Printable glycopeptide microgel / microsphere composite ink as well as preparation method and application thereof

Abstract

The invention relates to printable glycopeptide microgel / microsphere composite ink as well as a preparation method and application thereof, and belongs to the technical fields of biological medicines, daily chemicals and the like. The composition is prepared from a functional sodium alginate polymer, a functional sodium hyaluronate polymer, sodium carboxymethyl starch, sodium magnesium lithium silicate, calcium glycerophosphate, a plant cell exosome, a natural plant extract, diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide, sodium carboxymethyl starch, sodium magnesium silicate, sodium magnesium silicate, sodium magnesium silicate, calcium glycerophosphate, plant cell exosome, natural plant extract, sodium carboxymethyl starch, sodium carboxymethyl starch and sodium carboxymethyl starch. Hydrogel polysaccharide active microspheres prepared from diamino seleno hydrochloride, a multifunctional polyglutamic acid polymer and a multifunctional gelatin polymer are prepared into microgel, and the microgel is subjected to multiple cross-linking reaction. The integrated hydrogel microneedle prepared from the composite ink provided by the invention realizes deep programmed controllable release of active ingredients, and can be widely applied to the fields of scalp care, hair follicle nourishing, soft tissue repair and regeneration and the like.

Description

Technical Field

[0001] The invention relates to a printable glycopeptide microgel / microsphere composite ink and a preparation method and application thereof, belonging to the technical fields of biomedicine, daily chemicals and the like. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Hair loss is a common health problem and is associated with age, lack of sleep, stress, endocrine problems and other issues. Globally, hair loss has gradually become a serious disease due to stressful living environments and irregular lifestyle habits such as lack of sleep, heavy smoking, and alcohol abuse. However, there are many factors that cause hair loss, including increased sensitivity of scalp hair follicles to dihydrotestosterone, autoimmune reactions, hormonal imbalances, genetic problems, environmental factors, medications, nutritional deficiencies and stress. In the final analysis, hair follicles are dynamic micro-organs, and each hair follicle is a regenerative system that physiologically undergoes a continuous cycle of growth, catagen and resting phases. The growth phase is the active phase of the hair follicle and is the most important determinant of hair length and volume.

[0004] Currently, the only FDA-approved drugs for the treatment of hair loss are minoxidil and finasteride. However, due to their limited clinical efficacy, these two drugs require long-term use to observe significant improvements. In addition, minoxidil and finasteride can cause various side effects, including: alopecia areata, scalp itching, flaking, allergic reactions, abnormal liver function, and sexual dysfunction. Therefore, when patients have doubts about these two conventional therapies, they can consider using scalp-related products containing natural substances as an alternative to treating hair loss.

[0005] Hydrogel is a network composed of hydrophilic polymer chains derived from natural or synthetic sources. Due to its high water content, biocompatibility and degradability, hydrogel has been widely used in fields such as tissue engineering and regenerative medicine. However, as a carrier for drug and cell delivery, the limit penetration depth of traditional block hydrogels is generally considered to be 200 microns. The excessive volume of hydrogels will affect the absorption of nutrients and oxygen, and micron-level hydrogels (microgels) can effectively overcome this technical difficulty. In view of this, a microgel active system with excellent moisturizing, hair follicle tissue activation and scalp cell regeneration is developed by simulating the proteoglycan and collagen fiber network in skin tissue. It is used as a hair growth additive. It can enhance the transdermal moisture absorption and moisturizing ability at the site of hair loss, resist water loss, and activate the hair follicle tissue of the scalp, which is of great significance for promoting hair regeneration. Summary of the invention

[0006] In order to address the deficiencies in the prior art, the object of the present invention is to provide a printable glycopeptide microgel / microsphere composite ink, a preparation method and application thereof. The glycopeptide microgel / microsphere composite ink provided by the present invention utilizes the good injectability, high porosity, high specific surface area, tissue permeability and high tissue compatibility of the microgel / microsphere system to design a light / active oxygen dual-responsive active ingredient precise and efficient delivery system. The composite ink can be used to prepare integrated hydrogel microneedles through 3D printing technology. The integrated hydrogel microneedles prepared can reconstruct the dynamic cellular microenvironment of damaged tissues by improving the high permeability of the composite system, improve the vitality of endogenous cells, and guide cell homing to promote hair follicle regeneration, thereby achieving the effects of preventing hair loss and fixing hair.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, a method for preparing a printable glycopeptide microgel / microsphere composite ink comprises the following steps:

[0009] The polyglutamic acid was modified by acryloylation and cucurbituril to obtain a multifunctional polyglutamic acid polymer.

[0010] Multifunctional gelatin polymers were obtained by acryloylating and modifying gelatin with tryptophan.

[0011] The vicinal diol structure of sodium alginate is oxidized to obtain an oxidized sodium alginate polymer, and the oxidized sodium alginate polymer is modified with arginine to obtain a functionalized sodium alginate polymer;

[0012] The vicinal diol structure of hyaluronic acid is oxidized to obtain an oxidized sodium hyaluronate polymer, and the oxidized sodium hyaluronate polymer is modified with amino azobenzene to obtain a functionalized sodium hyaluronate polymer;

[0013] Dissolving sodium magnesium lithium silicate and calcium glycerophosphate to form a mixed solution, adding plant cell exosomes, natural plant extracts, diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, and diaminoseleno hydrochloride, and mixing evenly to obtain a composite solution, and then adding functionalized sodium alginate polymer, functionalized sodium hyaluronate polymer, and sodium carboxymethyl starch, and mixing evenly to obtain hydrogel polysaccharide active microspheres;

[0014] A multifunctional polyglutamic acid polymer, a multifunctional gelatin polymer and a photoinitiator are shear-mixed in a solution to form a peptide microgel, and then hydrogel polysaccharide active microspheres and amino-terminated polyethylene glycol are added and mixed evenly, and multiple cross-linking reactions are performed in the system to fix the microspheres to the microgel, thereby obtaining the composite ink; wherein the multiple cross-linking reactions include Schiff base reactions and supramolecular interactions between azobenzene groups and cucurbituril groups.

