A stimulus-responsive injectable composite drug-loaded hydrogel and preparation method thereof

Abstract

The present invention belongs to the field of drug carrier basic materials, and discloses a stimulus-responsive injectable composite drug-loaded hydrogel and a preparation method thereof. The composite drug-loaded hydrogel, as a drug delivery system, comprises a three-dimensional network injectable hydrogel composed of calcium chloride and sodium alginate as a carrier skeleton, and a polymer polyaniline nanofiber dispersed in a hydrogel matrix and a drug protein bee venom peptide as a therapeutic system. The present invention disperses nanofibers and drug polypeptides in an injectable hydrogel with excellent biocompatibility, achieving the effects of long-term drug release and synergistic treatment of multiple modes.

Description

Technical Field

[0001] The invention belongs to the field of drug carrier basic materials, and in particular relates to a stimulus-responsive injectable composite drug-loaded hydrogel and a preparation method thereof. Background Art

[0002] Modern cancer treatment methods include surgical treatment, radiotherapy, chemotherapy, etc., which are often accompanied by problems such as incurable, large toxic and side effects, easy metastasis and recurrence during the course of treatment. The reason is that these treatment methods have certain limitations and cannot specifically remove tumors. Some chemotherapy drugs such as anticancer antibiotics and immune preparations have a good inhibitory effect on cancer, but the efficacy of using them alone is poor. In order to achieve the ideal therapeutic effect, the construction of tumor drug delivery systems has been a hot topic in the biomedical field in recent years. Drug delivery systems have excellent characteristics in drug controlled release, drug targeting, stability regulation, etc. In cancer treatment, constructing an intelligent drug delivery system to deliver drugs and release them at a targeted point can effectively reduce the toxic and side effects of drugs and maximize the value of drugs. In this way, it not only reduces the damage to normal biological tissues, but also strengthens the release of drugs in the targeted area. Therefore, the construction of drug delivery systems is of great significance to human understanding and treatment of tumors.

[0003] As a high molecular polymer with a three-dimensional network structure, hydrogel has become one of the important platforms of drug delivery system. The preparation method of hydrogel is simple. It is generally formed by physical crosslinking (temperature sensitive gel, molecular self-assembly gel) or chemical crosslinking (crosslinking agent crosslinking gel, radiation crosslinking gel, photoinitiated polymerization gel) between polymer chains. It has the advantages of good biocompatibility, strong drug loading capacity, and good drug controlled release effect. As a member of hydrogel, injectable hydrogel has a fascinating attraction in biomedicine. In addition to the traditional advantages of hydrogel, it also has the characteristics of being designable and controllable. It has no obvious trauma to the patient's body during application and is simple to operate, which greatly reduces the pain of patient treatment.

[0004] The specific release of drugs from hydrogels is usually achieved through stimulus response, which mainly includes in vitro stimulation and in vivo stimulation. In vitro stimulation mainly utilizes external stimulation such as external magnetic field, light source, ultrasound, etc. to achieve drug release; in vivo stimulation mainly utilizes the natural microenvironment of the tumor. The tumor microenvironment, as the soil for cultivating cancer cells, has an important influence on the occurrence and growth of the tumor. Compared with normal tissue, the tumor microenvironment exhibits lower acidity, high levels of ATP, reactive oxygen and glutathione, overexpressed enzymes and hypoxia in the tumor area.

[0005] Although the local administration method in the prior art can greatly increase the local drug concentration, at the same time, a single administration method, such as chemotherapy, is prone to drug resistance in cells, resulting in chemotherapy failure. For another example, in recent years, researchers have gradually discovered that therapeutic systems that interfere with the tumor microenvironment play a key role in the growth, proliferation and apoptosis of tumor cells in the tumor microenvironment. Due to its own intelligent regulatory mechanism, the calcium ion content is dynamically balanced within a certain range. When the drug is applied and its content is too high, it will seriously affect the intracellular calcium content homeostasis and achieve "calcium ion overload". When too much calcium ions accumulate in the cell, it will seriously damage the function of the mitochondria, resulting in a decrease in cell respiration level, a decrease in ATP content, a decrease in mitochondrial membrane potential, and other effects, which will cause serious damage to the tumor system. See the open document Zheng Pan et al. A Multichannel Ca2+ Nanomodulator for Multilevel Mitochondrial Destruction-Mediated Cancer Therapy [J]. Advanced Materials, 2021, 33(15): e2007426-e2007426. A nanoplatform based on a calcium ion overload system is disclosed, and polyethylene glycol (PEG)-functionalized CaCO3 nanoparticles with acidic stimulus response ( PEG CaNM CUR+CDDP ), then loaded with curcumin (CUR) and cisplatin (CDDP), CUR inhibits intracellular Ca 2+ CDDP can induce mitochondrial dysfunction by causing mitochondrial damage and abnormalities in mitochondrial structure and metabolism. 2+ It accumulates in the cytoplasm, inducing a decrease in mitochondrial membrane potential, thereby affecting cellular respiration, reducing the production of intracellular ATP content, and promoting cell apoptosis (Advanced Materials 2021, 2007426). However, this treatment is performed by tail vein injection, which needs to circulate in the body for several days and is prone to systemic toxicity. Summary of the invention

