Hydrogel-microsphere composite drug-loaded material and preparation method thereof

Abstract

The invention discloses a hydrogel-microsphere composite drug-loading material. The hydrogel-microsphere composite drug-loading material is prepared from the following components in parts by mass: 5 to 10 parts of poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug, 5 to 10 parts of a second anti-tumor drug and 35 to 70 parts of temperature-sensitive hydrogel. After injection beside a tumor, the temperature-sensitive hydrogel is quickly converted into a gel state from a liquid state, the second anti-tumor drug is quickly released through diffusion, and the first anti-tumor drug is slowly released along with degradation of the hydrogel and the poly (lactic-co-glycolic acid) microspheres, so that precise space-time delivery of double drugs in local tumor is realized; the release speeds of the two drugs can be effectively controlled, and the difference of the release speeds of the two drugs within 5 days is about 15%; the release rate difference in 10 days is about 30%; the polyamino acid and the polylactic acid can be degraded in vivo, and degradation products can be directly discharged out of the body through the kidney and are harmless to the human body; the hydrogel-microsphere composite drug carrying material has a good anti-tumor effect and biocompatibility, and has a wide application prospect.

Description

Technical Field

[0001] This application relates to the field of polymer drug carriers, and particularly to a hydrogel-microsphere composite drug-loading material and a preparation method thereof. Background Art

[0002] Malignant tumors pose a great threat to human health, and the metastasis of malignant tumors symbolizes the deterioration of the disease and the increase in the difficulty of treatment. Bone is the third most common site of solid tumor metastasis, and lung cancer is one of the common primary tumors with bone metastasis. Approximately 30-40% of lung cancer patients develop bone metastasis. Lung cancer bone metastasis leads to bone-related events, including bone pain, hypercalcemia, and pathological fractures, severely reducing the quality of life. The treatment methods for lung cancer bone metastasis, such as radiotherapy, chemotherapy, and phototherapy, are usually palliative and have limited efficacy.

[0003] Combined therapy refers to the combination of two or more treatment methods, especially the combination of treatment methods based on bioactive drugs, to improve efficacy or reduce side effects. However, combined therapy often fails to achieve the expected effect. The main reason is that the bioavailability of orally administered small molecule bioactive drugs is usually low, and their spatio-temporal distribution in the body is not ideal. Therefore, there is an urgent need to find a method to precisely deliver bioactive drugs to the tumor site at specific time points. A variety of biomaterials have been developed as delivery systems for anti-tumor bioactive drugs. Among these biomaterials, nanoparticles can simultaneously deliver multiple bioactive drugs to the tumor microenvironment and even tumor cells, thus greatly improving the bioavailability and anti-tumor efficiency of bioactive drugs. For example, in order to maximize the ability of chemotherapy drugs to kill tumors, scientists prepared a manganese dioxide-based nanoparticle, encapsulated cisplatin, and modified Gly-Arg-Gly-Asp-Ser(TFA) on its surface. TFA is a bioactive drug that reverses cisplatin resistance by blocking the mTORC2 / AKT / NF-κB / MDR1 signaling pathway and can target osteopontin highly expressed in tumors. After intravenous administration, the nanoparticles precisely target the tumor and release cisplatin, while TFA improves the efficacy of cisplatin. The results showed that the nanoparticles could significantly block the spinal metastasis of non-small cell lung cancer in mice. However, this only achieved the delivery of a single drug.

[0004] Although many nanoparticle-based anti-tumor bioactive drug delivery systems have been developed, making multi-drug combination therapy and tumor targeting possible, it is still difficult to achieve precise control of drug release.

[0005] Therefore, providing a drug-loading material that can improve anti-tumor efficacy, reduce side effects, and precisely regulate the loading amount and spatio-temporal release of multiple bioactive drugs is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present application provides a hydrogel-microsphere composite drug-loading material, which can achieve sequential release of two anti-tumor drugs locally in tumors, and has good anti-tumor effects and biocompatibility.

