Poly-peptide-based microspheres with nanofiber structure and preparation method and application thereof
Polypeptide-based nanofiber microspheres prepared by gas shear microfluidics have solved the problems of insufficient osteoinductive activity and unstable drug release in bone defect repair, achieving efficient repair of bone defects and reduction of inflammation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI UNIV
- Filing Date
- 2026-06-05
- Publication Date
- 2026-07-31
AI Technical Summary
Existing nanofiber microspheres for bone defect repair suffer from problems such as insufficient osteoinductive activity, poor in vivo stability, cumbersome preparation process that is difficult to scale up, and lack of responsive drug release capability of traditional drug-loaded microspheres.
Polypeptide-based microspheres with nanofiber structures were prepared using gas shear microfluidics. By grafting polypeptides onto the surface of inorganic nanoparticles and modifying them with carboxylation, adenosine was loaded using pH-responsive borate ester bonds formed by phenylboronic acid groups, thus achieving controlled drug release.
It enhances osteoinductive activity and biocompatibility, avoids inorganic particle aggregation and burst release, achieves long-term stable drug release, and promotes efficient repair of bone defects and reduction of inflammation.
Smart Images
Figure CN122479199A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bone repair tissue engineering materials technology, and in particular relates to a polypeptide-based microsphere with a nanofiber structure, its preparation method and application. Background Technology
[0002] Bone defects are common clinical problems caused by trauma, infection, resection of bone tumors, or congenital malformations. Bone grafting is currently the "gold standard" for treating such injuries. However, autologous bone grafting remains the "gold standard" in clinical treatment, but it suffers from problems such as limited donors, secondary trauma to the bone harvesting site, infection, and pain. While allogeneic and xenogeneic bone grafts solve the source problem, they face challenges such as immune rejection, disease transmission risks, and insufficient osteogenic activity. Against this backdrop, bone tissue engineering offers a new direction for the treatment of bone defects.
[0003] The natural extracellular matrix (ECM) of bone cells is a fine network woven from collagen nanofibers. This nanoscale topology provides crucial physical signals for osteoblasts, significantly promoting cell adhesion, proliferation, and osteogenic differentiation. To mimic this natural structure, nanofiber membranes prepared by electrospinning have been extensively studied due to their ability to mimic the ECM structure. However, traditional electrospinning typically only produces two-dimensional thin film materials, exhibiting limitations such as restricted space filling and difficulty in cell infiltration when dealing with large or irregularly shaped three-dimensional bone defects. Nanofiber microspheres (NFMS), which combine nanofiber structures with microspheres, offer a novel therapeutic approach for bone defects. These microspheres possess high specific surface area, excellent stacking properties, and pore connectivity. NFMS provide a multifunctional and efficient new strategy for promoting bone defect repair.
[0004] In the field of bone tissue engineering, various materials have been used to prepare nanofiber microspheres for bone repair. Chinese invention patent CN109350768A discloses a method for preparing hydroxyapatite / modified polylactic acid composite microspheres with a network nanofiber structure using emulsification and thermally induced phase separation technology. However, the degradation products of polylactic acid are acidic, easily inducing local inflammatory reactions. Chinese invention patents CN113336977A and CN118307835A disclose preparation technologies for natural polymer nanofiber microspheres based on chitosan, gelatin, and silk fibroin, respectively. However, existing nanofiber microspheres for bone repair generally suffer from core defects such as uncontrollable degradation behavior, poor biocompatibility, and insufficient osteoinductive capacity, making it difficult to meet the application needs of clinical bone defect repair.
[0005] Synthetically synthesized peptides are chemically similar to natural collagen, and their degradation products are amino acids, exhibiting excellent biocompatibility. Currently, there are no patent reports on the preparation of fibrous microspheres using peptides via phase separation self-assembly. Summary of the Invention
[0006] Existing nanofiber microspheres for bone defect repair mainly suffer from the following technical defects: 1) Insufficient osteoinductive activity: Single-component polymer microspheres lack sufficient osteoinductive capacity; 2) Poor in vivo stability: Microspheres with inorganic active particles introduced through physical blending are prone to particle aggregation and burst release, and degradation products may induce local inflammatory reactions; 3) Limited preparation process: Existing nanofiber microsphere preparation processes are cumbersome, demanding, and have large batch-to-batch variations, making it difficult to achieve large-scale scaling.
[0007] In addition, traditional drug-loaded microspheres also have significant shortcomings: they lack responsive drug release capabilities, have short drug half-lives, cause significant side effects with systemic administration, and suffer from severe local burst release, making it impossible to achieve controllable and stable drug release.
[0008] To overcome the shortcomings of the existing technology, the present invention provides a polypeptide-based microsphere with a nanofiber structure, its preparation method and application.