[0006] In summary, it is indeed necessary to design and construct a new stimulus-responsive injectable composite hydrogel drug delivery system to achieve synergistic tumor treatment based on calcium ion overload, photothermal ablation and chemotherapy. The drug delivery system integrates the advantages of multiple treatment methods and reduces the problem of high-dose drugs causing damage to the body. Therefore, in response to the above-mentioned problems existing in the prior art, the present invention provides a nanofiber-injectable composite hydrogel drug delivery system, which uses injectable hydrogel as the main skeleton, and encapsulates polyaniline nanofibers and hydrophilic polypeptide drugs bee venom peptide in the hydrogel matrix. The injectable hydrogel has good biocompatibility and fluidity, and the drug is directly delivered to the tumor site by injection, forming a multi-drug synergistic treatment, and a long-term therapeutic effect of a single injection. Since the materials used in the present invention have good biocompatibility and low toxicity, and the synthesis process is simple, this material is expected to have good biological application prospects.

[0007] The technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides a stimulus-responsive injectable composite drug-loaded hydrogel, wherein the composite drug-loaded hydrogel is composed of calcium alginate hydrogel encapsulating polyaniline nanofibers PANI NF and bee venom peptide, wherein the polyaniline nanofibers are fibrous, 700-800 nm long and 100-150 nm in diameter; the calcium alginate hydrogel is gelled by mixing a calcium chloride solution and a sodium alginate solution in a volume ratio of 2:7, and before the two are mixed, the configuration concentration range of calcium chloride CaCl2 is 2-100 mM, and the configuration concentration range of sodium alginate ALG is 4-20 mg / mL; after gelling, the encapsulation concentration of the bee venom peptide is 50 μg / mL, and the encapsulation concentration of the PANI NF is 0.6 mg / mL.

[0009] Preferably, the polyaniline nanofibers are prepared by oxidative polymerization of aniline and ammonium persulfate in a perchloric acid solution.

[0010] The stimulus-responsive injectable composite drug-loaded hydrogel provided by the present invention can be degraded under acidic conditions. The method for using the injectable composite hydrogel is as follows: injecting the injectable composite hydrogel into the affected area with a syringe; under the stimulation of the slightly acidic environment of the tumor, the injectable hydrogel gradually degrades to release tumor therapeutic drugs such as bee venom peptide and polyaniline nanofibers; the encapsulated tumor therapeutic drugs and the calcium ions released after the degradation of the calcium alginate hydrogel act synergistically to promote the elimination of the tumor, and the stimulus-responsive injectable composite drug-loaded hydrogel has no biological toxicity after degradation.

[0011] In a second aspect, the present invention also provides a method for preparing the above-mentioned stimuli-responsive injectable composite drug-loaded hydrogel, the method comprising the following steps:

[0012] Selecting polyaniline nanofibers that are linear fibers with a length of 700-800 nm and a diameter of 100-150 nm;

[0013] A calcium chloride solution is prepared with ultrapure water, wherein the concentration of the prepared calcium chloride solution is in the range of 2-100 mM, and then polyaniline nanofibers are added to obtain a first mixed solution, wherein the concentration of the polyaniline nanofibers in the first mixed solution is 2.7 mg / mL;

[0014] A sodium alginate solution containing melittin is prepared with ultrapure water to obtain a second mixed solution, wherein the second mixed solution is a 4-20 mg / mL sodium alginate solution containing 64.5 μg / mL melittin;

[0015] The first mixed solution and the second mixed solution were taken in a volume ratio of 2:7, and were shaken and fully mixed in a turbine shaker to obtain the stimulus-responsive nanofiber-injectable composite hydrogel.

