Drug-loaded interpenetrating network hydrogel based on rada16-i and pegda, and preparation method and application thereof

Abstract

This application discloses a drug-loaded interpenetrating network hydrogel based on RADA16-I and PEGDA, its preparation method, and its applications. The hydrogel consists of a three-dimensional interpenetrating network structure composed of polyethylene glycol diacrylate (PEGDA) and the self-assembled short peptide RADA16-I, and is loaded with the osteogenic inducing drug icariin (ICA) and the anti-infective drug vancomycin (VAN). PEGDA forms a first network structure through cross-linking, and RADA16-I forms a second network structure through self-assembly. The two interweave to form a confined porous structure, thereby achieving sustained drug release. This hydrogel has both bone defect filling and local drug delivery functions and can be used for the repair of infected bone defects.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and bone tissue engineering technology, specifically to a drug-loaded interpenetrating network hydrogel based on RADA16-I and PEGDA, its preparation method, and its applications. Background Technology

[0002] Infected bone defects are often caused by severe trauma, surgical debridement, or tumor resection. Infection at the site of the defect significantly increases the difficulty of treatment. If the infection is not effectively controlled, it often leads to delayed bone healing, nonunion, or even amputation. Currently, clinical treatment for infected bone defects typically involves debridement, removal of necrotic bone tissue, and systemic antibiotic therapy. However, debridement and removal of residual bone often further expand the bone defect, creating larger gaps. Therefore, bone defect filling materials are usually required. Common filling materials include autologous bone grafts or allogeneic bone grafts, but these materials are difficult to sustain local antibacterial drug release. Antibiotic-loaded bone cement has also been used for local anti-infection treatment, but it has problems such as inducing foreign body reactions, affecting osteoblast activity, and requiring secondary surgery for removal. Therefore, developing a material system that can simultaneously achieve bone defect filling, anti-infection, and bone regeneration promotion is of great significance.

[0003] Interpenetrating network hydrogels (IPN hydrogels) consist of two or more polymer networks that are interwoven at the molecular scale but not covalently bonded. Due to their multi-network structure, IPN hydrogels typically possess good mechanical properties, structural stability, and tunable pore structures, making them suitable for constructing microenvironments conducive to cell growth and drug release. Therefore, they have attracted widespread attention in the field of bone tissue engineering.

[0004] The self-assembled short peptide RADA16-I is a typical ion-complementary self-assembled peptide with good biocompatibility, biodegradability, and low immunogenicity. It can form a three-dimensional nanofiber network structure under ionic conditions, but its mechanical properties are relatively weak, making it difficult to maintain a stable morphology under load-bearing or semi-load-bearing conditions in vivo. Polyethylene glycol diacrylate (PEGDA) is a commonly used photocrosslinked hydrogel material with good mechanical properties and structural stability, but its bioactivity and cell adhesion are poor, which is not conducive to cell migration, proliferation, and differentiation. Therefore, constructing an interpenetrating network hydrogel by combining RADA16-I and PEGDA is expected to achieve a balance between good biocompatibility and mechanical properties.

[0005] Repairing infected bone defects requires simultaneously achieving multiple functions, including defect filling, infection control, and promoting bone regeneration. Therefore, it is necessary to develop a composite hydrogel system capable of loading anti-infective drugs and osteogenic inducing drugs to meet the clinical needs of infected bone defect repair. Summary of the Invention

[0006] The present invention aims to provide a drug-loaded interpenetrating network hydrogel based on RADA16-I and PEGDA, its preparation method and its application, so as to achieve synergistic treatment of bone defect filling, local anti-infection and bone regeneration promotion.

[0007] To achieve the above objectives, the first aspect of this application provides the following technical solution: A drug-loaded interpenetrating network hydrogel based on RADA16-I and PEGDA includes: a three-dimensional interpenetrating network structure formed by polyethylene glycol diacrylate and self-assembled short peptide RADA16-I, and an anti-infective drug and an osteogenic induction drug loaded in the three-dimensional interpenetrating network structure.

