 2013.0; BCN-modified KLAK has a theoretical molecular weight of 1958 Da, with a major mass spectrum peak at m / z 1956.2; BCN-modified LTX-315 has a theoretical molecular weight of 1858 Da, with a major mass spectrum peak at m / z 1856.7; BCN-modified QK has a theoretical molecular weight of 2330 Da, with a major mass spectrum peak at m / z 2328.0. Compared with the corresponding unmodified active peptides, the mass spectrometry peaks of the BCN-modified products all showed an increase in molecular weight consistent with the introduction of the BCN group, indicating that KLAK, QK and LTX-315 were all successfully modified with the BCN group.

[0101] II. Preparation of a brush-like active peptide skin delivery system The azido-modified polythioctic acid backbone, azido-modified polyglutamic acid backbone, azido-modified thioctic acid-benzyl acrylate copolymer backbone, and azido-modified polyacrylic acid backbone prepared in Example 1 were respectively subjected to strain-promoted azido-cycloalkyne click reactions with the corresponding BCN-modified active peptides.

[0102] The amount of BCN-modified active peptides used in each reaction was calculated based on approximately 80% of the azide sites on the corresponding polymer backbone. 80% represents the proportion of target azide sites occupied by the peptides. The specific amounts of the four brush-like active peptide skin delivery systems are shown in Table 3.

[0103] Table 3. Reaction feed amounts for the brush-type active peptide skin delivery system

[0104] All of the active peptides were modified with BCN.

[0105] 1. Preparation of the KLAK brush-shaped active peptide skin delivery system Approximately 2.1 mg of azide-modified polythioctic acid backbone and 10 mg of BCN-modified KLAK were dissolved in 2 mL of DMF and stirred overnight at room temperature to allow the azide groups on the backbone to undergo a strain-promoted azide-cycloalkyne click reaction with the BCN groups in the BCN-modified KLAK, thus obtaining a KLAK brush-like active peptide skin delivery system.

[0106] 2. Preparation of LTX-315 brush-shaped active peptide skin delivery system Approximately 2.2 mg of azide-modified polyglutamic acid backbone and 10 mg of BCN-modified LTX-315 were dissolved in 2 mL of DMF and stirred overnight at room temperature to allow the azide groups on the backbone to undergo a strain-promoted azide-cycloalkyne click reaction with the BCN groups in the BCN-modified LTX-315, thus obtaining the LTX-315 brush-like active peptide skin delivery system.

[0107] 3. Preparation of QK brush-shaped active peptide skin delivery system Approximately 6.0 mg of the azido-modified thioctic acid-benzyl acrylate copolymer backbone and 10 mg of BCN-modified QK were dissolved in 2 mL of DMF and stirred overnight at room temperature. This allowed the azido groups on the thioctic acid structural units in the copolymer backbone to undergo a strain-promoted azido-cycloalkyne click reaction with the BCN groups in the BCN-modified QK, resulting in the QK brush-like active peptide skin delivery system.

[0108] Among them, the benzyl acrylate structural unit is mainly used to adjust the hydrophilicity / hydrophobicity, segment properties, steric hindrance, colloidal stability, surfactant tolerance and biological interface interaction of the copolymer backbone and its brush delivery system.

[0109] 4. Preparation of a QK brush-shaped active peptide skin delivery system based on a carbon-carbon polymer backbone Approximately 2.9 mg of azide-modified polyacrylic acid backbone and 10 mg of BCN-modified QK were dissolved in 2 mL of DMF and stirred overnight at room temperature to allow the azide groups on the backbone to undergo a strain-promoted azide-cycloalkyne click reaction with the BCN groups in the BCN-modified QK, thus obtaining a QK brush-like active peptide skin delivery system based on a carbon-carbon polymer backbone.

[0110] 5. Purification of the brush-like active peptide skin delivery system After the four click-coupling reactions described above were completed, 10 mL of diethyl ether was added to the reaction solution to precipitate the brush-shaped active peptide skin delivery system. The precipitate was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the precipitate was collected. The precipitate was redissolved in a small amount of DMF, and then 10 mL of diethyl ether was added again for reprecipitation. The precipitate was collected after centrifugation at 5000 rpm for 5 min. This redissolution and ether precipitation process was repeated once, resulting in a total of three ether precipitation cycles for each brush-shaped active peptide skin delivery system.

[0111] After removing residual ether, the obtained products were lyophilized to obtain the following skin delivery systems: KLAK brush-shaped active peptide skin delivery system based on azido-modified polythioctic acid backbone, LTX-315 brush-shaped active peptide skin delivery system based on azido-modified polyglutamic acid backbone, QK brush-shaped active peptide skin delivery system based on azido-modified thioctic acid-benzyl acrylate copolymer backbone, and QK brush-shaped active peptide skin delivery system based on azido-modified polyacrylic acid backbone.