[0015] In the preparation process of the present invention, sodium magnesium lithium silicate / calcium glycerophosphate is prepared with functionalized sodium alginate polymer, functionalized sodium hyaluronate polymer and sodium carboxymethyl starch to encapsulate plant cell exosomes, natural plant extracts, diaminopyrimidine oxides, and pyrrolidine diaminopyrimidine oxides, and then diaminoselenohydrochloride is reacted with the Schiff base between the aldehyde groups to form active microspheres with controllable release of active ingredients. Finally, a multifunctional polyglutamic acid polymer and a multifunctional gelatin polymer are mixed, and the aromatic residues and tryptophan in the side chain of the multifunctional gelatin polymer interact with the host-guest supramolecular interaction of cucurbituril in the multifunctional polyglutamic acid polymer to form a peptide microgel under continuous shearing. Then, hydrogel polysaccharide active microspheres and amino-terminated polyethylene glycol are added and mixed evenly. Then, the microspheres are fixed to the microgel system by multiple cross-linking reactions such as the Schiff base reaction of the amino group, guanidine group and other groups in the microgel system with the aldehyde group in the microspheres and the host-guest supramolecular interaction of the azobenzene group in the microspheres with the cucurbituril group in the microgel system to prevent the microspheres from depositing, thereby preparing a printable glycopeptide microgel / microsphere composite ink.

[0016] In some embodiments, polyglutamic acid is acrylated and modified by cucurbituril using glycidyl methacrylate and aminated cucurbituril. Specifically, the modification degree of glycidyl methacrylate is 3:1-1:3, and the modification degree of aminated cucurbituril is 3:1-1:3. The modification degree is the degree of chemical substitution.

[0017] In some embodiments, gelatin is acrylated and modified by using glycidyl methacrylate and tryptophan. Specifically, the modification degree of glycidyl methacrylate is 3:1-1:2, and the modification degree of tryptophan is 3:1-1:2.

[0018] In some embodiments, NaIO4 is used to oxidize the vicinal diol structure of sodium alginate. Specifically, the oxidation degree of sodium alginate is 0.5-5%. The oxidation degree is the proportion of aldehyde groups after sodium alginate is oxidized by sodium periodate.

[0019] In some embodiments, NaIO4 is used to oxidize the vicinal diol structure of hyaluronic acid. Specifically, the oxidation degree of sodium hyaluronate is 1-10%.

[0020] In some embodiments, a functionalized sodium alginate polymer, a functionalized sodium hyaluronate polymer, and sodium carboxymethyl starch are added to a composite solution by a piezoelectric spray method. This method can achieve efficient loading of active ingredients such as plant cell exosomes, natural plant extracts, diaminopyrimidine oxides, and pyrrolidine diaminopyrimidine oxides. Specifically, during the process of adding to the composite solution, the composite solution is continuously stirred. Specifically, when preparing hydrogel polysaccharide active microspheres, a functionalized sodium alginate polymer, a functionalized sodium alginate polymer, and sodium carboxymethyl starch are made into a mixed solution, and the mixed solution is added to the composite solution by a piezoelectric spray method. More specifically, the volume ratio of the mixed solution to the composite solution is 1:9 to 11.

[0021] In some embodiments, the plants in the plant cell exosomes include but are not limited to at least one of Polygonum multiflorum, Codonopsis pilosula, Millettia reticulata, Cuscuta australis, and Platycladus orientalis leaves.

[0022] In some embodiments, the source of the natural plant extract is at least one of safflower, Centella asiatica, aloe, mint, angelica, ligusticum chuanxiong, and atractylodes macrocephala.

[0023] In some embodiments, the mass concentration of plant cell exosomes in the composite solution is 0.1-10 mg / mL.

[0024] In some embodiments, the mass ratio of plant cell exosomes, natural plant extracts, diaminopyrimidine oxide, and diaminoselenohydrochloride is 1-100:10-500:10-200:1-200.

[0025] In some embodiments, the mass ratio of diaminopyrimidine oxide to pyrrolidino diaminopyrimidine oxide is 2:1-1:3.

[0026] In some embodiments, when preparing hydrogel polysaccharide active microspheres, the mass concentration of the functionalized sodium alginate polymer solution added is 1-10 mg / mL.

[0027] In some embodiments, when preparing hydrogel polysaccharide active microspheres, the mass concentration of sodium carboxymethyl starch solution added is 2.5-100 mg / mL.

[0028] In some embodiments, when preparing hydrogel polysaccharide active microspheres, the mass concentration of the functionalized sodium alginate polymer solution added is 1-50 mg / mL.

[0029] In some embodiments, the diameter of the prepared hydrogel polysaccharide active microspheres is 10-100 μm.

[0030] In some embodiments, a multifunctional polyglutamic acid polymer is added to a solution of a photoinitiator and mixed evenly to obtain a polyglutamic acid solution, a multifunctional gelatin polymer is added to a solution of a photoinitiator and mixed evenly to obtain a gelatin solution, and the polyglutamic acid solution and the gelatin solution are mixed and shear mixed to form a polypeptide microgel.

[0031] In some embodiments, the mass concentration of the multifunctional polyglutamic acid polymer solution is 5-100 mg / mL.

[0032] In some embodiments, the mass concentration of the multifunctional gelatin polymer solution is 10-200 mg / mL.

[0033] In some embodiments, the mass of hydrogel polysaccharide active microspheres added to each milliliter of peptide microgel is 5 to 100 mg.

[0034] In some embodiments, the ratio of amino-terminated polyethylene glycol added to each milliliter of peptide microgel is 5 to 100 mg.

[0035] In some embodiments, the particle size of the peptide microgel is 30-1000 μm.