[0008] Furthermore, the polyethylene glycol diacrylate forms a first network structure through cross-linking; the self-assembled short peptide RADA16-I forms a second network structure through self-assembly; the first network structure and the second network structure intertwine to form a three-dimensional interpenetrating network structure.

[0009] Furthermore, the anti-infective drug is vancomycin, and the osteogenic inducing drug is icariin.

[0010] Furthermore, the concentration of vancomycin in the drug-loaded interpenetrating network hydrogel is 0.5-10 mg / mL; the concentration of icariin in the drug-loaded interpenetrating network hydrogel is 100-1000 μg / mL.

[0011] Furthermore, the concentration of the self-assembled short peptide RADA16-I is 1% (w / v); the concentration of the polyethylene glycol diacrylate is 4% (w / v).

[0012] Furthermore, the polyethylene glycol diacrylate forms a first network structure through photocrosslinking; the self-assembled short peptide RADA16-I self-assembles under ionic conditions to form a second network structure.

[0013] A third aspect of this application provides an alternative preparation method that differs from the preparation method of the second aspect, comprising the following steps: S1. Dissolve the self-assembled short peptide RADA16-I and polyethylene glycol diacrylate in an aqueous solution containing a photoinitiator; S2. Add the anti-infective drug and osteogenic induction drug, and then sequentially irradiate with ultraviolet light and add ion solution to form a drug-loaded interpenetrating network hydrogel.

[0014] The fourth aspect of this application provides the use of the drug-loaded interpenetrating network hydrogel as described in the first aspect in the preparation of medical materials for repairing infected bone defects.

[0015] Furthermore, the drug-loaded interpenetrating network hydrogel serves as a drug delivery system for the continuous release of anti-infective drugs and osteogenic induction drugs.

[0016] Working principle and beneficial effects of the present invention: This invention constructs an interpenetrating network hydrogel composed of RADA16-I and PEGDA, and loads the anti-infective drug vancomycin and the osteogenic inducing drug icariin into this system, forming a composite drug-loaded hydrogel system that combines bone defect filling, anti-infection, and bone regeneration promotion functions. This interpenetrating network structure can improve the insufficient mechanical properties of single hydrogels and provides a structural basis for stable drug loading and sustained release.

[0017] Therefore, the present invention has the following beneficial effects: (1) Interpenetrating network structure improves the structural stability and mechanical properties of hydrogel; (2) It can simultaneously load anti-infective drugs and osteogenic induction drugs to achieve synergistic effects of infection control and bone regeneration; (3) Hydrogels can fill bone defect areas and provide a scaffold environment for cell growth; (4) To achieve local continuous release of drugs and improve drug utilization efficiency. Attached Figure Description

[0018] Figure 1 Preparation of drug-loaded interpenetrating network hydrogels and their repair process in infected bone defects; Figure 2 Characterization of the physicochemical properties of interpenetrating network hydrogels; Figure 3 The main biological functions of IPN hydrogels were characterized (Control group is the control group without gel). Figure 4 Characterization of the in vitro drug release performance of the drug-loaded IPN hydrogel (n=3, mean ± standard deviation); Figure 5 To demonstrate the in vivo antibacterial effect of drug-loaded IPN gel; Figure 6 HE staining of organs in each group of rabbit infectious bone defect models after drug administration (scale bar = 20 μm); Figure 7 General observation of the repair of tibial defects in rabbits (A) Control, (B) VAN+IPN, (C) VAN+ICA+IPN; Figure 8Micro-CT scan results of rabbit tibia (scale bar = 10 mm); Figure 9 The bone volume fraction (BV / TV) and bone mineral density (BMD) at the bone defects in each group of rabbits were calculated. Detailed Implementation

[0019] The following detailed description illustrates the specific implementation method: Figure 1 The preparation of drug-loaded interpenetrating network hydrogels and their repair process in infected bone defects were demonstrated. Figure 1 (A) The preparation process of hydrogel, (B) Local injection of hydrogel into infected bone defect sites to promote defect repair, and (C) The three key roles of hydrogel in the repair process of infected bone defects.