[0112] III. PAGE characterization of the brush-like active peptide skin delivery system Four purified brush-like active peptide skin delivery systems were characterized by polyacrylamide gel electrophoresis, with the corresponding free active peptides serving as controls. After electrophoresis, the gels were stained and destained with Coomassie Brilliant Blue. Free KLAK, free LTX-315, and free QK showed corresponding free peptide bands in the gel. No identifiable bands corresponding to the migration positions of the corresponding free peptides were observed in any of the four purified brush-like active peptide skin delivery system samples, indicating that no residual free active peptides were detected in the obtained brush-like active peptide skin delivery systems after three ether precipitation purifications. Representative PAGE and Coomassie Brilliant Blue staining results for the four brush-like active peptide skin delivery systems are shown in the figure. Figure 13 .

[0113] IV. Other Characterizations of Brush-like Active Peptide Skin Delivery Systems The obtained brush-like active peptide skin delivery system was further characterized by dynamic light scattering and zeta potential. The hydrated particle size and particle size distribution of the brush-like active peptide skin delivery system were determined by dynamic light scattering; its surface charge was evaluated by zeta potential measurement.

[0114] Table 4

[0115] The particle size and zeta potential characterization results of the brush-like active peptide skin delivery system (polyglutamic acid LTX-315, polythioctic acid-KLAK, polythioctic acid-benzyl acrylate copolymer backbone-QK) are shown in [reference needed]. Figure 14 See Table 4. Example 3: Stability evaluation of a brush-like active peptide skin delivery system in a surfactant-containing system. This embodiment evaluates the stability of the following three brush-type active peptide skin delivery systems in surfactant-containing systems: (1) A KLAK brush-like active peptide skin delivery system based on an azide-modified polythioctic acid backbone; (2) LTX-315 brush-like active peptide skin delivery system based on azide-modified polyglutamic acid backbone; (3) QK brush-like active peptide skin delivery system based on azido-modified thioctic acid-benzyl acrylate copolymer backbone.

[0116] Three brush-like active peptide skin delivery systems were prepared to achieve a final concentration of 1 μmol / L for each brush-like polymer chain. Each system was mixed with PBS, 2% (w / v) sodium lauryl ether sulfate, 2% (w / v) cocamidopropyl betaine, and 2% (w / v) decyl glucoside, or a mixed surfactant system consisting of 4% (w / v) sodium lauryl ether sulfate, 2% (w / v) cocamidopropyl betaine, and 1% (w / v) decyl glucoside, and incubated at 37 °C for 12 h. After incubation, each sample was diluted 1000-fold with PBS, and its diffusion behavior and apparent hydration radius were immediately determined using fluorescence correlation spectroscopy. Five independent samples were included in each group.

[0117] The results showed that the apparent hydration radius of the three brush-like active peptide skin delivery systems did not change significantly compared with the PBS control group after treatment with different surfactant systems, indicating that the brush-like active peptide skin delivery systems have good dimensional stability under the above surfactant-containing conditions. Representative results are shown in […]. Figure 15 .

[0118] Example 4: Evaluation of skin and hair follicle delivery using the QK brush-shaped active peptide skin delivery system This embodiment evaluates the delivery and enrichment capabilities of the QK brush-like active peptide skin delivery system in skin and hair follicles. The tested QK brush-like active peptide skin delivery system is the one prepared in Example 2 based on the azide-modified thioctic acid-benzyl acrylate copolymer backbone. Live BALB / c mouse back skin was used as a skin delivery model. Before the experiment, the mouse back skin surface was cleaned with a cleaning solution to simulate the skin cleansing scenario in actual topical care and to remove residual cleaning solution from the skin surface. Subsequently, Cy5-labeled free QK and the Cy5-labeled QK brush-like active peptide skin delivery system were applied to the cleansed skin surface, with each group using 1 nmol of Cy5. After 4 h of treatment, residual samples were removed from the skin surface and the skin surface was cleaned. Skin tissue was then taken, fixed, and sectioned. The distribution of Cy5 fluorescence in the skin tissue and hair follicle structure was observed using a confocal laser scanning microscope, and cell nuclei were stained with DAPI.

[0119] The results showed that, compared with free QK, the QK brush-like active peptide skin delivery system exhibited a stronger Cy5 fluorescence signal in skin tissue and showed significant enrichment in the hair follicle region, indicating that the brush-like structure can improve the delivery and enrichment of QK in the skin and hair follicles. Representative results are shown in [link to relevant data]. Figure 16 .