[0007] As a popular biomaterial, hydrogels and microspheres are also widely used in the delivery and controlled release of anti-tumor bioactive drugs. Hydrogels have a hydrophilic three-dimensional network structure and rapidly swell in water. During the swelling process, the hydrogels retain a large amount of water without dissolving. This unique property enables hydrogels to meet various biomedical applications, such as drug or cell delivery carriers, tissue filling materials, and artificial tissue mimics. At the same time, hydrogels have strong plasticity and can be modified by introducing new injectable or thermosensitive functional groups or components, such as injectable thermosensitive polyamino acid hydrogels. Polyamino acid hydrogels have good biocompatibility and appropriate degradation rates, and can achieve slow release and sustained release of bioactive drugs at the tumor site. However, with the degradation of hydrogels, various bioactive substances are usually released in a diffusive manner, and the release order is uncontrollable. Microspheres have the characteristics of degradability, simple preparation methods, good drug-loading performance, high stability, etc., and are widely used as delivery platforms for anti-tumor bioactive drugs. Among these microspheres, poly(lactic-co-glycolic acid) (PLGA) microspheres have attracted much attention due to their high safety and controllable mechanical properties. By adjusting the monomer ratio, molecular weight, concentration, terminal modification, and preparation methods, PLGA microspheres can meet complex spatio-temporal delivery requirements. However, it is difficult to precisely control the drug-loading ratio and release rate of multiple bioactive drugs in microspheres, resulting in low combined treatment efficiency. Therefore, in the combined treatment of tumors, the composite of hydrogels and microspheres is a promising strategy that can precisely regulate the loading amount and spatio-temporal release of multiple bioactive drugs.

[0008] The present application provides a hydrogel-microsphere composite drug-loading material, which includes 5 to 10 parts of poly(lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug, 5 to 10 parts of a second anti-tumor drug, and 35 to 70 parts of a thermosensitive hydrogel.

[0009] The present application synthesizes a hydrogel-microsphere composite drug-loading material using poly(lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug, a second anti-tumor drug, and a thermosensitive hydrogel as raw materials. It has the effect of controlling the spatio-temporal release of two anti-tumor drugs, and the difference in the release rates of the two drugs is controlled above 15%, and it has good anti-tumor effects and biocompatibility. In some specific implementation manners, the drug-loading rate of the poly(lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug is 4% to 6%.

[0010] The hydrogel-microsphere composite drug-loading material described in this application includes poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug. In some specific implementation manners, the particle size of the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug is 0.2 - 2.0 μm, preferably 0.77 ± 0.16 μm; the first anti-tumor drug includes but is not limited to one or more of SB525334, LY2157299, LY2109761, EPZ005687, Tazemetostat, GSK343, Lirametostat, Dactolisib or LY294002. There are no special requirements for the selection of the first anti-tumor drug in this application, and preferably it is SB525334. In some specific implementation manners, the mass ratio of the poly (lactic-co-glycolic acid) to the first anti-tumor drug is 5:1 to 20:1. Microspheres have the characteristics of biodegradability, simple preparation method, good drug-loading performance, high stability, etc., and are widely used as a delivery platform for anti-tumor bioactive drugs. The first anti-tumor drug is coated with poly (lactic-co-glycolic acid), and the first anti-tumor drug is slowly released as the temperature-sensitive hydrogel and poly (lactic-co-glycolic acid) microspheres degrade. The mass fraction of the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug is 5 parts to 10 parts, and can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts.

[0011] The hydrogel-microsphere composite drug-loading material described in this application includes a second anti-tumor drug. In some specific implementation manners, the second anti-tumor drug includes but is not limited to one or more of Anlotinib, Gefitinib, Erlotinib, Afatinib, Osimertinib, Imatinib, Dasatinib, Pazopanib, Regorafenib or Vandetanib. There are no special requirements for the selection of the second anti-tumor drug in this application. The mass fraction of the second anti-tumor drug is 5 parts to 10 parts, and can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts.

[0012] The hydrogel-microsphere composite drug-loading material described in this application includes a temperature-sensitive hydrogel. In some specific implementation manners, the temperature-sensitive hydrogel has the structure of Formula 1;

[0013]

[0014] n is 20 to 30.

[0015] In some specific implementation manners, the temperature-sensitive hydrogel has the structure of Formula 2;

[0016]

[0017] The present application also provides a method for preparing a hydrogel-microsphere composite drug-loading material, including:

[0018] Mixing a temperature-sensitive hydrogel, a second anti-tumor drug, and poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug to obtain a hydrogel-microsphere composite drug-loading material.