[0016] Further, the concentration of the calcium chloride solution is preferably 18 mM;

[0017] Preferably, the polyaniline nanofiber is prepared by oxidative polymerization of aniline and ammonium persulfate in a perchloric acid solution. Specifically, the preparation method of the polyaniline nanofiber is as follows: aniline solution and ammonium persulfate solid are added to a perchloric acid solution to prepare an aniline mixed solution and an ammonium persulfate mixed solution; the aniline mixed solution and the ammonium persulfate mixed solution are subjected to ultrasonic treatment and magnetic stirring, and then the ammonium persulfate mixed solution is added dropwise to the aniline mixed solution, and then reacted under ice-water bath conditions for at least 24 hours, and then washed twice with ethanol and ultrapure water, respectively, and then freeze-dried to obtain polyaniline nanofiber. Further preferably, the aniline solution is added to the perchloric acid solution by stirring evenly under a magnetic stirrer at 1000 rpm to form an aniline mixed solution; further preferably, the molar ratio of the aniline to the ammonium persulfate is 3:2; further preferably, the magnetic stirring is specifically stirring under a magnetic stirrer at 210 rpm.

[0018] Principle of the invention: In the prior art, calcium ion overload is achieved by intravenous injection of nano-drugs such as nano-calcium carbonate, which travel through the blood circulation to reach the tumor area to exert its effects, which can easily cause systemic toxicity; when bee venom peptide is used alone as an anticancer drug, its inhibition of cancer cell activity depends on a higher concentration, which will also produce greater toxic side effects on the body. In order to reduce the toxic side effects of anticancer drugs on normal cells and achieve local treatment by injection in the tumor area, the present invention uses injectable hydrogels loaded with bee venom peptide, polyaniline nanofibers with photothermal properties and oxidase-like properties, and selects calcium alginate hydrogel as the hydrogel carrier. On the one hand, calcium alginate is used as the hydrogel of the alginate system, compared with Compared with other hydrogels, it has good biocompatibility, fast degradation rate, and acidic stimulus response. It releases calcium ions, bee venom peptide, and positively charged polyaniline nanofibers in the microenvironment of tumor cells. On the other hand, after calcium alginate is degraded, calcium ions are continuously released in the extracellular fluid of cancer cells, forming a calcium ion overload. At the same time, the membrane-breaking effect of bee venom peptide allows large-particle polyaniline to quickly flow into cancer cells, and calcium ions can also flow in quickly, impairing the self-regulation mechanism of tumor cells, changing the tumor microenvironment, and causing serious damage to cancer cells. In addition, the polyaniline that enters the cancer cells further accelerates the apoptosis of cancer cells due to its excellent photothermal properties under the irradiation of external near-infrared light.

[0019] The present invention has the following effects:

[0020] 1. The preparation method of the stimulus-responsive injectable composite drug-loaded hydrogel provided by the present invention comprises the following steps: firstly synthesizing polyaniline nanofibers with excellent photothermal properties and oxidase-like properties, and then dispersing them in a calcium chloride solution, and finally preparing a sodium alginate solution containing bee venom peptide, and the two are used to prepare a calcium alginate hydrogel according to ion exchange; the polyaniline nanofibers are adsorbed in the calcium chloride solution by physical adsorption, and then form an injectable composite hydrogel with the sodium alginate solution containing the anticancer polypeptide drug bee venom peptide, thereby achieving uniform dispersion of the nanofibers and the polypeptide drug.

[0021] 2. The stimulus-responsive injectable composite drug-loaded hydrogel provided by the present invention has a lethal mechanism consisting of the membrane-breaking effect of bee venom peptide, the calcium ion overload effect, and the photothermal ablation effect of polyaniline nanofibers; it can realize multiple treatment methods including chemotherapy, ion therapy, and photothermal therapy, and utilizes the advantages of the properties of injectable hydrogels to solve the limitations of single therapy, and drug combination therapy improves the treatment effect;

[0022] 3. The encapsulation efficiency of polyaniline nanofibers and bee venom peptide is high, and the degradation rate of the injectable composite hydrogel is very fast. In the PBS buffer solution with pH = 6.0, the degradation efficiency reaches 92.3% in 12 hours, and the cumulative release of bee venom peptide is 50.1% in 48 hours and 72.8% in 168 hours, which can be administered for a long time. The drug delivery system is at 1064nm, 1.0W / cm 2 Under near-infrared light irradiation, the temperature can rise to 50.3°C within 10 minutes, and it has the characteristics of strong penetration into the tumor area and good photothermal performance.