[0036] The following steps are preferred:

[0037] (1) The side chains of polyglutamic acid were sequentially acrylated and modified with cucurbituril using glycidyl methacrylate and amino cucurbituril to obtain a multifunctional polyglutamic acid polymer that was methacrylated and cucurbituril-modified;

[0038] (2) Using glycidyl methacrylate and tryptophan to sequentially acryloylate and tryptophan-modify the side chains of gelatin to obtain a multifunctional gelatin polymer modified with acryloylate and tryptophan;

[0039] (3) Using NaIO4 to oxidize the vicinal diol structure of sodium alginate and hyaluronic acid to prepare oxidized sodium alginate polymer and oxidized sodium hyaluronate polymer, and then using arginine to modify the side chain carboxyl group of the oxidized sodium alginate to prepare a functionalized sodium alginate polymer; using amino azobenzene to functionalize the oxidized sodium hyaluronate to prepare a functionalized sodium hyaluronate polymer;

[0040] (4) Dissolve the sodium magnesium lithium silicate / calcium glycerophosphate to form a uniform mixed solution, then disperse and dissolve the plant cell exosomes, natural plant extracts, diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, and diaminoselenohydrochloride in the sodium magnesium lithium silicate / calcium glycerophosphate solution to form a uniform composite solution, then use a piezoelectric nozzle to dissolve the functionalized sodium alginate polymer, the functionalized sodium hyaluronate polymer, and sodium carboxymethyl starch and spray them into the continuously stirred composite solution, and use the intermolecular force to load the plant cell exosomes, natural plant extracts, diaminopyrimidine oxide, and pyrrolidino diaminopyrimidine oxide into the hydrogel to form hydrogel polysaccharide active microspheres, which are finally collected and freeze-dried for later use;

[0041] (5). The multifunctional polyglutamic acid polymer and multifunctional gelatin polymer prepared in steps (1) and (2) are dissolved in the photoinitiator solution to form a polyglutamic acid solution and a gelatin solution respectively. The polyglutamic acid and gelatin solutions are fully mixed with a homogenizer under high-speed stirring. The aromatic residues and tryptophan in the side chain of the multifunctional gelatin polymer and the host-guest supramolecular interaction of cucurbituril in the multifunctional polyglutamic acid polymer are used to form a peptide microgel under continuous shearing. Then, the hydrogel polysaccharide active microspheres prepared in step (4) and the amino-terminated polyethylene glycol are added and mixed evenly. The microspheres are fixed to the microgel system by the Schiff base reaction of the amino group, guanidine group and other groups in the microgel system with the aldehyde group in the microspheres and the multiple cross-linking reaction formed by the host-guest supramolecular interaction of the azobenzene group in the microspheres with the cucurbituril group in the microgel system to prevent the microspheres from sedimentation.

[0042] In a second aspect, a printable glycopeptide microgel / microsphere composite ink is obtained by the above preparation method.

[0043] In a third aspect, an integrated hydrogel microneedle is made of the above-mentioned printable glycopeptide microgel / microsphere composite ink.

[0044] In some embodiments, the photocuring 3D printing method is used. Specifically, the photocuring 3D printing method is pneumatic extrusion.

[0045] In some embodiments, the length of the hydrogel microneedle is 30-700 μm.

[0046] In a fourth aspect, a use of the above-mentioned printable glycopeptide microgel / microsphere composite ink or integrated hydrogel microneedle in the preparation of anti-hair loss or hair solidification products.

[0047] Specifically, the product can be daily chemicals (such as cosmetics, toiletries, etc.), medicines, etc.

[0048] Specifically, the anti-hair loss or hair strengthening product has the functions of scalp care, hair follicle nourishment, soft tissue repair and regeneration, etc.

[0049] The beneficial effects of the present invention are:

[0050] The present invention utilizes the good injectability, high porosity, high specific surface area, tissue permeability and high tissue compatibility of the microgel / microsphere system to design a light / active oxygen dual-responsive active ingredient precise and efficient delivery system, namely, printable microgel / microsphere composite glycopeptide ink, and an integrated hydrogel microneedle prepared by 3D printing technology, which improves the high permeability of the composite system to reconstruct the dynamic cell microenvironment of damaged tissues, reshapes the damaged dynamic cell microenvironment for damaged tissues and organs from a physiological perspective, and provides a physical barrier for them. The high specific surface area and permeability of microgels enhance the delivery of nutrients such as oxygen, growth factors, external active ingredients, and the transportation of carbon dioxide and cell metabolic waste, providing sufficient 3D space for cell attachment, proliferation and migration, increasing the retention of cells in damaged tissues, and improving the interaction between cells and between cells and matrix. The azobenzene groups in the host-guest molecules are broken by ultraviolet light irradiation, thereby breaking the cross-links formed by the host-guest chemical reaction, achieving microsphere dissociation, and promoting the breaking of diselenide on the surface of the microspheres by overexpressed reactive oxygen species under physiological conditions to achieve programmed release of encapsulated substances, ultimately improving the vitality of endogenous cells, guiding cell homing, promoting hair follicle regeneration, and achieving the effects of preventing hair loss and strengthening hair. In addition, by modifying the hydrophilic and hydrophobic groups of the gelatin of the microgel skeleton material, it is possible to avoid the blockage of the nozzle due to the thermosensitive behavior of gelatin and other behaviors that hinder the operation process. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0052] Example 1

[0053] Preparation of functionalized polymers:

[0054] Preparation of multifunctional polyglutamic acid polymer: 1g polyglutamic acid is completely dissolved in an aqueous solution to form an aqueous solution with a mass concentration of 1% and adjusted to pH 5 using 1M dilute hydrochloric acid, 3g amino cucurbituril is added and reacted at room temperature for 24 hours under the action of carboxyl coupling agent EDC / NHS, so that cucurbituril is modified into the side chain of polyglutamic acid, and then heated to 60°C, and then 2g glycidyl methacrylate is added and reacted for 8 hours to achieve acrylation of the residual carboxyl groups of the side chain of polyglutamic acid, and then the reaction solution is dialyzed (dialysis bag cutoff 3500Da, dialysis for 72 hours) and then freeze-dried to obtain a multifunctional polyglutamic acid polymer double-modified by methacrylylation and cucurbituril. The chemical substitution degree of methacrylylation and cucurbituril is calculated using nuclear magnetic resonance hydrogen spectrum.