[0009] This invention utilizes gas shear microfluidics to prepare peptide-based microspheres with nanofiber structures. First, peptide macromolecular chains are covalently grafted onto the surface of osteoinductively active nanoparticles. After carboxylation modification, aminophenylboronic acid is covalently grafted onto the peptide macromolecular chains to obtain osteoinductively active peptide raw materials. These modified peptide raw materials are then formulated into a solution, and nanofiber microspheres are prepared using gas shear microfluidics. The resulting nanofiber microspheres are loaded with osteogenic drugs via borate ester bonds, and after reaction and washing, pH-responsive drug-releasing peptide-based nanofiber microspheres are obtained.
[0010] The objective of this invention can be achieved through the following technical solutions: This invention first provides a method for preparing peptide-based microspheres with a nanofiber structure, comprising the following steps: (1) Preparation of polypeptides grafted onto inorganic nanoparticles; Aminated inorganic nanoparticles initiate ring-opening polymerization of polypeptide monomers to obtain polypeptides grafted onto inorganic nanoparticles. (2) Carboxylation-modified polypeptides grafted onto inorganic nanoparticles; The prepared polypeptide grafted onto inorganic nanoparticles was modified by carboxylation, and then aminophenylboronic acid was grafted onto it by chemical bonds to prepare a phenylboronic acid-modified polypeptide grafted onto inorganic nanoparticles. (3) Preparation of polypeptide-based microspheres with nanofiber structures: A polymer solution was prepared by grafting phenylboronic acid onto inorganic nanoparticles to form a polypeptide. The polymer solution was then dispersed into an ethanol collection phase using microfluidics combined with gas shearing to obtain polypeptide-based microspheres with nanofiber structures.
[0011] In one embodiment of the present invention, in step (1), the inorganic nanoparticles are zinc-doped mesoporous silica inorganic nanoparticles.
[0012] In one embodiment of the present invention, the zinc-doped mesoporous silica inorganic nanoparticles are prepared by dissolving hexadecyltrimethylammonium bromide and NaOH in deionized water, injecting a mixture of zinc nitrate hexahydrate, anhydrous ethanol, and tetraethyl orthosilicate using an injection pump, centrifuging, washing, and drying the product obtained from the reaction, and then calcining it at high temperature to obtain zinc-doped mesoporous silica inorganic nanoparticles.
[0013] In one embodiment of the present invention, the ratio of hexadecyltrimethylammonium bromide, NaOH and deionized water is 1g:(0.005~0.010)g:(400~600)mL, preferably 1g:0.007g:480mL.
[0014] In one embodiment of the present invention, the stirring temperature when hexadecyltrimethylammonium bromide and NaOH are dissolved in deionized water is 70~90°C, preferably 80°C; the stirring time is 20~40 min, preferably 30 min.
[0015] In one embodiment of the present invention, the molar concentration of zinc nitrate hexahydrate relative to tetraethyl orthosilicate is 2-25 mol%, preferably 5-20 mol%; based on 480 mL of deionized water, the amount of tetraethyl orthosilicate used is 3-8 mL, preferably 5 mL.
[0016] In one embodiment of the present invention, the injection rate of the injection pump for the mixture is 0.2~1.0 mL / min, preferably 0.5 mL / min; the reaction temperature is 70~90℃, preferably 80℃; and the reaction time is 1.5~3h, preferably 2h.
[0017] In one embodiment of the present invention, the calcination temperature is 500~600℃, preferably 550℃; the calcination time is 5~8 h, preferably 6 h.
[0018] In one embodiment of the present invention, step (1) involves preparing a polypeptide grafted onto inorganic nanoparticles, specifically including the following steps: (1-1) Modify inorganic nanoparticles by amylation; Inorganic nanoparticles were ultrasonically dispersed in anhydrous ethanol, and then 3-aminopropyltriethoxysilane (APTES) was added under stirring. The product was collected by centrifugation and washed repeatedly with anhydrous ethanol to completely remove unreacted reagents. After vacuum drying, surface-aminated inorganic nanoparticles were finally obtained. (1-2) Aminated inorganic nanoparticles and polypeptide monomers are dissolved in dichloromethane. The inorganic nanoparticles initiate the ring-opening polymerization of the polypeptide monomers to obtain polypeptides grafted onto the inorganic nanoparticles.
[0019] In one embodiment of the present invention, in step (1-1), based on 500 mg of inorganic nanoparticles, the amount of anhydrous ethanol used is 20-100 mL, and the amount of APTES used is 1-5 mL.
[0020] In one embodiment of the present invention, in step (1-1), the amination modification time is 12~72 h.
[0021] In one embodiment of the present invention, in step (1-2), the inorganic nanoparticles are dissolved in dichloromethane, and the mass fraction is 1% to 20%.
[0022] In one embodiment of the present invention, in step (1-2), the reaction temperature of the inorganic nanoparticles and the polypeptide monomer is 10~25 °C, and the reaction time is 3 days.
[0023] In one embodiment of the present invention, in step (1-2), the polypeptide monomer is selected from one or more of L-glutamic acid-γ-benzyl ester, aspartic acid, and phenylalanine.