[0019] The present application first synthesizes poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug. The preparation method includes: mixing poly (lactic-co-glycolic acid), a first anti-tumor drug, a surfactant, and an organic solvent, centrifuging, and freeze-drying to obtain poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug. In some specific implementation manners, poly (lactic-co-glycolic acid) and a first anti-tumor drug are dissolved in an organic solvent, mixed with a high-concentration surfactant, emulsified, and then added to a low-concentration surfactant, stirred, centrifuged, and freeze-dried to obtain poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug. In some specific implementation manners, the rotation speed of the centrifugation is 2000 r / min to 9000 r / min, preferably 3000 r / min; the mass ratio of poly (lactic-co-glycolic acid) to the first anti-tumor drug is 5:1 to 20:1, preferably 5:1; the organic solvent includes but is not limited to dichloromethane, and the present application has no special requirements for the selection of the organic solvent; the surfactant includes but is not limited to Tween 80, and the present application has no special requirements for the selection of the surfactant.

[0020] The present application then synthesizes a temperature-sensitive hydrogel. The preparation method includes: reacting L-methionine-N-carboxyanhydride, an initiator, and a solvent to obtain a temperature-sensitive hydrogel.

[0021] In some specific implementation manners, using end-aminated monomethoxy polyethylene glycol (Formula 3) as an initiator, reacting with L-methionine-N-carboxyanhydride (Formula 4) in dimethyl sulfoxide, dialyzing, and freeze-drying to obtain a temperature-sensitive hydrogel. In some specific implementation manners, the molar ratio of the initiator to L-methionine-N-carboxyanhydride is 1:20 to 1:30, preferably 1:25; the solvent includes but is not limited to dimethylformamide, and the present application has no special requirements for the selection of the solvent; the initiator includes but is not limited to end-aminated monomethoxy polyethylene glycol, and the present application has no special requirements for the selection of the initiator. The present application has no special restrictions on the source of end-aminated monomethoxy polyethylene glycol, and it can be prepared by the resin preparation method of those skilled in the art or a commercially available product. In some specific implementation manners, the reaction time is 2 days to 6 days, preferably 3 days. In some specific implementation manners, the dialysis time is 2 days to 6 days, preferably 3 days.

[0022]

[0023] In some specific implementation manners, triphosgene method is adopted to prepare L-methionine-N-carboxyanhydride. The water in the flask is removed by heating with a hot air gun, and then 120.0 mL of anhydrous tetrahydrofuran, 15.0 g of L-methionine and 23.0 g of triphosgene are added. The reaction is carried out in an oil bath at 60.0 °C and maintained in a nitrogen environment. After about 1 hour, the flask is taken out of the oil bath, the nitrogen flow rate is increased, and the nitrogen flow is stopped when the remaining liquid is about 20.0 mL. It is immediately precipitated with ice n-hexane, then dissolved with a small amount of ethyl acetate, and washed three times with ice sodium chloride solution. Anhydrous magnesium sulfate is added to remove water overnight, and then vacuum is applied to obtain solid L-methionine-N-carboxyanhydride.

[0024] In this application, the thermosensitive hydrogel is dissolved in a buffer solution to obtain a thermosensitive hydrogel solution. In some specific implementation manners, the buffer solution includes but is not limited to PBS, and this application has no special requirements for the selection of the buffer solution. In some specific implementation manners, the temperature of the mixing is 0 °C to 8 °C, preferably 4 °C; the time of the mixing is 2 days to 6 days, preferably 3 days.

[0025] Then, in this application, the thermosensitive hydrogel solution, the second anti-tumor drug and the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug are mixed to obtain a hydrogel-microsphere composite drug-loading material. In some specific implementation manners, the time of the mixing is 20 min to 50 min, preferably 30 min. In some specific implementation manners, the mass concentration of the thermosensitive hydrogel in the thermosensitive hydrogel solution is 5 wt% to 10 wt%, preferably 7 wt%.