[0055] Preparation of multifunctional gelatin polymer:

[0056] 10g of gelatin was completely dissolved in MES buffer (pH 5.5) at 40°C to form a gelatin solution with a mass concentration of 10%. Under this condition, 5g of tryptophan and carboxyl coupling agent EDC / NHS (4g each) were added. After complete dissolution, the reaction was continued for 24 hours to modify the tryptophan to the side chain of gelatin. Then, 5g of glycidyl methacrylate was added to react for 8 hours at 60°C, so that the glycidyl methacrylate acrylated the residual carboxyl groups of the gelatin side chain functionalized with tryptophan, and then the reaction solution was dialyzed (dialysis bag cutoff 3500Da, dialysis for 72 hours) and freeze-dried to obtain a multifunctional gelatin polymer double-modified with methacrylylation and tryptophan. The chemical substitution degree of methacrylylation and tryptophan was calculated using nuclear magnetic resonance hydrogen spectrum.

[0057] Preparation of functionalized sodium alginate polymer and functionalized sodium hyaluronate polymer: 2% mass concentration NaIO4 solution (10 mL) was added to 1% mass concentration sodium alginate solution (100 mL) and 0.5% mass concentration sodium hyaluronate solution (100 mL), respectively, and reacted at room temperature for 3 hours to oxidize the vicinal diol groups in the structure, thereby preparing oxidized sodium alginate polymer and oxidized sodium hyaluronate polymer; 8 g of arginine was added to the solution after the oxidation reaction of sodium alginate to dissolve, and then carboxyl coupling agent EDC / NHS (the mass ratio of sodium alginate, EDC and NHS was 1:1:1) was added in sequence, and the pH was adjusted to 5, and then reacted at room temperature for 24 hours. The modification of the carboxyl group of the side chain of the oxidized sodium alginate is achieved to prepare a functionalized sodium alginate polymer; similarly, 10g of amino azobenzene is added to the solution after the hyaluronic acid oxidation reaction to dissolve, and then the carboxyl coupling agent EDC / NHS (the mass ratio of sodium hyaluronate, EDC and NHS is 1:1:1) is added in sequence, and the pH is adjusted to 5, and the reaction is carried out at room temperature for 24 hours to achieve the functional modification of the oxidized sodium hyaluronate, thereby preparing a functionalized sodium hyaluronate polymer; finally, the reaction solution is dialyzed (dialysis bag cutoff 3500Da, dialysis 72 hours) and freeze-dried to obtain a functionalized sodium alginate polymer and a functionalized sodium hyaluronate polymer. The oxidation degree of sodium alginate and hyaluronic acid is tested using the hydroxylamine hydrochloride method, and the chemical substitution degree of arginine and azobenzene is calculated using nuclear magnetic resonance hydrogen spectrum.

[0058] Embodiment 2-8

[0059] The preparation process is the same as that in Example 1. By reasonably adjusting the feed ratios of key chemical molecules such as glycidyl methacrylate, amino cucurbituril, tryptophan, arginine, and NaIO4 in Example 1, the programmed regulation of the degree of substitution is achieved. The specific degree of substitution is shown in Table 2:

[0060] Table 1 Feeding amount of functional molecules of functionalized polymers

[0061]

[0062] Table 2 Chemical substitution degree of functionalized polymers

[0063]

[0064]

[0065] The microsphere system was prepared using functionalized polymers with different chemical substitution degrees described in Examples 1-8 in Table 1. The specific preparation method is as follows:

[0066] Embodiment 9:

[0067] Sodium magnesium lithium silicate / calcium glycerophosphate (the mass ratio of sodium magnesium lithium silicate to calcium glycerophosphate is 7:3) was dissolved to form a 2 mg / mL mixed solution, and then Polygonum multiflorum plant cell exosomes (Nanomedicine: Nanotechnology, Biology and Medicine, 2023, 47: 102625. The following exosome preparation methods all adopt this method), Millettia spatholobi plant cell exosomes, Centella asiatica extract (Guilin Yitiancheng Biotechnology Co., Ltd.), Angelica sinensis extract (Shaanxi Baichuan Biotechnology Co., Ltd.), diaminopyrimidine oxide, pyrrolidinodiaminopyrimidine oxide, selenocystamine dihydrochloride and sequentially dissolved to form a uniform composite solution. The mass concentrations of all components in the final composite solution were 1.4 mg / mL sodium magnesium lithium silicate, 0.6 mg / mL calcium glycerophosphate, 0.1 mg / mL Polygonum multiflorum plant cell exosomes, 0.2 mg / mL Millettia spatholobi plant cell exosomes, 5 mg / mL Centella asiatica extract, 2 mg / mL Angelica sinensis extract, 5 mg / mL diaminopyrimidine oxide, 5 mg / mL pyrrolidinodiaminopyrimidine oxide, and 1 mg / mL selenocystamine dihydrochloride. Then, a piezoelectric nozzle was used to spray 10 mL of the prepared functionalized sodium alginate polymer (Example 1), functionalized sodium hyaluronate polymer (Example 1) and sodium carboxymethyl starch polysaccharide mixed solution (the mass concentrations of all components in the final polysaccharide mixed solution were 5 mg / mL of functionalized sodium alginate polymer (Example 2), 5 mg / mL of functionalized sodium hyaluronate polymer (Example 3) and 10 mg / mL of sodium carboxymethyl starch) into the continuously stirred 100 mL composite solution, and the active ingredients were loaded into the hydrogel by the intermolecular force to form hydrogel polysaccharide active microspheres. Finally, a microscope was used to take pictures to record the particle size of the microspheres, and a Zeta potential analyzer was used to test the surface charge of the microspheres.