[0024] In one embodiment of the present invention, in steps (1-2), the carboxyl content of the polypeptide is 10% to 100%. The polypeptide monomer is selected from BLG-NCA. BLG-NCA is an abbreviation for γ-benzyl-L-glutamate-N-carboxyanhydride, which is an amino acid derivative intracyclic anhydride monomer used to synthesize polyγ-benzyl-L-glutamate (PBLG).
[0025] In one embodiment of the present invention, in step (1-2), after the reaction is completed, the reaction solution settles in a large amount of methyl tert-butyl ether, and the volume ratio of the reaction solution to methyl tert-butyl ether is 1:10.
[0026] In one embodiment of the present invention, the preparation method of the phenylboronic acid-modified grafted polypeptide onto inorganic nanoparticles in step (2) is as follows: (2-1) Carboxylation modification of peptides grafted onto inorganic nanoparticles: The polypeptide grafted onto inorganic nanoparticles was dissolved in anhydrous dichloromethane, and trimethyliodosilane was added. The reaction was carried out under nitrogen atmosphere in the dark. After the reaction was completed, the reaction solution was washed with methyl tert-butyl ether, anhydrous ethanol and water, respectively. The precipitate was collected and washed repeatedly with water and ethanol until the washing solution was colorless and then dried under vacuum to obtain the carboxylated modified polypeptide grafted onto inorganic nanoparticles. (2-2) Preparation of phenylboronic acid-modified polypeptides grafted onto inorganic nanoparticles: The prepared carboxylated polypeptides grafted onto inorganic nanoparticles were dissolved in N,N-dimethylformamide, and N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added and reacted with aminophenylboronic acid to obtain phenylboronic acid-modified polypeptides grafted onto inorganic nanoparticles.
[0027] In one embodiment of the present invention, in step (2-1), based on 100 mL of anhydrous dichloromethane, the amount of peptide grafted onto inorganic nanoparticles is 0.5-5 g; the amount of trimethyliodosilane is 0.2-1 mL; the concentration of trimethyliodosilane is 0.1-1%; the reaction temperature is 40°C; and the reaction is carried out in the dark for 3-9 h.
[0028] In one embodiment of the present invention, in step (2-1), the carboxyl group ratio of the polypeptide grafted onto the inorganic nanoparticles is 10-50%.
[0029] In one embodiment of the present invention, in step (2-2), the carboxylated modified polypeptide grafted onto inorganic nanoparticles is dissolved in N,N-dimethylformamide; N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and aminophenylboronic acid are added, and the reaction is carried out at room temperature for 24 h.
[0030] In one embodiment of the present invention, in step (2-2), phenylboronic acid is modified and grafted onto the polypeptide of inorganic nanoparticles, and the grafting rate of phenylboronic acid groups is 5% to 60%.
[0031] In one embodiment of the present invention, the specific steps for preparing peptide-loaded drug-eluting nanofiber microspheres in step (3) are as follows: The obtained phenylboronic acid was modified and grafted onto inorganic nanoparticles and dissolved in 1,4-dioxane as the inner phase fluid of the coaxial needle. Nitrogen gas was used as the outer phase of the coaxial needle. Using gas shear microfluidics, the polymer solution was dispersed into anhydrous ethanol collecting phase (receiving bath). The polymer droplets formed nanofiber microspheres through phase separation. The microspheres were then sieved, washed, and freeze-dried to obtain peptide-based microspheres with nanofiber structures.
[0032] In one embodiment of the present invention, phenylboronic acid is modified and grafted onto inorganic nanoparticles dissolved in 1,4-dioxane, and its solid content is 1~3 wt%.
[0033] In one embodiment of the present invention, the polymer solution propulsion speed is 2~10 mL / h, and the receiving distance is 5~30 cm.
[0034] This invention further provides polypeptide-based microspheres with nanofiber structures prepared by the above method. The microspheres have a nanofiber structure, with the nanofibers on the surface and inside the microspheres arranged randomly or in an ordered manner. The particle size of the microspheres is 50–300 micrometers. The diameter of the nanofibers is 50–500 nm.
[0035] This invention further provides a drug-loaded nanofiber microsphere based on modified polypeptides, which is a polypeptide-based microsphere with a nanofiber structure loaded with drug. Because phenylboronic acid groups are grafted onto the polypeptide macromolecular chain, pH-responsive drug release from the microspheres is achieved through the formation of borate ester bonds between the drug and the phenylboronic acid groups. In one embodiment of this invention, the drug loading per 15 mg of microspheres is 200 μg to 600 μg.
[0036] The present invention further provides a method for preparing drug-loaded nanofiber microspheres based on modified polypeptides, wherein polypeptide-based microspheres with nanofiber structures are added to a solution containing drugs, soaked, and washed to obtain drug-loaded nanofiber microspheres based on modified polypeptides.
[0037] In one embodiment of the present invention, the drug is adenosine. The solution containing the drug is an adenosine solution of 0.5~1 mg / mL.