[0026] After the hydrogel-microsphere composite drug-loading material provided by this application is injected beside the tumor, the thermosensitive hydrogel rapidly changes from a liquid state to a gel state, the second anti-tumor drug is rapidly released by diffusion, and the first anti-tumor drug is slowly released along with the degradation of the hydrogel and the poly (lactic-co-glycolic acid) microspheres, so as to realize the precise spatio-temporal delivery of the two drugs at the tumor local area, and the release rates of the two drugs can be effectively controlled. The release rates of the two drugs differ by about 15% in 5 days; and by about 30% in 10 days; polyamino acid and polylactic acid can be degraded in vivo, and the degradation products can be directly excreted from the body through the kidneys without harm to the human body; the hydrogel-microsphere composite drug-loading material has good anti-tumor effects and biocompatibility, and has broad application prospects. Description of the Drawings

[0027] Figure 1 It is the scanning electron microscope image of the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug prepared in Example 1 of this application;

[0028] Figure 2 It is the particle size distribution diagram of the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug prepared in Example 1 of this application;

[0029] Figure 3 1H NMR spectrum of the thermosensitive hydrogel prepared in Example 2 of the present application;

[0030] Figure 4 Solution-gel phase transition temperature diagram of the thermosensitive hydrogel prepared in Example 2 of the present application;

[0031] Figure 5 Circular dichroism spectrum of the thermosensitive hydrogel prepared in Example 2 of the present application;

[0032] Figure 6 In vitro drug release diagram of the hydrogel-microsphere composite drug delivery material prepared in Example 3 of the present application;

[0033] Figure 7 Tumor inhibition curve obtained from the in vivo tumor inhibition experiment of the hydrogel-microsphere composite drug delivery material prepared in Example 3 of the present application;

[0034] Figure 8 1H NMR spectrum of the thermosensitive hydrogel prepared in Comparative Example 2 of the present application;

[0035] Figure 9 In vitro drug release diagram of the hydrogel-microsphere composite drug delivery material prepared in Comparative Example 2 of the present application. Detailed Description of the Invention

[0036] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0037] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open-ended and non-limiting, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0038] It should be understood that as long as the present application is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be performed simultaneously.

[0039] The use of any and all examples or exemplary language such as "for example" or "including" herein is merely intended to better illustrate the present application and does not limit the scope of the present application unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present application.

[0040] In addition, the numerical ranges and parameters used to define the present application are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently and inevitably contains standard deviations caused by individual testing methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, numerical values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific numerical value or range.

[0041] The present application provides a hydrogel-microsphere composite drug-loading material, which includes, by mass parts: 5 to 10 parts of poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug, 5 to 10 parts of a second anti-tumor drug, and 0.1 to 50 parts of a temperature-sensitive hydrogel.

[0042] After the hydrogel-microsphere composite drug-loading material is injected beside the tumor, the temperature-sensitive hydrogel rapidly changes from a liquid state to a gel state, and the second anti-tumor drug is rapidly released by diffusion, while the first anti-tumor drug is slowly released along with the degradation of the hydrogel and poly (lactic-co-glycolic acid) microspheres, thereby realizing the precise spatio-temporal delivery of the two drugs at the tumor site, effectively controlling the release rates of the two drugs. The release rates of the two drugs differ by about 15% at 5 days; and by about 30% at 10 days; polyamino acids and polylactic acid are biodegradable in vivo, and the degradation products can be directly excreted from the body through the kidneys, which is harmless to the human body; the hydrogel-microsphere composite drug-loading material has good anti-tumor effects and biocompatibility, and has broad application prospects.

[0043] The following further elaborates on the present application in conjunction with embodiments. The protection scope of the present application is not limited by the following embodiments.

[0044] Example 1

[0045] This example provides poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug. The preparation method of the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug includes:

[0046] Dissolve 100.0 mg of poly (lactic-co-glycolic acid) and 20.0 mg of SB525334 in 10.0 mL of dichloromethane. Add 20.0 mg (about the mass of one drop) of Tween 80 and sonicate for 10 minutes to dissolve. Mix the above solution with 10.0 mL of PVA aqueous solution (1.5 wt%) to obtain a mixture. The sonication was performed using a Y92-IIN ultrasonic emulsifier (Ningbo Xinzhi Biotechnology Co., Ltd., China), and the working parameters were set as follows: the power was 50%, the total working time was 2 minutes, the sonication on-time was 3.0 seconds, and the sonication off-time was 2.0 seconds in a constant cycle. The mixture was ultrasonically emulsified, and then the resulting product was poured into 45.0 mL of PVA aqueous solution (0.5 wt%), and magnetically stirred for 6 hours. Centrifuge at 3000 r / min for 10 minutes, discard the supernatant, add ultrapure water to resuspend the product and wash it 3 times. Lyophilize to obtain poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug. The yield of poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug was 89.5%, the drug loading rate was 5.0%, and the drug loading efficiency was 30.1%.