[0023] 4. The materials used in the present invention have the characteristics of low toxicity and good biocompatibility, simple preparation and synthesis process, low cost of raw materials, which is very conducive to large-scale promotion and research, and has broad application prospects in the field of biological applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure is a diagram showing the preparation and action mechanism of the stimulus-responsive injectable composite drug-loaded hydrogel of the present invention;

[0025] Figure 2a is a SEM image of the polyaniline nanofibers prepared in Example 1;

[0026] Figure 2b This is a SEM image of the injectable composite hydrogel, the final product prepared in Example 1;

[0027] Figure 2c This is a photo of the final injectable composite hydrogel product prepared in Example 1 stored in a glass container;

[0028] Figure 2d The photo shows the injectable behavior of the injectable composite hydrogel finally prepared in Example 1;

[0029] Figure 3a This is a degradation curve diagram of the injectable composite hydrogel prepared in Example 1;

[0030] Figure 3b This is the release curve of the anticancer drug melittin in the injectable composite hydrogel prepared in Example 1;

[0031] Figure 4 The photothermal temperature rise curve of the injectable composite hydrogel prepared in Example 1;

[0032] Figure 5 This is a diagram showing the effect of reducing glutathione GSH of the injectable composite hydrogel prepared in Example 1;

[0033] Figure 6 The synergistic effect of melittin and calcium ions in the injectable composite drug-loaded hydrogel prepared in Example 1;

[0034] Figure 7 This is a MTT test chart of the biocompatibility of the injectable hydrogel prepared in Example 1 as a drug carrier;

[0035] Figure 8 This is a diagram showing the cell therapy effect of the injectable composite hydrogel drug delivery system prepared in Example 1. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below with reference to the embodiments.

[0037] like Figure 1 As shown, the present invention provides a stimulus-responsive injectable composite drug-loaded hydrogel and a preparation method thereof, wherein calcium chloride and sodium alginate solution constitute an injectable hydrogel skeleton structure, and bee venom peptide and polyaniline nanofibers are coated in a hydrogel matrix. A mixture of calcium chloride and polyaniline and a mixture of bee venom peptide and sodium alginate form a three-dimensional network hydrogel structure according to an appropriate ratio, which is injected into the affected area with a syringe. Under the stimulation of the tumor microenvironment, the injectable hydrogel gradually degrades, releasing bee venom peptide and polyaniline nanofibers, wherein bee venom peptide has a membrane-breaking effect, thereby promoting high-concentration calcium ions and polyaniline in the extracellular fluid to enter the cell, achieving a calcium ion overload effect, inducing mitochondrial dysfunction, reducing mitochondrial membrane potential, inhibiting intracellular ATP levels, and promoting cell apoptosis. Under the action of external near-infrared light, the excellent photothermal performance of polyaniline is demonstrated, and the temperature in the tumor area rises to 50°C, accelerating tumor cell death.

[0038] Example 1

[0039] The specific preparation process of a stimulus-responsive injectable composite drug-loaded hydrogel provided in Example 1 is as follows:

[0040] Step S1, preparing polyaniline nanofibers: taking 0.365 ml of 0.1M aniline solution in 20 ml of 0.1M perchloric acid solution, stirring evenly under a magnetic stirrer at 1000 rpm to prepare an aniline mixed solution; taking 0.609 g of ammonium persulfate and dissolving it in 20 ml of 0.1M perchloric acid solution to prepare an ammonium persulfate mixed solution; ultrasonically treating the aniline mixed solution and the ammonium persulfate mixed solution for 5 min, respectively, and then stirring them under a magnetic stirrer at 210 rpm, respectively, and then adding the ammonium persulfate mixed solution dropwise to the aniline mixed solution, and then reacting in an ice-water bath for 24 h; after the reaction is completed, separating under the action of a centrifuge at 10,000 rpm, and washing with 20 mL of ethanol and ultrapure water, respectively, and repeating three times to obtain purified polymer nanofibers;

[0041] Step S2, preparing an injectable composite drug-loaded hydrogel: using ultrapure water to prepare a calcium chloride solution with a concentration of 18 mM, and then adding 2.7 mg / mL of polyaniline nanofibers to obtain a first mixed solution; using ultrapure water to prepare a sodium alginate solution containing 64.3 μg / mL bee venom peptide and the concentration of the sodium alginate is 4 mg / mL, to obtain a second mixed solution; taking 100 μL of the first mixed solution and 350 μL of the second mixed solution respectively, adding them to 2 mL centrifuge tubes, and mixing them thoroughly in a turbine oscillator to obtain the product of this embodiment, namely the stimulus-responsive injectable composite drug-loaded hydrogel of the present invention.