[0038] The present invention further provides the application of peptide-based microspheres with nanofiber structures and drug-loaded nanofiber microspheres based on modified peptides in the preparation of bone defect repair materials.
[0039] In this application, the peptide-based nanofiber microspheres exhibit good biocompatibility and self-assemble into nanofibers through hydrogen bonding and π-π stacking of aromatic groups, demonstrating the ability to mimic collagen fiber structures. However, research on applying peptide self-assembly to the production of nanofiber microspheres, especially further multifunctionalization for bone defect repair, has not been reported.
[0040] This invention utilizes self-synthesized polypeptides as the matrix material to prepare nanofiber microspheres. First, polypeptides are grafted onto inorganic nanoparticles to prepare an organic-inorganic hybrid material. Then, the polypeptide macromolecular chains are modified with carboxyl groups, and further covalently linked to aminophenylboronic acid via amide bonds to obtain a phenylboronic acid-modified polypeptide-based material. Using microfluidics combined with gas shearing technology, the final modified polypeptide-based material is prepared into microspheres with a nanofiber morphology to mimic the structure of extracellular matrix collagen fibers. The obtained nanofiber microspheres are then immersed in an adenosine solution to load adenosine.
[0041] On the other hand, adenosine, as an endogenous nucleoside molecule, promotes osteoblast and angiogenesis by activating A2A and A2B adenosine receptors; by activating A2A receptors, it can reduce the production of inflammatory factors such as TNF-α and increase anti-inflammatory factors such as IL-10, thus exhibiting a certain anti-inflammatory effect. After bone defects occur, the local pH may drop to a slightly acidic state due to factors such as inflammatory responses. Given the important role of adenosine in promoting bone regeneration and maintaining bone homeostasis, this invention utilizes peptide nanofiber microspheres containing phenylboronic acid groups to load adenosine. The pH-responsive borate ester bonds enable the loading and release of adenosine molecules, promoting bone tissue regeneration and reducing inflammation. This application verifies the bone defect repair effect of drug-loaded peptide nanofiber microspheres using animal models. This invention provides a new therapeutic strategy for bone defect repair.
[0042] This application provides a polypeptide-based nanofiber microsphere with a biomimetic nanofiber structure and bone regeneration function, as well as its preparation method and application.
[0043] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention uses covalent grafting to construct organic-inorganic hybrid peptide materials, which stably combine nanoparticles with osteoinductive activity with peptides. Compared with physical blending systems, it effectively avoids the aggregation, burst release and shedding of inorganic particles, and can achieve long-term sustained release of zinc ions, significantly improving the osteoinductive activity of the material. At the same time, the peptides have excellent biocompatibility, and the degradation products are non-immunogenic and non-acidic, which can effectively avoid the local inflammatory response induced by the degradation products and adapt to the in vivo environment for bone defect repair.
[0044] (2) This invention uses gas shear microfluidics combined with phase separation self-assembly to prepare nanofiber microspheres. By adjusting the fluid parameters, the particle size and fiber morphology of the microspheres can be precisely controlled. The preparation and self-assembly of microspheres can be completed under mild conditions of room temperature and pressure. This overcomes the limitations of traditional thermal phase separation and electrospinning methods, which are complicated, have harsh conditions, and are difficult to scale up. The process is stable and operable, and has the potential for industrial application.
[0045] (3) The microspheres prepared by the present invention have a porous structure of collagen nanofibers that mimics the natural extracellular matrix of osteoblasts, which can provide cells with an excellent microenvironment for adhesion, proliferation and differentiation, effectively promote the adhesion and spread of osteoblasts, and improve osteogenic differentiation efficiency.
[0046] (4) This invention utilizes the functionalization modification of phenylboronic acid to form a pH-responsive borate ester bond with the cis-ortho-dihydroxy structure of adenosine, thereby achieving efficient loading and pH-responsive release of adenosine. In the acidic environment of local inflammation in bone defects, the borate ester bond breaks, and adenosine is released precisely, effectively solving the problems of short half-life, large side effects of systemic administration, and severe local burst release of traditional adenosine, and achieving long-term, stable and controllable release of the drug. At the same time, adenosine has osteogenic, angiogenic and anti-inflammatory activities, which can work synergistically with inorganic active ingredients to achieve efficient repair of bone defects. Attached Figure Description
[0047] Figure 1 : Stereoscopic morphology of the polypeptide-based nanofiber microspheres prepared in Example 1; Figure 2 Scanning electron microscope images of the polypeptide-based nanospheres prepared in Example 1; (a) low-magnification overall morphology of the polypeptide-based nanofiber microspheres; (b) high-magnification morphology of the nanofibers on the surface of the polypeptide-based nanofiber microspheres. Figure 3 Example 1: In vivo repair effect of peptide-based nanofiber microspheres. Detailed Implementation
[0048] To clearly illustrate the technical solution of the present invention, the following uses poly(L-glutamic acid benzyl ester) (PBLG) as an example to prepare peptide-based nanofiber microspheres, and provides a detailed explanation in conjunction with specific embodiments.