[0047] Perform scanning electron microscopy and particle size measurement on poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug:

[0048] Resuspend the microspheres with ultrapure water, drop them on a silicon wafer and let them dry naturally, and then sputter gold. Take microsphere images at different magnifications under a JSM-7900F thermal field emission scanning electron microscope. The scanning electron micrograph of poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug prepared in Example 1 of this application is as Figure 1 shown; Measure the diameters of at least 100 microspheres through ImageJ software and calculate the average particle size and variance. The particle size distribution diagram of poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug prepared in Example 1 of this application is as Figure 2 shown.

[0049] Example 2

[0050] This example provides a thermosensitive polyamino acid hydrogel: poly (ethylene glycol)-poly-L-methionine-N-carboxyanhydride (structure of formula 2).

[0051]

[0052] The preparation method of the thermosensitive polyamino acid hydrogel includes:

[0053] 1.0 g of the terminal amino-functionalized monomethoxypolyethylene glycol was added to a reaction flask, and water was removed by azeotropic distillation with toluene. 30.0 mL of anhydrous dimethylformamide was added to dissolve the terminal amino-functionalized monomethoxypolyethylene glycol, and then 2.2 g of L-methionine-N-carboxyanhydride was added. The reaction was carried out at room temperature for 3 days, dialyzed with ultrapure water for 3 days, and freeze-dried to obtain a powdery product, which was the thermosensitive polyamino acid hydrogel.

[0054] The nuclear magnetic resonance (NMR) test was performed on the obtained polyamino acid hydrogel poly(ethylene glycol)-poly(L-methionine-N-carboxyanhydride). The 1H NMR spectrum of the thermosensitive hydrogel prepared in Example 2 of this application is as Figure 3 shown.

[0055] The solution-gel transition experiment was carried out on the obtained polyamino acid hydrogel poly(ethylene glycol)-poly(L-methionine-N-carboxyanhydride): The solution-gel transition temperature of the hydrogel was determined by the test tube inversion method. Four different hydrogel solutions with mass concentrations of 6 wt%, 7 wt%, 8 wt%, and 9 wt% were prepared with phosphate buffered saline (PBS) at pH = 7.4 and stirred at 4 °C for 3 days. 300.0 μL of the hydrogel was taken respectively and loaded into glass bottles with a diameter of 11 mm, and then placed in a water bath box set to start heating from 0 °C. Each time the temperature was increased by 2 °C and stabilized for 5 minutes, the vial was tried to be inverted. If the hydrogel did not flow within 30 seconds, it was considered that it had transitioned from the liquid state to the gel state. The solution-gel phase transition temperature diagram of the thermosensitive hydrogel prepared in Example 2 of this application is as Figure 4 shown.

[0056] The circular dichroism (CD) test was performed on the obtained poly(ethylene glycol)-poly(L-methionine-N-carboxyanhydride). The test results are as Figure 5 shown. As the temperature increased, the secondary structure of the polymer continuously changed, providing physical cross-linking for the polymer network and promoting the sol-gel transition.

[0057] Example 3

[0058] This example provides a hydrogel-microsphere composite drug carrier material. The preparation method of the hydrogel-microsphere composite drug carrier material includes:

[0059] A solution of the thermosensitive hydrogel provided in Example 2 with a mass concentration of 7 wt% was prepared with PBS and stirred at 4 °C for 3 days. Subsequently, 9.0 mg of Anlotinib and the poly(D,L-lactide-co-glycolide) microspheres loaded with 9.0 mg of the first anti-tumor drug provided in Example 1 were added to each 1.0 mL of the thermosensitive hydrogel solution, and stirring was continued for 30 min to mix them, obtaining a mixed solution.