[0042] Figure 2a This is a SEM image of the polyaniline nanofibers prepared in Example 1. It can be seen that the prepared polyaniline nanofibers are linear fibers with uniform size. The length of a single polyaniline nanofiber is in the range of 700-800 nm and the diameter is in the range of 100-150 nm.

[0043] Figure 2b This is a SEM image of the injectable composite hydrogel, the final product prepared in Example 1. It can be seen from the image that the injectable composite hydrogel presents a three-dimensional network structure, and the drug is encapsulated in the hydrogel matrix.

[0044] Figure 2c This is a photo of the final injectable composite hydrogel prepared in Example 1 stored in a glass container. The hydrogel has a good macroscopic shape;

[0045] Figure 2d The injectable composite hydrogel finally prepared in Example 1 is shown in a photo showing the injectable behavior, which can be easily injected from a syringe with a diameter of 0.45 mm to present any shape. The injectable composite hydrogel can be injected from a syringe with a diameter of less than or equal to 0.45 mm, and has excellent injectable properties.

[0046] Figure 3aThe degradation of the injectable composite hydrogel prepared in Example 1 under two PBS solutions of pH = 7.4 and pH = 6.0, respectively. Once the gel is degraded, the calcium ions therein will be released. Therefore, the degradation of the gel can be characterized according to the content of calcium ions in the external fluid. According to the tumor microenvironment, the comparative degradation under two different pH conditions was designed. When pH = 7.4, the release of calcium ions is very slow, and the cumulative release rates at 6h and 12h are 11.2% and 28.6%, respectively. This is because there is a small amount of calcium inside the gel. The ions do not serve as the hydrogel skeleton, but are coated in the hydrogel matrix like drugs. Therefore, in the solution environment, this small amount of calcium ions will be released. After 12 hours, the cumulative release curve of calcium ions tends to be flat. Therefore, the hydrogel degrades in a small amount in the environment of pH = 7.4. When the external PBS solution pH = 6.0, the hydrogel degradation rate is faster. The cumulative release rate of calcium ions at 6h and 12h is 58.7% and 97.6%, respectively. The release rate is much greater than that of the simulated body fluid environment. Therefore, the injectable composite hydrogel has an acidic pH stimulation response.

[0047] Specifically, the above Figure 3a The drawing and calculation methods of the calcium ion release curve are as follows: first, a calcium ion release standard curve is drawn using a calcium ion reagent detection kit; a dialysis bag with a molecular weight of 3000D is pre-soaked in hot water for 20 minutes, 900 μL of the prepared injectable composite hydrogel is transferred to the dialysis bag with a syringe, and then the dialysis bag is placed in a glass bottle containing 7 mL of a buffer solution containing two pH values; a suitable magnet is placed in the glass bottle, and the solution is stirred at 300 rpm on a magnetic stirrer; after the experiment starts, 50 μL of the dialysis solution is taken out at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 hours, respectively, and new buffer is added to keep the volume of the solution in the glass bottle unchanged; the ultraviolet absorbance of the dialysis solution is measured under an ELISA instrument, and the release rate can be calculated by substituting it into the standard curve equation.

[0048] Figure 3bThe release curve of the anticancer drug melittin in the injectable composite hydrogel prepared in Example 1; Similarly, two different pH conditions were designed to test whether the acidic stimulus response of the injectable composite hydrogel can accelerate the release of the drug, and the drug can minimize its side effects on normal tissues. When pH = 7.4, the release of melittin is very slow, and the cumulative release rates at 24h and 48h are 23.1% and 25.6%, respectively. It can be explained that the early release is slightly faster, and then the release is slow. Continue to monitor its release curve, and it is found that the release of melittin is flat, and the cumulative release rate of 168h is 26.0%. Considering the influence of experimental error, it can be determined that melittin is released slowly in the simulated body fluid environment, and when the external PBS solution pH = 6.0, due to the fast degradation rate of the hydrogel, the cumulative release rates of melittin at 24h and 48h are 38.1% and 54.6%. Continue to monitor, the cumulative release of melittin is still increasing, and finally released to 72.7% at 168h, achieving the effect of long-term administration and controlled release.