[0049] It should be clarified that these embodiments are only used to exemplify and explain the concept and method of the present invention, and are not intended to limit the scope of protection of the present invention. Any other technical solutions that can be obtained by those skilled in the art through conventional analysis, deduction, or simple modifications based on the technical principles of the present invention should be considered to fall within the scope of protection of the claims of the present invention.
[0050] Example 1: This embodiment provides a method for preparing drug-loaded nanofiber microspheres based on modified polypeptides (drug-loaded polypeptide-based nanofiber microspheres), and the steps are as follows: (1) Preparation of amination-modified inorganic nanoparticles: 300 mg of dried inorganic nanoparticles were weighed and dispersed in 20.0 mL of anhydrous ethanol. The nanoparticles were sonicated for 15 min to ensure complete dispersion and form a uniform suspension. 1 mL of APTES was slowly added dropwise to the suspension at room temperature with rapid stirring, and the mixture was stirred at a constant temperature for 24 h. After the reaction was completed, the product was collected by centrifugation (10,000 rpm) and repeatedly resuspended and washed three times with anhydrous ethanol to completely remove free APTES. Finally, the collected solid product was dried in a vacuum drying oven to prepare surface-aminated inorganic nanoparticles.
[0051] (2) Preparation of PBLG grafted onto inorganic nanoparticles: BLG-NCA and amino-modified inorganic nanoparticles were dissolved in anhydrous DCM at a ratio of 10:1 and stirred with a magnetic stirrer for 3 days under nitrogen protection at room temperature. After the reaction was completed, the reaction solution was poured into a large amount of methyl tert-butyl ether (MTBE) for precipitation. The precipitate was washed several times with 1,4-dioxane and MTBE, filtered, and vacuum dried for 24 h to obtain PBLG grafted onto inorganic nanoparticles.
[0052] (3) PBLG grafted onto inorganic nanoparticles modified with phenylboronic acid: 1 g of PBLG grafted onto inorganic nanoparticles was dissolved in 100 mL of DMF, and 0.4 mL of trimethyliodosilane was added. The reaction was carried out under nitrogen atmosphere in the dark for 6 h at a reaction temperature of 40 °C. After the reaction was completed, the reaction solution was washed with MTBE, anhydrous ethanol and water, respectively. The precipitate was collected and washed repeatedly with water and ethanol until the washing solution was colorless. Then, it was dried under vacuum to obtain carboxyl-modified PBLG grafted onto inorganic nanoparticles. The obtained product was dissolved in 100 mL of DMF, and 0.8 g of NHS and 1.4 g of EDC·HCl were added. 0.6 g of aminophenylboronic acid was added, and the reaction was carried out at room temperature for 24 h. The reaction solution was settled in 1 L of deionized water and filtered. After washing several times, the product was transferred to a 3.5 kDa dialysis bag, dialyzed in deionized water for 3 days, filtered, and freeze-dried to obtain the final modified PBLG-based raw material.
[0053] (4) The final obtained preferred modified PBLG raw material was dissolved in 1,4-dioxane to prepare a microsphere precursor solution with a concentration of 2 wt%. Anhydrous ethanol was used as the receiving bath. A 15G / 21G coaxial needle, a 10mL syringe, and a microfluidic device were selected. The nitrogen flow rate was adjusted to 5 L / min, the injection speed to 5 ml / h, and the receiving distance to 15 cm. The precursor solution was dispersed into the anhydrous ethanol receiving bath by gas shearing to form nanofiber microspheres. Finally, the microspheres were collected by filtration through a sieve, washed, and freeze-dried.
[0054] (5) The nanofiber microspheres prepared above are immersed in a 0.5 mg / mL adenosine solution and loaded with adenosine through borate ester bonds. The unloaded drug on the surface is removed by washing with deionized water to obtain drug-loaded peptide-based nanofiber microspheres.
[0055] The overall morphology of the polypeptide-based nanofiber microspheres prepared in this embodiment under a stereomicroscope is as follows: Figure 1 As shown, the scanning electron microscope image of the polypeptide-based nanospheres prepared in this embodiment is as follows. Figure 2 As shown, Figure 2In the figures, (a) shows the low-magnification overall morphology of the polypeptide-based nanofiber microspheres; and (b) shows the high-magnification morphology of the nanofibers on the surface of the polypeptide-based nanofiber microspheres. The drug-loaded polypeptide-based nanofiber microspheres obtained in this embodiment have a PBLG grafting rate of 80%–90% and a microsphere particle size of 150–350 μm.