[0060] In vitro drug release experiment of the hydrogel-microsphere composite drug-loading material for spatio-temporal delivery of anti-tumor drugs: Take 300.0 μL of the mixed solution of the hydrogel-microsphere composite material in a glass bottle with a diameter of 16 mm, and place it in a 37 °C water bath for 30 min to form a stable gel state. Then, add 1.0 mL of PBS and 1.0 mL of PBS containing 0.2 mg / mL elastase to the vials respectively, and collect the upper layer solution at the preset time points, and then add the corresponding new solution. The concentration of the drug contained in the removed solution was measured by high performance liquid chromatography, and the cumulative release rate of the drug was calculated. The in vitro drug release diagram of the hydrogel-microsphere composite drug-loading material prepared in Example 3 of this application is as Figure 6 shown. The release rate of the second anti-tumor drug is relatively fast, while the first anti-tumor drug is slowly released with the degradation of the hydrogel and poly (lactic-co-glycolic acid) microspheres, so as to achieve the precise spatio-temporal delivery of the two drugs locally in the tumor. The release rates of the two drugs differ by about 15% at 5 days; and by about 30% at 10 days. Within 30 days, the cumulative release amounts of the second anti-tumor drug and the first anti-tumor drug in PBS reach 70.4% and 42.7% respectively, while the cumulative release amounts in PBS plus elastase (simulating the tumor tissue microenvironment) are 92.4% and 58.5% respectively. Sustained release ensures the effective concentration of the drug at the tumor site and reduces the toxic side effects. The release rate of the second anti-tumor drug is always faster than that of the first anti-tumor drug. Therefore, the hydrogel-microsphere composite system achieves the expected goal of sequential release of the two drugs and can be used for subsequent animal experiments.

[0061] In vivo anti-tumor experiment of the hydrogel-microsphere composite drug-loading material: Male C57 mice (6-8 weeks old) were selected and a lung cancer bone metastasis model was constructed for them. The specific method: When the LLC cells were cultured to the logarithmic growth phase, the cells were digested and configured into a cell suspension with a concentration of 1.0×10 7 cells / mL. We aspirated the cell suspension with a 1.0 mL syringe and placed it on ice for standby. The mice were anesthetized with isoflurane, and then the skin of the right hind limb of the C57 mice was prepared. We broke through the tibial plateau of the right hind limb with a syringe, entered the bone marrow cavity, and injected 50.0 μL of the cell suspension. Six days later, when the tumor broke through the tibial plateau and the tumor volume was about 100 mm 3 , in-situ treatment could be carried out.

[0062] Mice were grouped according to different treatment methods: PBS group (control), dual-free drug group (second anti-tumor drug + first anti-tumor drug), hydrogel-loaded Anlotinib group (hydrogel / second anti-tumor drug), hydrogel-loaded Anlotinib + SB525334 group (hydrogel / (second anti-tumor drug + first anti-tumor drug)), and hydrogel-loaded Anlotinib + poly(lactic-co-glycolic acid) microspheres loaded with SB525334 group (hydrogel / (second anti-tumor drug + poly(lactic-co-glycolic acid) microspheres / second anti-tumor drug)). The final dosing dose was Anlotinib: 9.0 mg / mouse, SB525334: 9.0 mg / mouse. Treatments were all administered by peritumoral injection and single-dose administration.

[0063] The major and minor axes of the tumor were measured every other day, and the tumor volume was calculated using the following formula:

[0064] Tumor volume = major axis × minor axis × minor axis / 2.

[0065] After 10 days of measurement, the mice were sacrificed and the tumor inhibition curves were plotted. The tumor inhibition curve obtained from the in vivo tumor inhibition experiment of the hydrogel-microsphere composite drug-loading material prepared in Example 3 of this application is as Figure 7 shown. On the 10th day of treatment, the anti-tumor effect of the hydrogel-microsphere composite material group (397.1 ± 116.5 mm 3 ) was the best, less than one-third of the average tumor volume of the control group; and its tumor volume was significantly smaller than that of the hydrogel / (second anti-tumor drug + poly(lactic-co-glycolic acid) microspheres / second anti-tumor drug) group (667.7 ± 85.7 mm 3 ), which proved that the composite material achieved a better anti-tumor effect through the sequential release of drugs.

[0066] Example 4

[0067] This example provides poly(lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug. The difference from Example 1 is that the centrifugation speed of the drug-loaded poly(lactic-co-glycolic acid) microspheres is 9000 r / min.