[0068] Embodiment 10:

[0069] After dissolving sodium magnesium lithium silicate / calcium glycerophosphate to form a mixed solution of 1 mg / mL, exosomes from Polygonum multiflorum plant cells, exosomes from Millettia spatholobi plant cells, Centella asiatica extract, Angelica sinensis extract, diaminopyrimidine oxide, pyrrolidinodiaminopyrimidine oxide, and selenocystamine dihydrochloride were added to the mixed solution in sequence and dissolved in sequence to form a uniform composite solution. The mass concentrations of all components in the final composite solution were 0.7 mg / mL sodium magnesium lithium silicate, 0.3 mg / mL glycerophosphate, 0.1 mg / mL Polygonum multiflorum plant cell exosomes, 0.1 mg / mL Millettia spatholobi plant cell exosomes, 1 mg / mL Centella asiatica extract, 2 mg / mL Angelica sinensis extract, 5 mg / mL diaminopyrimidine oxide, 10 mg / mL pyrrolidinodiaminopyrimidine oxide, and 1 mg / mL selenocystamine dihydrochloride. Then, a piezoelectric nozzle was used to spray 10 mL of the prepared functionalized sodium alginate polymer (Example 2), functionalized sodium hyaluronate polymer (Example 3) and sodium carboxymethyl starch polysaccharide mixed solution (the mass concentrations of all components in the final polysaccharide mixed solution were 10 mg / mL of functionalized sodium alginate polymer (Example 2), 5 mg / mL of functionalized sodium hyaluronate polymer (Example 3) and 15 mg / mL of sodium carboxymethyl starch) into the continuously stirred 100 mL composite solution, and the active ingredients were loaded into the hydrogel by intermolecular forces to form hydrogel polysaccharide active microspheres. Finally, a microscope was used to take pictures to record the particle size of the microspheres, and a Zeta potential analyzer was used to test the surface charge of the microspheres.

[0070] Embodiment 11:

[0071] After dissolving sodium magnesium lithium silicate / calcium glycerophosphate to form a mixed solution of 5 mg / mL, exosomes of Polygonum multiflorum plant cells, exosomes of Millettia spatholobi plant cells, Centella asiatica extract, Angelica sinensis extract, diaminopyrimidine oxide, and pyrrolidinodiaminopyrimidine oxide were added to the mixed solution in sequence and dissolved in sequence to form a uniform composite solution. The mass concentrations of all components in the final composite solution were 3.5 mg / mL sodium magnesium lithium silicate, 1.5 mg / mL glycerophosphate, 0.1 mg / mL Polygonum multiflorum plant cell exosomes, 0.2 mg / mL Millettia spatholobi plant cell exosomes, 5 mg / mL Centella asiatica extract, 2 mg / mL Angelica sinensis extract, 5 mg / mL diaminopyrimidine oxide, and 5 mg / mL pyrrolidinodiaminopyrimidine oxide. Then, a piezoelectric nozzle was used to spray 10 mL of the prepared functionalized sodium alginate polymer (Example 4), functionalized sodium hyaluronate polymer (Example 5) and sodium carboxymethyl starch polysaccharide mixed solution (the mass concentrations of all components in the final polysaccharide mixed solution were 5 mg / mL of functionalized sodium alginate polymer (Example 4), 10 mg / mL of functionalized sodium hyaluronate polymer (Example 5) and 10 mg / mL of sodium carboxymethyl starch) into the continuously stirred 100 mL composite solution, and the active ingredients were loaded into the hydrogel by the intermolecular force to form hydrogel polysaccharide active microspheres. Finally, the microsphere particle size was recorded by taking pictures with a microscope, and the surface charge of the microspheres was tested by a Zeta potential analyzer.

[0072] Embodiment 12:

[0073] After dissolving sodium magnesium lithium silicate / calcium glycerophosphate to form a mixed solution of 2 mg / mL, Atractylodes macrocephala extract (Shaanxi New Horizon Biotechnology Co., Ltd.), diaminopyrimidine oxide, pyrrolidinodiaminopyrimidine oxide, and selenocystamine dihydrochloride were added to the mixed solution in sequence and dissolved in sequence to form a uniform composite solution. The mass concentrations of all components in the final composite solution were 1.4 mg / mL sodium magnesium lithium silicate, 0.6 mg / mL glycerolphosphate, 5 mg / mL Atractylodes macrocephala extract, 5 mg / mL diaminopyrimidine oxide, 5 mg / mL pyrrolidinodiaminopyrimidine oxide, and 1 mg / mL selenocystamine dihydrochloride. Then, a piezoelectric nozzle was used to spray 10 mL of the prepared functionalized sodium alginate polymer (Example 5), functionalized sodium hyaluronate polymer (Example 4) and sodium carboxymethyl starch polysaccharide mixed solution (the mass concentrations of all components in the final polysaccharide mixed solution were 5 mg / mL of functionalized sodium alginate polymer (Example 4), 5 mg / mL of functionalized sodium hyaluronate polymer (Example 5) and 10 mg / mL of sodium carboxymethyl starch) into 100 mL of the continuously stirred composite solution, and the active ingredients were loaded into the hydrogel by intermolecular forces to form hydrogel polysaccharide active microspheres. Finally, a microscope was used to take pictures to record the particle size of the microspheres, and a Zeta potential analyzer was used to test the surface charge of the microspheres.

[0074] Embodiment 13:

[0075] After dissolving sodium magnesium lithium silicate / calcium glycerophosphate to form a mixed solution of 2 mg / mL, exosomes from Polygonum multiflorum plant cells, exosomes from Millettia spatholobi plant cells, Centella asiatica extract, Angelica sinensis extract, diaminopyrimidine oxide, pyrrolidinodiaminopyrimidine oxide, and selenocystamine dihydrochloride were added to the mixed solution in sequence and dissolved in sequence to form a uniform composite solution. The mass concentrations of all components in the final composite solution were 1.4 mg / mL sodium magnesium lithium silicate, 0.6 mg / mL glycerophosphate, 0.1 mg / mL Polygonum multiflorum plant cell exosomes, 0.2 mg / mL Millettia spatholobi plant cell exosomes, 5 mg / mL Centella asiatica extract, 2 mg / mL Angelica sinensis extract, 5 mg / mL diaminopyrimidine oxide, 5 mg / mL pyrrolidinodiaminopyrimidine oxide, and 1 mg / mL selenocystamine dihydrochloride. Then, a piezoelectric nozzle was used to spray 10 mL of the prepared functionalized sodium hyaluronate polymer (Example 6) and sodium carboxymethyl starch polysaccharide mixed solution (the mass concentrations of all components in the final polysaccharide mixed solution were 5 mg / mL of functionalized sodium hyaluronate polymer (Example 6) and 5 mg / mL of sodium carboxymethyl starch, respectively) into the 100 mL composite solution that was constantly stirred, and the active ingredients were loaded into the hydrogel by intermolecular forces to form hydrogel polysaccharide active microspheres. Finally, a microscope was used to take pictures to record the particle size of the microspheres, and a Zeta potential analyzer was used to test the surface charge of the microspheres.