[0056] Healthy 8-week-old 250g SD rats were selected and anesthetized via intraperitoneal injection. The hind limb knee joint area was prepared and routinely disinfected. The skin was longitudinally incised along the lateral side of the knee joint, and the subcutaneous fascia was dissected layer by layer. The lateral muscle groups and connective tissue were bluntly dissected to fully expose the femoral condyle. A bone defect (2 mm in diameter, 3 mm in depth) was constructed on the lateral side of the femoral condyle using a micro-bone drill. Rats were randomly divided into a Blank defect group (blank group) and a polypeptide-based nanofiber microsphere group, with three rats in each group. The microspheres were tightly packed into the bone defect; the bone defect in the Blank defect group was left untreated. The muscles and skin were then sutured layer by layer. Antibiotics were administered for 3 consecutive days postoperatively to prevent infection. The recovery was assessed at 4 weeks using Micro-CT 3D reconstruction. The repair effect was as follows: Figure 3 As shown, the polypeptide-based nanofiber microsphere group exhibited superior bone remodeling effects compared to the control group.
[0057] Example 2: This embodiment provides a method for preparing drug-loaded nanofiber microspheres based on modified polypeptides (drug-loaded polypeptide-based nanofiber microspheres), and the steps are as follows: (1) Preparation of amination-modified inorganic nanoparticles: 300 mg of dried inorganic nanoparticles were weighed and dispersed in 20.0 mL of anhydrous ethanol. The nanoparticles were sonicated for 15 min to ensure complete dispersion and form a uniform suspension. 1 mL of APTES was slowly added dropwise to the suspension at room temperature with rapid stirring, and the mixture was stirred at a constant temperature for 24 h. After the reaction was completed, the product was collected by centrifugation (10,000 rpm) and repeatedly resuspended and washed three times with anhydrous ethanol to completely remove free APTES. Finally, the collected solid product was dried in a vacuum drying oven to prepare surface-aminated inorganic nanoparticles.
[0058] (2) Preparation of PBLG grafted onto inorganic nanoparticles: BLG-NCA and amino-modified inorganic nanoparticles were dissolved in anhydrous DCM at a ratio of 10:1 and stirred with a magnetic stirrer for 3 days under nitrogen protection at room temperature. After the reaction was completed, the reaction solution was poured into a large amount of MTBE for precipitation. The precipitate was washed several times with 1,4-dioxane and MTBE, filtered, and vacuum dried for 24 h to obtain PBLG grafted onto inorganic nanoparticles.
[0059] (3) PBLG grafted onto inorganic nanoparticles modified with phenylboronic acid: 1 g of PBLG grafted onto inorganic nanoparticles was dissolved in 100 mL of DMF, and 0.4 mL of trimethyliodosilane was added. The reaction was carried out under nitrogen atmosphere in the dark for 6 h at a reaction temperature of 40 °C. After the reaction was completed, the reaction solution was washed with MTBE, anhydrous ethanol and water, respectively. The precipitate was collected and washed repeatedly with water and ethanol until the washing solution was colorless. Then, it was dried under vacuum to obtain carboxyl-modified PBLG grafted onto inorganic nanoparticles. The obtained product was dissolved in 100 mL of DMF, and 0.8 g of NHS and 1.4 g of EDC·HCl were added. 0.6 g of aminophenylboronic acid was added, and the reaction was carried out at room temperature for 24 h. The reaction solution was settled in 1 L of deionized water and filtered. After washing several times, the product was transferred to a 3.5 kDa dialysis bag and dialyzed in deionized water for 3 days. After filtration, the product was freeze-dried to obtain the final modified PBLG-based raw material.
[0060] (4) The final obtained preferred modified PBLG-based raw material was dissolved in 1,4-dioxane to prepare a microsphere precursor solution with a concentration of 2 wt%. Anhydrous ethanol was used as the receiving bath. A 15G / 21G coaxial needle, a 10mL syringe, and a microfluidic device were selected. The nitrogen flow rate was adjusted to 7 L / min, the propulsion speed to 5 ml / h, and the receiving distance to 15 cm. The precursor solution was dispersed into the anhydrous ethanol receiving bath by gas shearing to form nanofiber microspheres. Finally, the microspheres were collected by filtration through a sieve, washed, and freeze-dried.
[0061] (5) The nanofiber microspheres prepared above were immersed in a 0.5 mg / mL adenosine solution and loaded with adenosine via borate ester bonds. The unloaded drug on the surface was removed by washing with deionized water to obtain drug-loaded peptide-based nanofiber microspheres. The PBLG grafting rate was 80%~90%; the particle size of the obtained microspheres was 50~250 μm.
[0062] Example 3 This embodiment provides a method for preparing drug-loaded nanofiber microspheres based on modified polypeptides (drug-loaded polypeptide-based nanofiber microspheres), and the steps are as follows: (1) Preparation of amination-modified inorganic nanoparticles: 300 mg of dried inorganic nanoparticles were weighed and dispersed in 20.0 mL of anhydrous ethanol. The nanoparticles were sonicated for 15 min to ensure complete dispersion and form a uniform suspension. 1 mL of APTES was slowly added dropwise to the suspension at room temperature with rapid stirring, and the mixture was stirred at a constant temperature for 24 h. After the reaction was completed, the product was collected by centrifugation (10,000 rpm) and repeatedly resuspended and washed three times with anhydrous ethanol to completely remove free APTES. Finally, the collected solid product was dried in a vacuum drying oven to prepare surface-aminated inorganic nanoparticles.