[0120] Example 5: Evaluation of cellular uptake by the QK brush-shaped active peptide skin delivery system This example evaluates the cellular uptake capability of the QK brush-like active peptide skin delivery system. The QK brush-like active peptide skin delivery system tested is the one prepared in Example 2 based on the azide-modified thioctic acid-benzyl acrylate copolymer backbone.

[0121] Cy5-labeled free QK and the Cy5-labeled QK brush-like active peptide skin delivery system were co-incubated with human umbilical vein endothelial cells (HUVECs), with untreated cells serving as a blank control. The final Cy5 concentration was 1 μmol / L for both systems, and the incubation time was 4 h. After incubation, the cells were washed, and the intracellular Cy5 fluorescence distribution was observed using confocal laser scanning microscopy, while the fluorescence intensity was detected by flow cytometry. The results showed that compared to free QK, the QK brush-like active peptide skin delivery system exhibited significantly enhanced intracellular Cy5 fluorescence signal and higher flow cytometry fluorescence intensity, indicating that the brush structure can improve QK uptake by cells. Representative confocal images, flow cytometry histograms, and quantitative fluorescence intensity results are shown below. Figure 17 .

[0122] Example 6: Enzymatic stability evaluation of a brush-like active peptide skin delivery system This example evaluates the stability of QK grafted with a brush-like structure against protease degradation. The tested QK brush-like active peptide skin delivery system is the QK brush-like active peptide skin delivery system based on the azide-modified thioctic acid-benzyl acrylate copolymer backbone prepared in Example 2.

[0123] Free QK and QK brush-like active peptide skin delivery systems were prepared separately, with a final QK concentration of 1 mmol / L in both groups of samples. α-chymotrypsin was added to each group of samples to a final enzyme concentration of 5 U / mL, and incubated at 37 ℃. Samples were taken at 0 min, 15 min, 30 min, 1 h, 2 h, and 4 h, and immediately frozen at -80 ℃ after sampling to terminate the enzymatic digestion reaction.

[0124] After thawing at all time points, intact QK was detected by polyacrylamide gel electrophoresis, and quantification was performed by the gray level of the corresponding band of intact QK. The gray level of the intact QK band at 0 min was defined as 100%, and the remaining proportion of intact QK at each time point was calculated.

[0125] The results showed that free QK was rapidly degraded by α-chymotrypsin, and after 15 min of incubation, almost no intact QK was detectable. In contrast, the QK brush-like active peptide skin delivery system exhibited significantly improved enzyme stability, with a degradation half-life of approximately 1 h. These results indicate that covalently grafting QK as a side chain onto the azide-modified lipoic acid-benzyl acrylate copolymer backbone can significantly improve its stability against α-chymotrypsin degradation. Representative results are shown below. Figure 18 .

[0126] Example 7: Evaluation of the ABTS radical cation scavenging ability of the azide-modified thioctic acid-benzyl acrylate copolymer backbone This embodiment evaluates the free radical scavenging ability of the polydisulfide copolymer backbone containing thioctic acid structural units used to construct the QK brush-like active peptide skin delivery system. The tested sample was specifically the azide-modified thioctic acid-benzyl acrylate copolymer backbone prepared in Example 1. The ABTS free radical cation system was used for evaluation. The sample was added to the ABTS free radical cation working solution to final concentrations of 1, 2, 4, and 8 mg / mL, with a blank control group containing no sample. Each group had three parallel wells, incubated at room temperature in the dark, and the absorbance at 734 nm was measured within 0–240 min.

[0127] The ABTS radical cation scavenging rate is calculated using the following formula: in, The absorbance is that of the blank control group. The absorbance is the value at the corresponding time point after the sample is added.

[0128] The results showed that the scavenging effect of the azide-modified thioctic acid-benzyl acrylate copolymer backbone on ABTS radical cations was significantly concentration- and time-dependent. After 240 min of incubation, the scavenging rates of the 1, 2, 4, and 8 mg / mL groups were approximately 14%, 27%, 63%, and close to 100%, respectively. As the sample concentration increased, the blue-green color of the reaction solution gradually faded, with the 8 mg / mL group essentially fading after 240 min.

[0129] The above results demonstrate that the azide-modified thioctic acid-benzyl acrylate copolymer backbone, in addition to providing active peptide grafting sites, also possesses in vitro ABTS radical cation scavenging and antioxidant properties, laying the foundation for further applications in oxidative stress regulation. Representative scavenging kinetic curves and well plate images before and after the reaction are shown below. Figure 19 .