[0076] Embodiment 14:

[0077] After sodium magnesium lithium silicate / calcium glycerophosphate was dissolved to form a 2 mg / mL mixed solution, Polygonum multiflorum plant cell exosomes and selenocystamine dihydrochloride were added to the mixed solution in sequence and dissolved in sequence to form a uniform composite solution. The mass concentrations of all components in the final composite solution were 1.4 mg / mL sodium magnesium lithium silicate, 0.6 mg / mL glycerophosphate, 0.2 mg / mL Polygonum multiflorum plant cell exosomes, and 2 mg / mL selenocystamine dihydrochloride. Then, a piezoelectric nozzle was used to spray 10 mL of the prepared sodium carboxymethyl starch solution of 10 mg / mL into the 100 mL composite solution that was constantly stirred, and the active ingredients were loaded into the hydrogel by the intermolecular force to form hydrogel polysaccharide active microspheres. Finally, a microscope was used to take pictures to record the size of the microspheres, and a Zeta potential analyzer was used to test the surface charge of the microspheres.

[0078] Table 3 Particle size, potential and spherical properties of hydrogel microspheres

[0079]

[0080] Table 3 shows that spherical hydrogel polysaccharide active microspheres can only be prepared by adding functionalized sodium alginate polymer and functionalized sodium hyaluronate polymer at the same time.

[0081] The microgel system was prepared using functionalized polymers with different chemical substitution degrees described in Examples 1-8 in Table 1. The specific preparation method is as follows:

[0082] Embodiment 15:

[0083] The multifunctional polyglutamic acid polymer (Example 1) and the multifunctional gelatin polymer (Example 1) were dissolved in a photoinitiator solution (LAP 0.2 mg / mL) to form a polyglutamic acid solution (concentration of 20 mg / mL) and a gelatin solution (concentration of 50 mg / mL), and the polyglutamic acid and gelatin solutions were mixed in equal volumes with a homogenizer under high-speed stirring (8000 rad / min) for 20 minutes. During the mixing process, the aromatic residues and tryptophan in the side chain of the multifunctional gelatin polymer interacted with the host-guest supramolecular interaction of the cucurbituril in the multifunctional polyglutamic acid polymer to form a peptide microgel under continuous shearing. The particle size and potential of the microgel were measured by a nanoparticle size and Zeta potential analyzer, and then the rheological properties of the microgel were tested by a rheometer to evaluate the physicochemical properties of the microgel.

[0084] Embodiment 16:

[0085] 10 mg / mL of multifunctional polyglutamic acid polymer (Example 2) and 50 mg / mL of multifunctional gelatin polymer (Example 3) were dissolved in a photoinitiator solution (LAP 0.2 mg / mL) to form a polyglutamic acid solution and a gelatin solution, and a homogenizer was used to fully mix the polyglutamic acid and gelatin solutions in equal volumes under high-speed stirring (5000 rad / min) for 20 minutes. During the mixing process, the aromatic residues and tryptophan in the side chain of the multifunctional gelatin polymer and the host-guest supramolecular interaction of cucurbituril in the multifunctional polyglutamic acid polymer were used to form a peptide microgel under continuous shearing. The particle size and potential of the microgel were measured by a nanoparticle size and Zeta potential analyzer, and then the rheological properties of the microgel were tested by a rheometer to evaluate the physicochemical properties of the microgel.

[0086] Embodiment 17:

[0087] 30 mg / mL of multifunctional polyglutamic acid polymer (Example 2) and 80 mg / mL of multifunctional gelatin polymer (Example 6) were dissolved in a photoinitiator solution (LAP 0.2 mg / mL) to form a polyglutamic acid solution and a gelatin solution, and a homogenizer was used to fully mix the polyglutamic acid and gelatin solutions in equal volumes under high-speed stirring (10000 rad / min) for 20 minutes. During the mixing process, the aromatic residues and tryptophan in the side chain of the multifunctional gelatin polymer and the host-guest supramolecular interaction of cucurbituril in the multifunctional polyglutamic acid polymer were used to form a peptide microgel under continuous shearing. The particle size and potential of the microgel were measured by a nanoparticle size and Zeta potential analyzer, and then the rheological properties of the microgel were tested by a rheometer to evaluate the physicochemical properties of the microgel.

[0088] Embodiment 18:

[0089] 30 mg / mL of multifunctional polyglutamic acid polymer (Example 5) and 50 mg / mL of multifunctional gelatin polymer (Example 4) were dissolved in a photoinitiator solution (LAP 0.2 mg / mL) to form a polyglutamic acid solution and a gelatin solution, and a homogenizer was used to fully mix the polyglutamic acid and gelatin solutions in equal volumes under high-speed stirring (10000 rad / min) for 20 minutes. During the mixing process, the aromatic residues in the side chains of the multifunctional gelatin polymer and the host-guest supramolecular interaction of cucurbituril in the multifunctional polyglutamic acid polymer were used to form a peptide microgel under continuous shearing. The particle size and potential of the microgel were measured by a nanoparticle size and Zeta potential analyzer, and then the rheological properties of the microgel were tested by a rheometer to evaluate the physicochemical properties of the microgel.

[0090] Embodiment 19:

[0091] 10 mg / mL of multifunctional polyglutamic acid polymer (Example 6) and 80 mg / mL of multifunctional gelatin polymer (Example 6) were dissolved in a photoinitiator solution (LAP 0.2 mg / mL) to form a polyglutamic acid solution and a gelatin solution, and a homogenizer was used to fully mix the polyglutamic acid and gelatin solutions in equal volumes under high-speed stirring (6000 rad / min) for 20 minutes. During the mixing process, the aromatic residues and tryptophan in the side chain of the multifunctional gelatin polymer and the host-guest supramolecular interaction of cucurbituril in the multifunctional polyglutamic acid polymer were used to form a polypeptide microgel under continuous shearing. The particle size and potential of the microgel were measured by a nanoparticle size and Zeta potential analyzer, and then the rheological properties of the microgel were tested by a rheometer to evaluate the physicochemical properties of the microgel.