[0068] The preparation method includes:

[0069] Dissolve 100.0 mg of poly (lactic-co-glycolic acid) and 20.0 mg of SB525334 in 10.0 mL of dichloromethane. Add 20.0 mg (about the mass of one drop) of Tween 80 and sonicate for 10 minutes to dissolve. Mix the above solution with 10.0 mL of PVA aqueous solution (1.5 wt%) to obtain a mixture. The sonication is carried out using a Y92-IIN ultrasonic emulsifier (Ningbo Xinzhi Biotechnology Co., Ltd., China), and the working parameters are set as follows: the power is 50%, the total working time is 2 minutes, the ultrasonic startup time is 3.0 seconds, and the ultrasonic shutdown time is 2.0 seconds for a constant cycle. Ultrasonically emulsify the mixture, then pour the obtained product into 45.0 mL of PVA aqueous solution (0.5 wt%) and stir magnetically for 6 hours. Centrifuge at 9000 r / min for 10 minutes, discard the supernatant, add ultrapure water to resuspend the product and wash it 3 times. Lyophilize to obtain poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug. The yield of poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug is 79.1%, the drug loading rate is 3.8%, and the drug loading efficiency is 41.8%.

[0070] Comparative Example 1

[0071] This comparative example provides a hydrogel-microsphere composite drug-loading material, which is different from Example 3 in that: the nanoparticles loaded with the first anti-tumor drug are used.

[0072] The preparation method includes:

[0073] The nanoparticles used are polymers of glutamic acid and phenylalanine initiated by end-aminated monomethoxy polyethylene glycol (the polymerization degree is 20:10). Dissolve 17.6 mg of the nanoparticle polymer powder in 1.0 mL of dimethylformamide, and dissolve 0.6 mg of SB525334 in 0.6 mL of dimethyl sulfoxide. Then mix the two and slowly drop them into 5.0 mL of PBS using a micro syringe pump. Stir for 3 hours, then dialyze with ultrapure water for 3 hours and lyophilize to obtain nanoparticles loaded with the first anti-tumor drug. The yield of nanoparticles loaded with the first anti-tumor drug is 54.9%, the drug loading rate is 0.62%, and the drug loading efficiency is 18.7%. Since the drug loading rate and drug loading efficiency of the nanoparticles are too low, they are not considered for use in the subsequent composite system.

[0074] Comparative Example 2

[0075] This comparative example provides a hydrogel-microsphere composite drug-loading material, which is different from Example 3 in that: the hydrogel used is a poly (L-norleucine-N-carboxyanhydride) hydrogel (polyethylene glycol-poly (L-norleucine-N-carboxyanhydride), structure of Formula 5) initiated by end-aminated monomethoxy polyethylene glycol.

[0076]

[0077] The preparation method of the temperature-sensitive polyamino acid hydrogel includes:

[0078] Add 1.0 g of end-aminated monomethoxy poly(ethylene glycol) to a reaction flask, and remove water by azeotropic distillation with toluene. Add 30.0 mL of anhydrous N,N-dimethylformamide to dissolve the end-aminated monomethoxy poly(ethylene glycol), and then add 1.96 g of L-norleucine-N-carboxyanhydride. The reaction is carried out at room temperature for 3 days, dialyzed with ultrapure water for 3 days, and freeze-dried to obtain a powdery product, which is the temperature-sensitive polyamino acid hydrogel.

[0079] Perform nuclear magnetic resonance (NMR) testing on the obtained polyamino acid hydrogel poly(ethylene glycol)-poly(L-norleucine-N-carboxyanhydride). The 1H NMR spectrum of the temperature-sensitive hydrogel prepared in Comparative Example 2 of this application is as Figure 8 shown.

[0080] This comparative example provides a hydrogel-microsphere composite drug carrier material. The difference between the preparation method of the hydrogel-microsphere composite drug carrier material and that of Example 3 is that the hydrogel used is the one provided in Comparative Example 2.

[0081] Prepare a solution of the temperature-sensitive hydrogel provided in Comparative Example 2 with a mass concentration of 7 wt% in PBS and stir it at 4 °C for 3 days. Subsequently, add 9.0 mg of Anlotinib and the poly(D,L-lactide-co-glycolide) microspheres loaded with 9.0 mg of the first anti-tumor drug provided in Example 4 to each 1.0 mL of the temperature-sensitive hydrogel solution, and continue to stir for 30 min to mix them to obtain a mixed solution.