[0063] (2) Preparation of PBLG grafted onto inorganic nanoparticles: BLG-NCA and amino-modified inorganic nanoparticles were dissolved in anhydrous DCM at a ratio of 5:1 and stirred with a magnetic stirrer for 3 days under nitrogen protection at room temperature. After the reaction was completed, the reaction solution was poured into a large amount of MTBE for precipitation. The precipitate was washed several times with 1,4-dioxane and MTBE, filtered, and vacuum dried for 24 h to obtain PBLG grafted onto inorganic nanoparticles.
[0064] (3) PBLG grafted onto inorganic nanoparticles modified with phenylboronic acid: 1 g of PBLG grafted onto inorganic nanoparticles was dissolved in 100 mL of DMF, and 0.4 mL of trimethyliodosilane was added. The reaction was carried out under nitrogen atmosphere in the dark for 6 h at a reaction temperature of 40 °C. After the reaction was completed, the reaction solution was washed with MTBE, anhydrous ethanol and water, respectively. The precipitate was collected and washed repeatedly with water and ethanol until the washing solution was colorless. Then, it was dried under vacuum to obtain carboxyl-modified PBLG grafted onto inorganic nanoparticles. The obtained product was dissolved in 100 mL of DMF, and 0.8 g of NHS and 1.4 g of EDC·HCl were added. 0.6 g of aminophenylboronic acid was added, and the reaction was carried out at room temperature for 24 h. The reaction solution was settled in 1 L of deionized water and filtered. After washing several times, the product was transferred to a 3.5 kDa dialysis bag, dialyzed in deionized water for 3 days, filtered, and freeze-dried to obtain the final modified PBLG-based raw material.
[0065] (4) The final obtained preferred modified PBLG-based raw material was dissolved in 1,4-dioxane to prepare a microsphere precursor solution with a concentration of 2 wt%. Anhydrous ethanol was used as the receiving bath. A 15G / 21G coaxial needle, a 10mL syringe, and a microfluidic device were selected. The nitrogen flow rate was adjusted to 6 L / min, the propulsion speed was 4 ml / h, and the receiving distance was 15 cm. The precursor solution was dispersed into the anhydrous ethanol receiving bath by gas shearing to form nanofiber microspheres. Finally, the microspheres were collected by filtration through a sieve, washed, and freeze-dried.
[0066] (5) The nanofiber microspheres prepared above were immersed in a 0.5 mg / mL adenosine solution and loaded with adenosine via borate ester bonds. The unloaded drug on the surface was removed by washing with deionized water to obtain drug-loaded peptide-based nanofiber microspheres. The PBLG grafting rate was 65%~85%; the particle size of the obtained microspheres was 150~350 μm.
[0067] It should be specifically noted that the preferred embodiments disclosed in this specification are merely exemplary examples illustrating the technical solution of the present invention and do not constitute a limitation on the scope of patent protection. Any person skilled in the art, upon fully understanding the technical concept of the present invention, may make equivalent substitutions, reorganize technical features, or adjust process parameters in the above embodiments. Such technical modifications, as long as they do not depart from the substantive technical features specified in the claims, shall be considered within the legal protection scope of the patent right of the present invention. The legal protection boundary of the present invention shall be subject to the content of the claims published by the State Intellectual Property Office. Any non-substantive changes based on the technical solution of the present invention shall not override the statutory protection effect of the patent right.
Claims
1. A method for preparing peptide-based microspheres with a nanofiber structure, characterized in that, Includes the following steps: (1) Preparation of polypeptides grafted onto inorganic nanoparticles; Aminated inorganic nanoparticles initiate ring-opening polymerization of peptide monomers to obtain peptides grafted onto inorganic nanoparticles. (2) Carboxylation-modified polypeptides grafted onto inorganic nanoparticles; The prepared polypeptide grafted onto inorganic nanoparticles was modified by carboxylation, and then aminophenylboronic acid was grafted onto it by chemical bonds to prepare a polypeptide modified by phenylboronic acid grafted onto inorganic nanoparticles. (3) Preparation of polypeptide-based microspheres with nanofiber structures: A polymer solution was prepared by grafting phenylboronic acid onto inorganic nanoparticles to form a polypeptide. The polymer solution was then dispersed into an ethanol collection phase using microfluidics combined with gas shearing to obtain polypeptide-based microspheres with nanofiber structures.
2. The method for preparing peptide-based microspheres with a nanofiber structure according to claim 1, characterized in that, In step (1), the inorganic nanoparticles are zinc-doped mesoporous silica inorganic nanoparticles.