Claims

1. A brush-like active polypeptide skin delivery system, characterized in that, It includes a multifunctional polymer backbone and multiple active polypeptide side chains covalently grafted onto the multifunctional polymer backbone; The multifunctional polymer backbone contains multiple reactive functional groups that can covalently couple with the active peptide or modified active peptide before grafting with the active peptide. The active peptide side chain is grafted onto the multifunctional polymer backbone through the covalent coupling reaction involving the reactive functional groups to form a brush-like or bottle-brush-like polymer coupling structure. The multifunctional polymer backbone is selected from one or more of the following: polyamino acid backbone, carbon-carbon polymer backbone, and polydisulfide backbone containing thiooctanoic acid structural units.

2. The brush-like active polypeptide skin delivery system according to claim 1, characterized in that, The polyamino acid backbone is selected from one or more of polyglutamic acid, polyaspartic acid, polylysine, polyornithine, glutamic acid-lysine copolymer, glutamic acid-aspartic acid copolymer or their derivatives.

3. The brush-like active polypeptide skin delivery system according to claim 2, characterized in that, The polyamino acid backbone contains repeating units as shown in Formula I before grafting with the active polypeptide: Equation I, In formula I, m is a natural number between 2 and 10000. R may be the same or different in each repeating unit, and R is selected from one or more amino acid side chains that are not modified or contain reactive functional groups.

4. The brush-like active polypeptide skin delivery system according to claim 1, characterized in that, The carbon-carbon polymer backbone, before grafting with the active polypeptide, comprises a carbon-carbon backbone formed by polymerization of one or more vinyl monomers and multiple side chain functional groups, at least some of which are reactive functional groups capable of covalently coupling with the active polypeptide or modified active polypeptide.

5. The brush-like active polypeptide skin delivery system according to claim 4, characterized in that, The carbon-carbon polymer backbone is selected from copolymers of polyacrylic acid, polymethacrylic acid, acrylic acid and / or methacrylic acid with one or more vinyl monomers, or one or more derivatives thereof. The vinyl monomer is selected from one or more of acrylate monomers, methacrylate monomers, acrylamide monomers, methacrylamide monomers, styrene monomers, vinylpyrrolidone monomers, vinyl ether monomers, vinyl ester monomers, or their derivatives.

6. The brush-like active polypeptide skin delivery system according to claim 5, characterized in that, The vinyl monomer is selected from one or more of the following: benzyl acrylate, benzyl methacrylate, alkyl acrylate, alkyl methacrylate, hydroxyalkyl acrylate, hydroxyalkyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, isobornyl acrylate, isobornyl methacrylate, 2,2,2-trifluoroethyl acrylate, 2,2,2-trifluoroethyl methacrylate, N-isopropylacrylamide, N,N-dimethylacrylamide, N-hydroxyethylacrylamide, vinylpyrrolidone, styrene, hydroxystyrene, sulfonated styrene, carboxystyrene, or their derivatives.

7. The brush-like active polypeptide skin delivery system according to claim 1, characterized in that, The polydisulfide backbone of the thioctic acid structural unit includes a polydisulfide backbone formed from thioctic acid or its derivatives, or the polydisulfide backbone of the thioctic acid structural unit includes a polydisulfide copolymer backbone formed by copolymerization of thioctic acid or its derivatives with vinyl monomers.

8. The brush-like active polypeptide skin delivery system according to claim 7, characterized in that, The polydisulfide backbone containing the thiooctanoic acid structural unit contains the structural unit shown in Formula II before grafting with the active polypeptide: Formula II, In formula II, a is a natural number between 1 and 10000. b is 0 or a natural number between 1 and 10000. R5 is hydrogen or methyl. R6 is a side group in the lipoic acid structural unit, selected from one or more reactive functional groups containing hydroxyl, alkoxy, amino, substituted amino, carboxyl, active ester, azide, alkynyl, cycloalkynyl, maleimide, mercapto, tetrazinyl, or transcyclooctenyl. Q is a side group retained in the repeating structural unit of the polymer after the polymerization of vinyl monomers, selected from one or more of alkyl, aryl, carboxyl, ester, amide, hydroxyl, amino, or polyether groups.

9. The brush-like active polypeptide skin delivery system according to claim 7, characterized in that, The polydisulfide backbone of the sulfur-containing octanoic acid structural unit has free radical scavenging and / or antioxidant properties.