[0092] Embodiment 20:

[0093] 30 mg / mL of multifunctional polyglutamic acid polymer (Example 8) and 30 mg / mL of multifunctional gelatin polymer (Example 8) were dissolved in a photoinitiator solution (LAP 0.2 mg / mL) to form a polyglutamic acid solution and a gelatin solution, and a homogenizer was used to fully mix the polyglutamic acid and gelatin solutions in equal volumes under high-speed stirring (5000 rad / min) for 20 minutes. During the mixing process, the aromatic residues and tryptophan in the side chain of the multifunctional gelatin polymer and the host-guest supramolecular interaction of cucurbituril in the multifunctional polyglutamic acid polymer were used to form a polypeptide microgel under continuous shearing. The particle size and potential of the microgel were measured by a nanoparticle size and Zeta potential analyzer, and then the rheological properties of the microgel were tested by a rheometer to evaluate the physicochemical properties of the microgel.

[0094] Embodiment 21:

[0095] 30 mg / mL of multifunctional polyglutamic acid polymer (Example 4) and 70 mg / mL of multifunctional gelatin polymer (Example 5) were dissolved in a photoinitiator solution (LAP 0.2 mg / mL) to form a polyglutamic acid solution and a gelatin solution, and a homogenizer was used to fully mix the polyglutamic acid and gelatin solutions in equal volumes under high-speed stirring (7000 rad / min) for 20 minutes. During the mixing process, the aromatic residues and tryptophan in the side chain of the multifunctional gelatin polymer and the host-guest supramolecular interaction of cucurbituril in the multifunctional polyglutamic acid polymer were used to form a peptide microgel under continuous shearing. The particle size and potential of the microgel were measured by a nanoparticle size and Zeta potential analyzer, and then the rheological properties of the microgel were tested by a rheometer to evaluate the physicochemical properties of the microgel.

[0096] Table 4 Particle size, potential and rheological properties of microgels

[0097]

[0098] The microsphere / microgel glycopeptide composite system was prepared using the different polysaccharide microsphere systems described in Examples 9-14 in Table 3 and the polypeptide microgel systems described in Examples 15-21 in Table 4. The specific preparation method is as follows:

[0099] Embodiment 22:

[0100] 20 mg of the polysaccharide microspheres prepared in Example 9 and 10 mg of amino-terminated polyethylene glycol were added to 1 mL of the polypeptide microgel system prepared in Example 15, and the mixture was mixed at room temperature for 20 minutes to mix evenly. The mass concentration of the microgel in the microgel / microsphere composite system was finally formed. During the mixing process, the microspheres were fixed to the microgel system by the Schiff base reaction of the amino group, guanidine group and other groups in the microgel system with the aldehyde group in the microspheres, and the multiple cross-linking reactions formed by the host-guest supramolecular interaction between the azobenzene group in the microspheres and the cucurbituril group in the microgel system to prevent the microspheres from depositing, thereby preparing a printable glycopeptide microgel / microsphere composite ink, and the integrated hydrogel microneedles were prepared by pneumatic extrusion using a photocurable 3D printer.

[0101] Embodiment 23:

[0102] 10 mg of the polysaccharide microspheres prepared in Example 12 and 20 mg of amino-terminated polyethylene glycol were added to 1 mL of the peptide microgel system prepared in Example 16, and the mixture was mixed at room temperature for 20 minutes to make it uniformly mixed. During the mixing process, the microspheres were fixed to the microgel system by the Schiff base reaction of the amino group, guanidine group and other groups in the microgel system with the aldehyde group in the microspheres, and the multiple cross-linking reactions formed by the host-guest supramolecular interaction between the azobenzene group in the microspheres and the cucurbituril group in the microgel system to prevent the microspheres from depositing, thereby preparing a printable glycopeptide microgel / microsphere composite ink, and using a light-curing 3D printer to prepare integrated hydrogel microneedles by pneumatic extrusion.

[0103] Embodiment 24:

[0104] 20 mg of the polysaccharide microspheres prepared in Example 14 and 20 mg of amino-terminated polyethylene glycol were added to 1 mL of the peptide microgel system prepared in Example 17, and the mixture was mixed at room temperature for 20 minutes to make it uniformly mixed. During the mixing process, the microspheres were fixed to the microgel system by the Schiff base reaction of the amino group, guanidine group and other groups in the microgel system with the aldehyde group in the microspheres, and the multiple cross-linking reactions formed by the host-guest supramolecular interaction between the azobenzene group in the microspheres and the cucurbituril group in the microgel system to prevent the microspheres from depositing, thereby preparing a printable glycopeptide microgel / microsphere composite ink, and the integrated hydrogel microneedles were prepared by pneumatic extrusion using a photocurable 3D printer.

[0105] Embodiment 25:

[0106] 20 mg of the polysaccharide microspheres prepared in Example 9 were added to 1 mL of the peptide microgel system prepared in Example 18, and the mixture was mixed at room temperature for 20 minutes to make it uniformly mixed. During the mixing process, the microspheres were fixed to the microgel system by the Schiff base reaction of the amino group, guanidine group and other groups in the microgel system with the aldehyde group in the microspheres, and the multiple cross-linking reactions formed by the host-guest supramolecular interaction between the azobenzene group in the microspheres and the cucurbituril group in the microgel system to prevent the microspheres from depositing, thereby preparing a printable glycopeptide microgel / microsphere composite ink, and using a light-curing 3D printer to prepare integrated hydrogel microneedles by pneumatic extrusion.