3. The method for preparing a polypeptide-based microsphere with a nanofiber structure according to claim 1, characterized in that, The preparation of polypeptides grafted onto inorganic nanoparticles specifically includes the following steps: (1-1) Modify inorganic nanoparticles by amylation; Inorganic nanoparticles were ultrasonically dispersed in anhydrous ethanol, and then 3-aminopropyltriethoxysilane was added under stirring. The product was collected by centrifugation and washed repeatedly with anhydrous ethanol to completely remove unreacted reagents. After vacuum drying, surface-aminated inorganic nanoparticles were finally obtained. (1-2) Aminated inorganic nanoparticles and polypeptide monomers were dissolved in dichloromethane. The inorganic nanoparticles initiated the ring-opening polymerization of the polypeptide monomers to obtain polypeptides grafted onto the inorganic nanoparticles. In steps (1-2), the inorganic nanoparticles are dissolved in dichloromethane, with a mass fraction of 1%~20%; In steps (1-2), the reaction temperature between inorganic nanoparticles and polypeptide monomers is 10~25 ℃; In steps (1-2), the polypeptide monomer is selected from one or more of L-glutamic acid-γ-benzyl ester, aspartic acid, and phenylalanine; the carboxyl content of the polypeptide is 10%~100%.
4. The method for preparing a polypeptide-based microsphere with a nanofiber structure according to claim 1, characterized in that, In step (2), the preparation method of the phenylboronic acid-modified grafted polypeptide onto inorganic nanoparticles is as follows: (2-1) Carboxylation modification of peptides grafted onto inorganic nanoparticles: The polypeptide grafted onto inorganic nanoparticles was dissolved in anhydrous dichloromethane, and trimethyliodosilane was added. The reaction was carried out under nitrogen atmosphere in the dark. After the reaction was completed, the reaction solution was washed with methyl tert-butyl ether, anhydrous ethanol and water, respectively. The precipitate was collected and washed repeatedly with water and ethanol until the washing solution was colorless and then dried under vacuum to obtain the carboxylated modified polypeptide grafted onto inorganic nanoparticles. (2-2) Preparation of phenylboronic acid-modified polypeptides grafted onto inorganic nanoparticles: The prepared carboxylated polypeptides grafted onto inorganic nanoparticles were dissolved in N,N-dimethylformamide, and N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added and reacted with aminophenylboronic acid to obtain phenylboronic acid-modified polypeptides grafted onto inorganic nanoparticles.
5. The method for preparing a polypeptide-based microsphere with a nanofiber structure according to claim 4, characterized in that, In step (2-1), based on 100 mL of anhydrous dichloromethane, the amount of peptide grafted onto inorganic nanoparticles added is 0.5~5 g; the amount of trimethyliodosilane added is 0.2~1 mL; the concentration of trimethyliodosilane is 0.1~1%; the reaction temperature is 40℃; and the reaction is carried out in the dark for 3~9 h. In step (2-1), the carboxyl group content of the polypeptide grafted onto the inorganic nanoparticles is 10-50%; In step (2-2), phenylboronic acid is modified and grafted onto the polypeptide of inorganic nanoparticles, with the grafting rate of phenylboronic acid groups being 5%~60%.
6. The method for preparing a polypeptide-based microsphere with a nanofiber structure according to claim 1, characterized in that, The specific steps for preparing peptide-loaded drug-eluting nanofiber microspheres in step (3) are as follows: The obtained phenylboronic acid was modified and grafted onto inorganic nanoparticles and dissolved in 1,4-dioxane as the inner phase fluid of the coaxial needle. Nitrogen gas was used as the outer phase of the coaxial needle. Using gas shear microfluidics, the polymer solution was dispersed into anhydrous ethanol collection phase (receiving bath). The polymer droplets formed nanofiber microspheres through phase separation. The microspheres were then sieved, washed, and freeze-dried to obtain peptide-based microspheres with nanofiber structures. After phenylboronic acid was modified and grafted onto inorganic nanoparticles and dissolved in 1,4-dioxane, its solid content was 1-3 wt%. The polymer solution propulsion speed is 2~10 mL / h, and the receiving distance is 5~30 cm.
7. A polypeptide-based microsphere with a nanofiber structure, characterized in that, The microspheres are prepared by any one of claims 1-6, and have a nanofiber structure, wherein the nanofibers on the surface and inside the microspheres are arranged randomly or in an ordered manner; the particle size of the microspheres is 50-300 micrometers; and the diameter of the nanofibers is 50-500 nm.
8. A drug-loaded nanofiber microsphere based on modified polypeptide, characterized in that, It is a drug-loaded peptide-based microsphere with a nanofiber structure, as described in claim 7, wherein the drug loading of each 15mg microsphere is 200μg~600μg.
9. The method for preparing drug-loaded nanofiber microspheres based on modified polypeptides as described in claim 8, characterized in that, Polypeptide-based microspheres with nanofiber structures were added to a solution containing drugs, soaked, and washed to obtain drug-loaded nanofiber microspheres based on modified polypeptides.
10. The application of the polypeptide-based microspheres with nanofiber structure according to claim 7 and the drug-loaded nanofiber microspheres based on modified polypeptides according to claim 8 in the preparation of bone defect repair materials.