CN120241623A

CN120241623A - A hydrogel-microsphere composite drug-carrying material and preparation method thereof

Info

Publication number: CN120241623A
Application number: CN202510454888.4A
Authority: CN
Inventors: 丁建勋; 刘师贤; 谢梦恬; 石旭东; 李昔纹; 陈学思
Original assignee: Changchun Institute of Applied Chemistry of CAS
Current assignee: Changchun Institute of Applied Chemistry of CAS
Priority date: 2025-04-11
Filing date: 2025-04-11
Publication date: 2025-07-04

Abstract

The present application discloses a hydrogel-microsphere composite drug-carrying material, which includes, by mass, 5 to 10 parts of poly(lactic acid)-co-glycolide 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. After injection near 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 as the hydrogel and poly(lactic acid)-co-glycolide microspheres degrade, thereby achieving precise spatiotemporal delivery of the two drugs in the local tumor, and can effectively control the release rate of the two drugs. The release rate of the two drugs differs by about 15% in 5 days; the release rate of 10 days differs by about 30%; polyamino acids and polylactic acid are degradable in the body, 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-carrying material has good anti-tumor effects and biocompatibility, and has broad application prospects.

Description

Hydrogel-microsphere composite medicine carrying material and preparation method thereof

Technical Field

The application relates to the field of polymer drug carriers, in particular to a hydrogel-microsphere composite drug-carrying material and a preparation method thereof.

Background

Malignant tumors are a great hazard to human health, and metastasis of malignant tumors is indicative of disease progression and increased difficulty in treatment. Bone is the third most common site of solid tumor metastasis, lung cancer is one of the common primary tumors of bone metastasis, and about 30-40% of lung cancer patients develop bone metastasis. Lung cancer bone metastasis results in bone related events including bone pain, hypercalcemia, and pathological fractures, severely degrading quality of life. Methods of treating bone metastases in lung cancer, such as radiotherapy, chemotherapy and phototherapy, are often palliative and have limited efficacy.

Combination therapy refers to the combination of two or more therapeutic methods, particularly those based on bioactive agents, to improve efficacy or reduce side effects. However, combination therapy often fails to achieve the desired effect, mainly because of the generally low bioavailability of small molecule bioactive drugs administered orally and the non-optimal spatial and temporal distribution in the body. Thus, there is an urgent need to find a way to precisely deliver bioactive drugs to tumor sites at specific points in time. A wide variety of biological materials have been developed as delivery systems for antitumor bioactive drugs. In the biological materials, the nano particles can simultaneously convey various bioactive medicaments to tumor microenvironments and even tumor cells, thereby greatly improving the bioavailability and anti-tumor efficiency of the bioactive medicaments. For example, to maximize the ability of chemotherapeutic drugs to kill tumors, scientists have prepared a manganese dioxide-based nanoparticle that encapsulates cisplatin and modifies Gly-Arg-Gly-Asp-Ser (TFA) on its surface. TFA is a bioactive agent that reverses cisplatin resistance by blocking the mTorrC 2/AKT/NF- κB/MDR1 signaling pathway and can target osteopontin that is highly expressed in tumors. After intravenous administration, the nanoparticles precisely target tumors to release cisplatin, while TFA improves the efficacy of cisplatin. The result shows that the nano particles can obviously block the spinal metastasis of the mouse non-small cell lung cancer. But this only enables delivery of a single drug.

Although many nanoparticle-based anti-tumor bioactive drug delivery systems have been developed to enable multi-drug combination therapy and tumor targeting, precise control of drug release remains difficult to achieve.

Therefore, the drug-carrying material which can improve the anti-tumor curative effect, reduce the side effect and accurately adjust the loading capacity and the space-time release of various bioactive drugs is a problem to be solved by the person skilled in the art.

Disclosure of Invention

In view of the above, the application provides a hydrogel-microsphere composite drug-carrying material, which can realize sequential release of two antitumor drugs on tumor parts and has good antitumor effect and biocompatibility.

Hydrogels and microspheres are also widely used as a popular biomaterial for the delivery and controlled release of antineoplastic bioactive drugs. Hydrogels have a hydrophilic three-dimensional network structure that rapidly swells in water, and during swelling, the hydrogels retain a significant amount of water without dissolution. The unique properties enable hydrogels to meet a variety of biomedical applications, such as drug or cell delivery vehicles, tissue filling materials, and artificial tissue mimics. Meanwhile, the hydrogel has strong plasticity, and can be modified by introducing new injectable or thermosensitive functional groups or components, such as injectable thermosensitive polyamino acid hydrogel. The polyamino acid hydrogel has good biocompatibility and proper degradation rate, and can realize slow release and slow release of bioactive drugs at tumor sites. However, as hydrogels degrade, a variety of bioactive substances are typically released in a diffuse manner, and the order of release is not controllable. The microsphere has the characteristics of degradability, simple preparation method, good drug carrying performance, high stability and the like, and is widely used as a delivery platform of antitumor bioactive drugs. Among these microspheres, poly (glycolide) (PLGA) microspheres have been attracting attention for their high safety and controllable mechanical properties. By adjusting the monomer proportion, molecular weight, concentration, end modification and preparation method, the polyglycolide-lactide microsphere can meet the complex space-time delivery requirement. However, the drug loading ratio and release rate of various bioactive drugs within microspheres are difficult to control accurately, resulting in lower efficacy of the combination therapy. Therefore, in the combined treatment of tumors, the complex of hydrogels and microspheres is a promising strategy that can precisely regulate the loading and space-time release of a variety of bioactive drugs.