[0082] In vitro drug release experiment of the hydrogel-microsphere composite drug carrier material for spatio-temporal delivery of anti-tumor drugs: Take 300.0 μL of the mixed solution of the hydrogel-microsphere composite material and place it in a glass bottle with a diameter of 16 mm, and place it in a 37 °C water bath for 30 min to form a stable gel state. Then add 1.0 mL of PBS to the vial, collect the upper solution at preset time points, and then add the corresponding new solution. Determine the concentration of the drug contained in the removed solution by high performance liquid chromatography (HPLC), and calculate the cumulative drug release rate. The in vitro drug release profile of the hydrogel-microsphere composite drug carrier material prepared in Comparative Example 2 of this application is as Figure 9As shown. The cumulative release rate of the second anti-tumor drug on the 10th day was 33.4%, and the cumulative release rate of the first anti-tumor drug on the 10th day was 15.1%, with a difference of 18.3% between the two. Under the same conditions, the release rate difference of the hydrogel-microsphere composite material system in Example 3 was 21.3%, which was higher than that in Comparative Example 2. Moreover, the release rates of the two anti-tumor drugs in Comparative Example 2 were both lower than those in Example 3. This may be because the side chain of norleucine is a straight-chain alkyl group with high hydrophobicity. This hydrophobicity makes norleucine more prone to form tight hydrophobic interactions in the hydrogel, which may lead to a denser network structure and slow down the drug release rate. Considering the gap in drug release rate and the characteristics of rapid growth of lung cancer bone metastasis tumors (requiring rapid release of anti-tumor drugs), the hydrogel-microsphere composite material system provided in Example 3 is a better choice.

[0083] As described above, the above are only the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present application, according to the technical solution of the present application and its application concept, makes equivalent substitutions or changes, and should be covered by the protection scope of the present application.

Claims

1. A hydrogel-microsphere composite drug-loading material, characterized in that, Comprising, by mass parts: 5 to 10 parts of poly (lactic-co-glycolic acid) microspheres loaded with a first anti-tumor drug, 5 to 10 parts of a second anti-tumor drug, and 35 to 70 parts of a temperature-sensitive hydrogel.

2. The hydrogel-microsphere composite drug-loading material according to claim 1, wherein The poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug have a particle size of 0.2 - 2.0 μm; the first anti-tumor drug includes one or more of SB525334, LY2157299, LY2109761, EPZ005687, Tazemetostat, GSK343, Lirametostat, Dactolisib, or LY294002; the mass ratio of poly (lactic-co-glycolic acid) to the first anti-tumor drug is 5:1 to 20:

1.

3. The hydrogel-microsphere composite drug-loading material according to claim 1, wherein The drug loading rate of the hydrogel-microsphere composite drug-loading material is 4% to 6%.

4. The hydrogel-microsphere composite drug-loading material according to claim 1, characterized in that The temperature-sensitive hydrogel has a structure of Formula 1; n is 20 to 30.

5. The hydrogel-microsphere composite drug-loading material according to claim 1, characterized in that, The second anti-tumor drug includes one or more of Anlotinib, Gefitinib, Erlotinib, Afatinib, Osimertinib, Imatinib, Dasatinib, Pazopanib, Regorafenib, or Vandetanib.

6. A preparation method of a hydrogel-microsphere composite drug-loading material, characterized in that, Comprising: Mixing the temperature-sensitive hydrogel, the second anti-tumor drug, and the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug to obtain a hydrogel-microsphere composite drug-loading material.

7. The preparation method according to claim 6, characterized in that, The preparation method of the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug includes: Mixing poly (lactic-co-glycolic acid), the first anti-tumor drug, a surfactant, and an organic solvent, centrifuging, and freeze-drying to obtain the poly (lactic-co-glycolic acid) microspheres loaded with the first anti-tumor drug.

8. The preparation method according to claim 7, characterized in that, The rotation speed of the centrifugation is 2000 r / min to 9000 r / min; The mass ratio of poly (lactic-co-glycolic acid) to the first anti-tumor drug is 5:1 to 20:1; The organic solvent includes dichloromethane; The surfactant includes Tween 80.

9. The preparation method according to claim 6, wherein, The preparation method of the temperature-sensitive hydrogel includes: Reacting L-methionine-N-carboxyanhydride, an initiator, and a solvent to obtain the temperature-sensitive hydrogel.

10. The preparation method according to claim 9, characterized in that, The molar ratio of the initiator to L-methionine-N-carboxyanhydride is 1:20 to 1:30; The solvent includes dimethylformamide; The initiator includes end-aminated monomethoxy polyethylene glycol.

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