[0049] Specifically, the drawing and calculation method of the above-mentioned bee venom peptide release curve is as follows: first prepare 0, 2, 4, 6, 8, 10 μg / mL standard solutions, and measure the corresponding fluorescence intensity under a fluorescence spectrometer (280 nm excitation wavelength, 356 nm absorption wavelength) to draw a standard curve of bee venom peptide; soak a dialysis bag with a molecular weight of 7000D in hot water for 20 minutes in advance, transfer 450 μL of the prepared injectable composite hydrogel into the dialysis bag with a syringe, and then put the dialysis bag into a 5 mL containing Two pH buffer solutions are placed in glass bottles; a suitable magnet is placed in the glass bottle, and the solution is stirred on a shaker at a rate of 100 rpm; 1 mL of the dialyzed solution is removed at time points of 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h, respectively, and new buffer under the same conditions is added to keep the volume of the solution in the glass bottle unchanged; the fluorescence intensity of the dialyzed solution is measured under a fluorescence spectrometer, and the release rate can be calculated by substituting it into the standard curve equation.

[0050] Figure 4 The photothermal temperature rise curve of the injectable composite hydrogel prepared in Example 1. Due to the excellent photothermal performance of polyaniline, the temperature rise effect of the injectable composite hydrogel doped with polyaniline nanofibers of different concentrations was compared. 2 Under the action of , the injectable composite hydrogel without polyaniline nanofiber doping hardly heated up within 10 min, while the groups doped with 0, 0.2, 0.6, and 1.0 mg / mL polyaniline heated up to 46.1°C, 50.3°C, and 52.5°C, respectively, showing a strong photothermal effect.

[0051] Figure 5This is a graph showing the glutathione GSH reduction experiment of the injectable composite hydrogel prepared in Example 1. The concentration of GSH is consistent with the concentration in tumor cells, which is 10mM. Since polyaniline has the ability to reduce GSH, a simulated GSH reduction experiment was conducted on the injectable hydrogel doped with polyaniline. After making injectable hydrogels containing 0 and 0.6 mg / mL polyaniline, 450 μL of gel was drawn into a 10 mL glass bottle. In order to simulate the physiological environment of the tumor microenvironment, 3 mL of pH = 6.0 PBS buffer solution was added to the bottle, and then placed on a shaker at 37°C and 200 rpm. 600 μL of supernatant was taken on the 1st, 2nd, 3rd, and 4th day, respectively, and mixed with 600 μL of 10mM GSH solution, and an equal volume of PBS buffer solution was added. After 2 days of blending, 180 μL of the mixture was taken into a 1.5 mL centrifuge tube, and then 314 μL Tris-HCl (pH = 8.5) and 6 μL DTNB solution were added. After being mixed evenly using a turbine shaker, the mixture was centrifuged at 10,000 r / min for 10 minutes, and 150 μL of the supernatant was taken into a 96-well plate. The ultraviolet absorption value was measured at a wavelength of 412 nm under an ELISA reader. When the concentration of polyaniline in the injectable hydrogel was 0.6 mg / mL, GSH could be reduced by more than 55% in 4-5 days, showing good GSH reduction properties.

[0052] Figure 6 The synergistic effect of melittin and calcium ions in the injectable composite drug-loaded hydrogel prepared in Example 1 was tested using 4T1 cells and the MTT test method. When only melittin was present during the incubation process, the cell survival rate exceeded 85% at 2 μg / mL, and when the system was doped with calcium ions at the same concentration as the extracellular fluid, the cell activity dropped sharply to less than 20%, which can effectively reduce the concentration of melittin alone during treatment and reduce side effects.

[0053] Figure 7 The results of the biocompatibility test of the injectable hydrogel prepared in Example 1 as a drug carrier are shown. For intelligent drug delivery systems, excellent biocompatibility of the carrier is crucial. In order to test the biocompatibility of injectable hydrogels with different gel contents, the MTT test method was used. According to the experimental results, the cell survival rates of the hydrogels with different gel contents in the range of 0.09wt%-1.42wt% were both above 95% at 24h and 48h compared with the Control group, with no obvious toxicity and good biocompatibility.