10. The brush-like active polypeptide skin delivery system according to claim 1, characterized in that, The reactive functional groups of the multifunctional polymer backbone before grafting with the active polypeptide are selected from one or more of the following groups: azide, alkynyl, cycloalkynyl, dibenzocyclooctynyl, maleimide, mercapto, amino, carboxyl, hydroxyl, aldehyde, ketone, hydrazine, hydroxylamine, tetraazine, transcyclooctenyl, norbornene, active ester, N-hydroxysuccinimide ester, isocyanate, isothiocyanate, or anhydride.

11. The brush-like active polypeptide skin delivery system according to claim 1, characterized in that, The active polypeptide side chain is grafted onto the multifunctional polymer backbone via one or more of the following reactions: click reaction, cycloaddition reaction, amidation reaction, active ester-amine reaction, carboxyl-amine condensation reaction, mercapto-maleimide reaction, mercapto-ene reaction, oxime bond formation reaction, hydrazone bond formation reaction, esterification reaction, carbonate reaction, disulfide bond exchange reaction, or isocyanate-amine reaction.

12. The brush-like active polypeptide skin delivery system according to claim 11, characterized in that, The active polypeptide side chain is grafted onto the multifunctional polymer backbone via azido-alkynyl click reaction, copper-free azido-cycloalkynyl click reaction, BCN-azido click reaction, DBCO-azido click reaction, tetrazine-trans-cyclooctene reaction, CuAAC reaction, mercapto-maleimide reaction, or active ester-amine reaction.

13. The brush-like active polypeptide skin delivery system according to claim 1, characterized in that, The grafting ratio of the active polypeptide to the reactive sites of the multifunctional polymer backbone before grafting is 5%-95%.

14. The brush-like active polypeptide skin delivery system according to claim 1, characterized in that, The active polypeptide is a polypeptide with biological activities related to the regulation of skin, scalp, hair follicles, skin appendages or local tissues. The biological activities of the active polypeptide include one or more of the following: skin care, scalp care, hair follicle regulation, tissue repair, anti-inflammation, antibacterial, antioxidant or cell regulation.

15. The brush-like active polypeptide skin delivery system according to claim 14, characterized in that, The active polypeptide includes one or more of QK, KLAK, or LTX-315.

16. The brush-like active polypeptide skin delivery system according to claim 15, characterized in that, The active polypeptide is selected from one or more of the following N-terminal cysteine-modified polypeptides: the amino acid sequence of KLAK modified with N-terminal cysteine ​​is CKALKLAKKLAKLAK; the amino acid sequence of QK modified with N-terminal cysteine ​​is CKLTWQELYQLKYKGI; the amino acid sequence of LTX-315 modified with N-terminal cysteine ​​is CKKWWKKWDipK-NH2, wherein Dip represents the non-natural amino acid residue 3,3-diphenylalanine.

17. The brush-like active polypeptide according to any one of claims 1 to 16 A method for preparing a skin delivery system, characterized by comprising the following steps: reacting the multifunctional polymer backbone with an active polypeptide or a modified active polypeptide in a solvent system, wherein the multifunctional polymer backbone contains a plurality of reactive functional groups capable of covalently coupling with the active polypeptide or the modified active polypeptide before grafting with the active polypeptide, and the side chains of the active polypeptide are grafted onto the multifunctional polymer backbone through a covalent coupling reaction involving the reactive functional groups to form a brush-like or bottle-brush-like polymer coupling structure.

18. The preparation method according to claim 17, characterized in that, When the reaction is a click reaction or a cycloaddition reaction, the reaction temperature is 0-100 °C and the reaction time is 0.5-96 h; when the reaction is an amidation reaction or an active ester-amine reaction, the reaction temperature is 0-60 °C and the reaction time is 0.5-72 h; the solvent system includes one or more of water, buffer solution, dimethyl sulfoxide, N,N-dimethylformamide, ethanol, methanol, acetonitrile, tetrahydrofuran, or mixtures thereof.

19. A topical skin composition, characterized in that, Includes the brush-like active peptide skin delivery system according to any one of claims 1 to 16 and an acceptable matrix for topical use.

20. The topical skin composition according to claim 19, characterized in that, It also includes surfactants, which are selected from one or more of anionic surfactants, amphoteric surfactants, nonionic surfactants, cationic surfactants, or amino acid surfactants.

21. The use of the brush-like active polypeptide skin delivery system according to any one of claims 1 to 16 in the preparation of topical skin products, characterized in that, The skin topical products are cosmetics, personal care products, skin topical preparations, or topical drug delivery preparations.

22. The application according to claim 21, characterized in that, The topical skin products are selected from shampoos, scalp care solutions, scalp serums, conditioners, hair masks, shower gels, facial cleansers, serums, skin lotions, gels, creams, ointments, sprays, or patches.

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