[0020] Example 1: Preparation of interpenetrating network hydrogels Weigh 40 mg of the photoinitiator 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (I2959), add it to 10 mL of double-distilled water, and sonicate it to dissolve it completely, to obtain an I2959 solution with a mass fraction of 0.4%.

[0021] Polyethylene glycol diacrylate (PEGDA, molecular weight 5000 DA) powder was weighed and dissolved in the above I2959 solution to prepare a PEGDA solution with a concentration of 16%. Self-assembled short peptide RADA16-I powder was weighed and added to the I2959 solution, then dissolved by sonication to prepare a RADA16-I solution with a concentration of 2%. (IPN hydrogel precursor solution) The 16% PEGDA solution and 2% RADA16-I solution were mixed in a specific ratio and diluted with I2959 solution to achieve a final concentration of 4% PEGDA and 1% RADA16-I in the system. The resulting mixed solution was added to a mold or reaction vessel and reacted under ultraviolet light for 5 min to allow PEGDA to undergo photocrosslinking and form a first network structure. Subsequently, phosphate buffered saline (PBS) was added to the system to induce RADA16-I self-assembly to form a second network structure, thereby obtaining an interpenetrating network hydrogel (IPN hydrogel).

[0022] Under the same conditions, RADA16-I self-assembled hydrogels can be obtained by using only RADA16-I solution (final concentration 1%) for PBS-induced self-assembly without the addition of PEGDA for photocrosslinking. (RADA hydrogel, denoted as R) Example 2: Preparation of IPN hydrogels with different drug loading methods Based on the IPN hydrogel precursor solution prepared in Example 1, different hydrogel samples were prepared by adding different drugs. The specific steps were as follows: the corresponding drug was added to the IPN hydrogel precursor solution, vortexed and stirred on a magnetic stirrer for 2 h (800 r / min) to ensure that the drug was fully dispersed. The resulting solution was then injected into a mold and reacted under ultraviolet light irradiation for 5 min to allow PEGDA to undergo photocrosslinking and form a first network structure. Subsequently, phosphate buffer solution was added dropwise to induce RADA16-I self-assembly to form a second network structure, thereby obtaining an interpenetrating network hydrogel.

[0023] Different hydrogel samples were prepared as follows: (1) No drugs were added to obtain blank IPN hydrogels, which were denoted as blank hydrogel group (IPN).

[0024] (2) Add vancomycin to make the final concentration of vancomycin in the system 5 mg / mL to obtain vancomycin-loaded hydrogel, which is denoted as VAN-loaded hydrogel group (VAN+IPN).

[0025] (3) Vancomycin and icariin were added simultaneously to make the final concentration of vancomycin in the system 5 mg / mL and the final concentration of icariin 250 μg / mL, so as to obtain a vancomycin and icariin-loaded hydrogel, which is denoted as the VAN and ICA-loaded hydrogel group (VAN+ICA+IPN).

[0026] (4) Vancomycin and icariin were added simultaneously to make the final concentration of vancomycin in the system 2.5 mg / mL and the final concentration of icariin 500 μg / mL, so as to obtain a hydrogel loaded with vancomycin and icariin, which was designated as the sustained-release experimental group (VAN+ICA+IPN-R).

[0027] Example 3: Physicochemical characterization of interpenetrating network hydrogels 1) Hydrogel rheological property testing The hydrogels prepared in Example 1 were subjected to rheological tests using a rotational rheometer. At room temperature, the hydrogel samples were placed between stainless steel flat plates with a diameter of 20 mm and a gap of 1000 μm. First, a frequency scan was performed under the following conditions: stress 0.5%, frequency range 0.1–10 rad / s. The changes in storage modulus (G') and loss modulus (G") as a function of frequency were recorded. Subsequently, a strain scan was performed under the following conditions: strain range 0.01%–500%, frequency 1 Hz. The changes in storage modulus (G') and loss modulus (G") were recorded.