[0054] Figure 8The cell therapy effect diagram of the injectable composite hydrogel drug-carrying system prepared in Example 1 is shown in Table 1. The experiment adopted the MTT test method and designed 6 different treatment groups. Among them, the drug-carrying group MP@HG+L had the strongest killing effect on tumor cells under the conditions of photothermal Laser. After incubation with tumor cells for 48 hours, more than 80% of tumor cells were killed; the specific experimental method is as follows: 6 groups of materials were prepared, among which the gelation ratio of the injectable hydrogel HG was consistent with the above, the concentration of polyaniline nanofiber PANI NF was 0.6 mg / mL, and the concentration of bee venom peptide Melittin was 50 μg / mL; two 96-well plates were taken, and 6 groups were set up respectively, with 5 duplicate wells in each group. 150 μL of cell suspension was added to each well and cultured in an incubator (37°C, 5% CO2) for 24 h; 50 μL of corresponding materials were added to each group of two 96-well plates and incubated in an incubator for 24 h and 48 h, respectively; after the incubation was completed, the upper mixed solution was aspirated, 50 μL of MTT (1×) solution was added to each well, and the cells were incubated in an incubator for another 4 h; the upper MTT was aspirated, 200 μL of DMSO solution was added to each well, and the cells were reacted on a shaker (110 rpm) for 15 min; the absorbance was measured at 490 nm on an ELISA reader and then compared with that of the Control group.

[0055] Table 1

[0056]

[0057] All test results show that the stimuli-responsive injectable composite drug-loaded hydrogel involved in the present invention adopts the form of encapsulating nanofibers and anticancer polypeptides into the hydrogel, which greatly improves the drug loading amount and drug loading form, and combines the tumor microenvironment stimuli-responsive characteristics to achieve controlled release of drugs, synergistic treatment, and improve the therapeutic effect. The synthesis process of this method is simple, the material is non-biotoxic, and the cost is low, which is conducive to large-scale promotion and research. In addition, it has important research significance for the preparation of drug delivery system materials.

Claims

1. A method for preparing a stimuli-responsive injectable composite drug-loaded hydrogel, characterized in that: The preparation method comprises the following steps: Selecting polyaniline nanofibers that are linear fibers with a length of 700-800 nm and a diameter of 100-150 nm; A calcium chloride solution is prepared with ultrapure water, wherein the concentration of the prepared calcium chloride solution is in the range of 2-100 mM, and then polyaniline nanofibers are added to obtain a first mixed solution, wherein the concentration of the polyaniline nanofibers in the first mixed solution is 2.7 mg / mL; A sodium alginate solution containing melittin is prepared with ultrapure water, i.e., a second mixed solution, wherein the second mixed solution is a 4-20 mg / mL sodium alginate solution containing 64.5 μg / mL melittin; The first mixed solution and the second mixed solution are taken in a volume ratio of 2:7, and the mixture is fully mixed by shaking in a turbine shaker to obtain the stimulus-responsive nanofiber-injectable composite hydrogel; The concentration of the calcium chloride solution is 18 mM; The preparation method of the polyaniline nanofiber comprises: adding an aniline solution and an ammonium persulfate solid into a perchloric acid solution respectively to prepare an aniline mixed solution and an ammonium persulfate mixed solution respectively; subjecting the aniline mixed solution and the ammonium persulfate mixed solution to ultrasonic treatment and magnetic stirring respectively, then adding the ammonium persulfate mixed solution dropwise into the aniline mixed solution, then reacting in an ice water bath for at least 24 hours, then washing twice with ethanol and ultrapure water respectively, and then freeze-drying to obtain the polyaniline nanofiber.

2. The method for preparing a stimulus-responsive injectable composite drug-loaded hydrogel according to claim 1, characterized in that: The aniline solution is added to the perchloric acid solution, and the mixture is stirred evenly under a magnetic stirrer at 1000 rpm to prepare an aniline mixed solution.

3. The method for preparing a stimulus-responsive injectable composite drug-loaded hydrogel according to claim 1, characterized in that: The molar ratio of the aniline to the ammonium persulfate is 3:

2.

4. The method for preparing a stimulus-responsive injectable composite drug-loaded hydrogel according to claim 1, characterized in that: The magnetic stirring is specifically stirring at 210 rpm with a magnetic stirrer.

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