[0107] Embodiment 26:

[0108] 20 mg of the polysaccharide microspheres prepared in Example 10 and 10 mg of amino-terminated polyethylene glycol were added to 1 mL of the peptide microgel system prepared in Example 20, and the mixture was mixed at room temperature for 20 minutes to make it uniformly mixed. During the mixing process, the microspheres were fixed to the microgel system by the Schiff base reaction of the amino group, guanidine group and other groups in the microgel system with the aldehyde group in the microspheres, and the multiple cross-linking reactions formed by the host-guest supramolecular interaction between the azobenzene group in the microspheres and the cucurbituril group in the microgel system to prevent the microspheres from depositing, thereby preparing a printable glycopeptide microgel / microsphere composite ink, and the integrated hydrogel microneedles were prepared by pneumatic extrusion using a photocurable 3D printer.

[0109] The length, cell safety, and antioxidant properties of the integrated hydrogel microneedles prepared in Examples 22-26 were evaluated, and their controlled release properties were evaluated by hydrogen peroxide stimulation and ultraviolet light irradiation for 10 minutes. The results are shown in Table 5.

[0110] Table 5 Printing performance, biological characteristics and physicochemical properties of microgel / microsphere composite system

[0111]

[0112]

[0113] The results in Table 5 show that the printable microgel / microsphere composite ink provided by the present invention has excellent printability, cell compatibility, oxidative stress regulation ability and controlled release performance of active ingredients. Among them, the microsphere skeleton of Example 24 does not have a functionalized sodium alginate polymer modified with active oxygen responsiveness, so it does not have active oxygen responsiveness; in Example 25, because the multifunctional polyglutamic acid polymer and the multifunctional polyglutamic acid polymer are not acrylated, there is no UV cross-linking to maintain the basic form during the printing process, and the microgel / microsphere system formed by the temperature response of gelatin and the side chain group is difficult to maintain a stable shape, so it cannot be formed into a needle; in the microsphere skeleton of Example 26, the functionalized hyaluronic acid polymer is not modified with azobenzene with UV responsiveness, so it does not have UV release performance.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a printable glycopeptide microgel / microsphere composite ink, characterized in that: The steps include: The polyglutamic acid was modified by acryloylation and cucurbituril to obtain a multifunctional polyglutamic acid polymer. Multifunctional gelatin polymers were obtained by acryloylating and modifying gelatin with tryptophan. The vicinal diol structure of sodium alginate is oxidized to obtain an oxidized sodium alginate polymer, and the oxidized sodium alginate polymer is modified with arginine to obtain a functionalized sodium alginate polymer; The vicinal diol structure of hyaluronic acid is oxidized to obtain an oxidized sodium hyaluronate polymer, and the oxidized sodium hyaluronate polymer is modified with amino azobenzene to obtain a functionalized sodium hyaluronate polymer; Dissolving sodium magnesium lithium silicate and calcium glycerophosphate to form a mixed solution, adding plant cell exosomes, natural plant extracts, diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, and diaminoseleno hydrochloride, and mixing evenly to obtain a composite solution, and then adding functionalized sodium alginate polymer, functionalized sodium hyaluronate polymer, and sodium carboxymethyl starch, and mixing evenly to obtain hydrogel polysaccharide active microspheres; A multifunctional polyglutamic acid polymer, a multifunctional gelatin polymer and a photoinitiator are shear-mixed in a solution to form a peptide microgel, and then hydrogel polysaccharide active microspheres and amino-terminated polyethylene glycol are added and mixed evenly, and multiple cross-linking reactions are performed in the system to fix the microspheres to the microgel, thereby obtaining the composite ink; wherein the multiple cross-linking reactions include Schiff base reactions and supramolecular interactions between azobenzene groups and cucurbituril groups.

2. The method for preparing the printable glycopeptide microgel / microsphere composite ink according to claim 1, characterized in that: Polyglutamic acid was acrylated and modified with cucurbituril using glycidyl methacrylate and amino cucurbituril. Alternatively, gelatin is acrylated and tryptophan modified using glycidyl methacrylate and tryptophan.

3. The method for preparing the printable glycopeptide microgel / microsphere composite ink according to claim 1, characterized in that: NaIO4 is used to oxidize the vicinal diol structure of sodium alginate; Alternatively, NaIO4 is used to oxidize the vicinal diol structure of hyaluronic acid.

4. The method for preparing the printable glycopeptide microgel / microsphere composite ink according to claim 1, characterized in that: The piezoelectric spray method was used to add plant cell exosomes, natural plant extracts, diaminopyrimidine oxide, pyrrolidino diaminopyrimidine oxide, and diaminoseleno hydrochloride.

5. The method for preparing the printable glycopeptide microgel / microsphere composite ink according to claim 1, characterized in that: The plants in the plant cell exosomes include but are not limited to at least one of Polygonum multiflorum, Codonopsis pilosula, Millettia reticulata, Cuscuta australis, and Platycladus orientalis leaves; Or, the source of the natural plant extract is at least one of safflower, asiatica glycoside, aloe, mint, angelica, ligusticum chuanxiong, and atractylodes.

6. The method for preparing the printable glycopeptide microgel / microsphere composite ink according to claim 1, characterized in that: A multifunctional polyglutamic acid polymer is added to a photoinitiator solution and mixed evenly to obtain a polyglutamic acid solution. A multifunctional gelatin polymer is added to a photoinitiator solution and mixed evenly to obtain a gelatin solution. The polyglutamic acid solution and the gelatin solution are mixed and shear-mixed to form a polypeptide microgel.

7. The method for preparing the printable glycopeptide microgel / microsphere composite ink according to claim 1, characterized in that: The diameter of the prepared hydrogel polysaccharide active microspheres is 10-100 μm; Alternatively, the particle size of the peptide microgel is 30-1000 μm.

8. A printable glycopeptide microgel / microsphere composite ink, characterized in that: The method is obtained by any one of claims 1 to 7.

9. An integrated hydrogel microneedle, characterized in that: Made of the printable glycopeptide microgel / microsphere composite ink of claim 8; Preferably, it is made by light-curing 3D printing method; Preferably, the length of the hydrogel microneedle is 30-700 μm.

10. Use of the printable glycopeptide microgel / microsphere composite ink according to claim 8 or the integrated hydrogel microneedle according to claim 9 in the preparation of anti-hair loss or hair solidification products.

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