[0028] (2) Characterization of hydrogel morphology The hydrogel sample prepared in Example 1 was placed on a glass slide and fixed with 5% glutaraldehyde solution for 12 h, followed by ethanol gradient dehydration and drying using the carbon dioxide critical point drying method. The morphology of the dried sample was observed using a scanning electron microscope (SEM).

[0029] (3) Hydrogel degradation performance test Proteinase K was weighed and dissolved in PBS to prepare a 0.3 mg / mL proteinase K solution. 500 μL of each group's gel precursor solution from Example 1 was gelled according to the method described in Example 1 and placed in a centrifuge tube. 10 mL of proteinase K solution was added, and degradation experiments were conducted at 37 °C. Samples were taken at 7, 14, 21, and 28 days, washed, and lyophilized. The mass change of the samples was measured to evaluate the hydrogel degradation performance.

[0030] Figure 2 To characterize the physicochemical properties of interpenetrating network hydrogels, Figure 2 (A) Frequency scanning test results of IPN hydrogel and R group hydrogel, (B) Strain scanning test results of R group hydrogel, (C) Scanning electron microscopy observation results of R group hydrogel (scale bar = 500 nm), (D) Strain scanning test results of IPN group hydrogel, (E) Scanning electron microscopy observation results of IPN hydrogel (scale bar = 500 nm), (F) Degradation performance test results of IPN hydrogel and R group hydrogel.

[0031] Rheological experiments showed that the storage modulus of the IPN hydrogel was significantly higher than that of the single-network hydrogel, indicating that the interpenetrating network structure effectively improves the mechanical properties of the hydrogel. SEM observations revealed that all hydrogel groups formed a continuous and uniform porous network structure without phase separation. Compared to group R, the IPN hydrogel had larger pore sizes, which is more conducive to the exchange of cellular nutrients and metabolites. Degradation experiments showed that the IPN hydrogel had a longer degradation time, exhibiting degradation characteristics that better match the drug release platform and bone growth.

[0032] Example 4: Characterization of the main biological functions of interpenetrating network hydrogels (1) Cell culture Mouse osteogenic progenitor cells MC3T3-E1 Subclone 14 were used for in vitro experiments. Cells were cultured in α-MEM complete medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37 ℃ and 5% CO2.

[0033] (2) Evaluation of hydrogel cytotoxicity MC3T3-E1 cells were seeded in 96-well plates and cultured for 24 h. Hydrogel samples were prepared according to the method in Example 1, and hydrogel extracts were prepared using the extraction method. The extracts were added to the cell culture system for further culture.

[0034] Cell viability was assessed using the CCK-8 assay at 24 h, 48 h, and 72 h, and absorbance was measured at 450 nm using an ELISA reader to evaluate the cytotoxicity of the hydrogel.

[0035] (3) Evaluation of cell adhesion performance Hydrogel samples were prepared according to the method in Example 1, and cells were seeded on the surface of the hydrogel for culture. Cell adhesion was observed at 10 min, 30 min, and 90 min, and the observations were recorded by microscopy to evaluate the cell adhesion properties of the hydrogel.

[0036] (4) Evaluation of the biocompatibility of hydrogels After MC3T3-E1 cells were seeded onto hydrogel samples from each group and cultured for 48 h, cell viability was detected by Calcein AM / PI staining, and cell survival was observed by fluorescence microscopy.

[0037] Figure 3 The main biological functions of IPN hydrogels were characterized (Control is the control group without gel). Figure 3 (A) Cytotoxicity results of IPN hydrogel and R group hydrogel, (B) Cell adhesion results of IPN hydrogel and R group hydrogel, (C) Biocompatibility evaluation of IPN hydrogel and R group hydrogel (scale bar = 100 μm). Note: Data are expressed as mean ± standard deviation (Mean ± SD). Compared with the control group, P<0.05, P<0.01.

[0038] Cytotoxicity results showed that all IPN hydrogels exhibited some toxicity to MC3T3-E1 Subclone 14 cells. According to the standards for biocompatibility certification of medical devices, a decrease in cell viability exceeding 30% when using the MTT assay to detect cytotoxicity indicates significant cytotoxicity. In this study, the cytotoxicity of all IPN hydrogel groups did not exceed this value, indicating compliance with the standard. In the biocompatibility experiment, the cells growing on the gel were in good condition, with the number of live cells significantly exceeding the number of dead cells, demonstrating the high safety of the prepared interpenetrating network hydrogel and its ability to meet the safety requirements for in vivo implants. Our experimental results indicate that the interpenetrating network hydrogel group showed a significantly higher number of adherent cells than the control group, demonstrating good cell adhesion and suitability as a scaffold for cell growth and for filling bone defects.

[0039] Example 5: Characterization of drug release performance of drug-loaded interpenetrating network hydrogel The sustained-release experimental group hydrogel (VAN+ICA+IPN-R) (1 mL of drug-loaded IPN hydrogel precursor solution volume) prepared in Example 2 was used for in vitro drug release experiments.

[0040] (1) Construction of the release system Cut the dialysis bag to an appropriate length and wash it with double-distilled water before use. Place the drug-loaded interpenetrating hydrogel into the dialysis bag and seal it. Then, place the dialysis bag into a 20 mL centrifuge tube. Add 12 mL of release medium, which is a mixture of methanol and PBS buffer (volume ratio 3:7), to the centrifuge tube. Place the centrifuge tube in a constant-temperature shaker and incubate at 37 °C with shaking (100 r / min) to conduct the drug release experiment.

[0041] (2) Sampling and testing Samples were taken at 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h. 3 mL of the release solution was collected each time, and an equal volume of fresh release medium was added to maintain a constant system volume. The collected samples were filtered through a microporous membrane, and the concentrations of vancomycin and icariin in the release solution were determined by high-performance liquid chromatography (HPLC). The drug concentrations were calculated based on the standard curve.

[0042] (3) Calculation of cumulative drug release rate The cumulative drug release rate is calculated using the following formula:

[0043] Where Q is the cumulative drug release rate, V0 is the total volume of the release medium (mL), Ci is the drug concentration of the sample taken at time point i, Cn is the drug concentration of the sample taken at time point n, Vi is the sampling volume (mL), and m is the total mass of the drug.

[0044] Figure 4 The in vitro drug release performance of the drug-loaded IPN hydrogel was characterized (n=3, mean ± standard deviation). The results showed that both drugs were initially released rapidly from the gel, then quickly entered a release plateau, and finally reached a cumulative release rate of approximately 50% at 5 days. This indicates that both drugs can be slowly released from the drug-loaded IPN hydrogel.

[0045] Example 6: Characterization of in vivo antibacterial activity and osteogenic repair effect of drug-loaded IPN gel (1) Preparation of experimental animals Fifteen male New Zealand rabbits were selected and acclimatized for one week before being used in the experiment. The animals were randomly divided into three groups of five each: a control group, a vancomycin-loaded hydrogel group (VAN+IPN), and a vancomycin-and-icariin-loaded hydrogel group (VAN+ICA+IPN). All animals were fasted for 12 hours prior to the experiment.

[0046] (2) Construction of infectious bone defect model A rabbit model of infectious bone defect was established using Staphylococcus aureus. New Zealand rabbits were first anesthetized and fixed in a prone position to expose the left tibial shaft. The skin and soft tissues were longitudinally incised along the tibia, and the fascia and periosteum were separated to fully expose the tibia. A circular bone defect approximately 3 mm in diameter was created at the tibial shaft using a bone drill, and Staphylococcus aureus bacterial solution (1×10⁻⁶) was injected into the defect area. 9 (CFU / mL, 10 μL) to establish an infectious bone defect model.

[0047] (3) Hydrogel drug delivery In the model group, only a bone defect model was established and the wound was sutured. In the VAN+IPN group and the VAN+ICA+IPN group, after the bacterial solution was injected, the corresponding drug-loaded IPN hydrogel precursor solution (about 40 μL) prepared in Example 2 was injected into the bone defect area. IPN hydrogel was formed by cross-linking triggered by ultraviolet irradiation and body fluid, thereby achieving local drug delivery.

[0048] (4) Evaluation of in vivo antibacterial effect One week after modeling, one New Zealand rabbit was randomly selected from each group and sacrificed. Tissue surrounding the bone defect was collected for bacterial culture. The tissue samples were diluted with PBS buffer and plated for culture. After incubation in an incubator for a certain period of time, colony growth was observed to evaluate the in vivo antibacterial effect of the hydrogel.

[0049] (5) In vivo safety evaluation Four weeks after the experiment, all New Zealand rabbits were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were harvested. Tissue samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned before being stained with hematoxylin and eosin (HE). Morphological changes in each tissue were observed under a microscope to evaluate the in vivo safety of the hydrogel drug delivery system.

[0050] (6) Evaluation of bone repair effect 1) General observation Four weeks into the experiment, tibial samples were removed, washed with PBS, and the repair of the bone defect area was observed and photographed.

[0051] 2) Micro-CT analysis Bone samples were subjected to Micro-CT scans, and the bone defect areas were analyzed using three-dimensional reconstruction software to calculate bone volume fraction (BV / TV) and bone mineral density (BMD) to evaluate the promoting effect of hydrogel on bone defect repair.

[0052] The results are as follows: like Figures 5-9 As shown, the results demonstrate that the prepared drug-loaded IPN hydrogel effectively killed bacteria and controlled infection within just one week, and completely repaired the bone defect within four weeks. Therefore, the IPN hydrogel loaded with vancomycin and icariin can significantly inhibit infection and promote new bone formation in the bone defect area, exhibiting good antibacterial and bone repair effects.

[0053] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the claims.

Claims

1. A drug-loaded interpenetrating network hydrogel based on RADA16-I and PEGDA, characterized in that, include: A three-dimensional interpenetrating network structure formed by polyethylene glycol diacrylate and self-assembled short peptide RADA16-I, and an anti-infective drug and an osteogenic inducing drug loaded in the three-dimensional interpenetrating network structure.

2. The drug-loaded interpenetrating network hydrogel according to claim 1, characterized in that, The polyethylene glycol diacrylate forms a first network structure through photocrosslinking; the self-assembled short peptide RADA16-I forms a second network structure through self-assembly; the first network structure and the second network structure intertwine to form a three-dimensional interpenetrating network structure.

3. The drug-loaded interpenetrating network hydrogel according to claim 2, characterized in that, The anti-infective drug is vancomycin, and the osteogenic inducing drug is icariin.

4. The drug-loaded interpenetrating network hydrogel according to claim 3, characterized in that, The concentration of vancomycin in the drug-loaded interpenetrating network hydrogel is 0.5-10 mg / mL; the concentration of icariin in the drug-loaded interpenetrating network hydrogel is 100-1000 μg / mL.

5. The drug-loaded interpenetrating network hydrogel according to any one of claims 1 to 4, characterized in that, The self-assembled short peptide RADA16-I self-assembles to form a second network structure under ionic conditions.

6. A method for preparing the drug-loaded interpenetrating network hydrogel as described in claim 5, characterized in that, Includes the following steps: S1. Dissolve the self-assembled short peptide RADA16-I and polyethylene glycol diacrylate in an aqueous solution containing a photoinitiator; S2. Add the anti-infective drug and osteogenic induction drug, and then sequentially irradiate with ultraviolet light and add ion solution to form a drug-loaded interpenetrating network hydrogel.

7. The method according to claim 6, characterized in that, The concentration of the self-assembled short peptide RADA16-I is 1% (w / v); the concentration of the polyethylene glycol diacrylate is 4% (w / v).

8. The use of a drug-loaded interpenetrating network hydrogel as described in any one of claims 1 to 4 in the preparation of medical materials for repairing infected bone defects.

9. The application according to claim 8, characterized in that, The drug-loaded interpenetrating network hydrogel serves as a drug delivery system for the continuous release of anti-infective drugs and osteogenic induction drugs.

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