The application provides a hydrogel-microsphere composite drug-carrying material, which comprises 5 to 10 parts of polyglycolide-lactide microsphere loaded with a first antitumor drug, 5 to 10 parts of a second antitumor drug and 35 to 70 parts of temperature-sensitive hydrogel.

The application synthesizes the hydrogel-microsphere composite drug-carrying material by taking the first anti-tumor drug loaded polyglycolide microsphere, the second anti-tumor drug and the temperature-sensitive hydrogel as raw materials, has the effect of controlling the space-time release of the two anti-tumor drugs, controls the difference of the release rates of the two drugs to be more than 15 percent, and has good anti-tumor effect and biocompatibility. In some specific implementations, the drug loading rate of the first antineoplastic drug loaded polyglycolide-lactide microsphere is from 4% to 6%.

The hydrogel-microsphere composite medicine carrying material comprises a polyglycolide-lactide microsphere loaded with a first antitumor medicine. In some specific embodiments, the particle size of the first antitumor drug-loaded polyglycolide microsphere is 0.2-2.0 μm, preferably 0.77+ -0.16 μm, and the first antitumor drug includes, but is not limited to, one or more of SB525334, LY2157299, LY2109761, EPZ005687, tazemetostat (tazistat), GSK343, lirametostat, dactolisib or LY294002, and the selection of the first antitumor drug is not particularly limited, preferably SB525334. In some specific implementations, the mass ratio of the polyglycolide to the first anti-neoplastic agent is from 5:1 to 20:1. The microsphere has the characteristics of degradability, simple preparation method, good drug carrying performance, high stability and the like, is widely used as a delivery platform of antitumor bioactive drugs, and uses the polyglycolide to coat the first antitumor drugs, and the first antitumor drugs are slowly released along with the degradation of the temperature-sensitive hydrogel and the polyglycolide-lactide microsphere. The mass parts of the polyglycolide-lactide microsphere loaded with the first antitumor drug are 5 to 10 parts, and can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts and 10 parts.

The hydrogel-microsphere composite medicine carrying material comprises a second anti-tumor medicine. In some specific implementations, the second anti-tumor drug includes, but is not limited to, one or more of Anlotinib (An Luoti) ni, gefitinib (Gefitinib), erlotinib (Erlotinib), afatinib (afatinib), osimertinib (ostinib), imatinib (Imatinib), dasatinib (Dasatinib), pazopanib (Pazopanib), regorafenib (regorafenib) or vanretanib (Mo De tanib), and the application does not have a particular requirement for the selection of the second anti-tumor drug. The mass fraction of the second anti-tumor drug is 5 to 10 parts, and can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts and 10 parts.

The hydrogel-microsphere composite medicine carrying material comprises temperature-sensitive hydrogel. In some specific implementations, the temperature-sensitive hydrogel has a structure of formula 1;

n is 20 to 30.

In some specific implementations, the temperature-sensitive hydrogel has a structure of formula 2;

the application also provides a preparation method of the hydrogel-microsphere composite drug-carrying material, which comprises the following steps:

And mixing the temperature-sensitive hydrogel, the second anti-tumor drug and the polyglycolide-lactide microsphere loaded with the first anti-tumor drug to obtain the hydrogel-microsphere composite drug-loaded material.

The preparation method comprises the steps of mixing the polyglycolide, the first antitumor drug, the surfactant and the organic solvent, centrifuging and freeze-drying to obtain the polyglycolide-lactide microsphere loaded with the first antitumor drug. In some specific implementations, the polyglycolide and the first antitumor drug are dissolved in an organic solvent, mixed with a high-concentration surfactant, emulsified, then added into a low-concentration surfactant, stirred, centrifuged and freeze-dried to obtain the polyglycolide-lactide microsphere loaded with the first antitumor drug. In some specific implementations, the rotational speed of the centrifugation is 2000r/min to 9000r/min, preferably 3000r/min, the mass ratio of the polyglycolide to the first antitumor drug is 5:1 to 20:1, preferably 5:1, the organic solvent comprises but is not limited to dichloromethane, no special requirements are imposed on the selection of the organic solvent according to the application, and the surfactant comprises but is not limited to tween 80.

The application then synthesizes temperature sensitive hydrogel, the preparation method comprises: and (3) reacting the L-methionine-N-cyclic anhydride, an initiator and a solvent to obtain the temperature-sensitive hydrogel.

In some specific implementation modes, terminal monomethoxy polyethylene glycol (formula 3) with terminal amination is used as an initiator to react with L-methionine-N-cyclic anhydride (formula 4) in dimethyl sulfoxide, dialysis is carried out, and freeze-drying is carried out, so that the temperature-sensitive hydrogel is obtained. In some embodiments, the molar ratio of the initiator to the L-methionine-N-cyclic anhydride is 1:20 to 1:30, preferably 1:25, the solvent includes but is not limited to dimethylformamide, the solvent is not particularly required to be selected, the initiator includes but is not limited to terminal amino-terminated monomethoxy polyethylene glycol, the initiator is not particularly required to be selected, the source of the terminal amino-terminated monomethoxy polyethylene glycol is not particularly limited, and the resin can be prepared by a preparation method of a resin by a person skilled in the art or a commercial product. In some specific embodiments, the reaction is carried out for a period of 2 days to 6 days, preferably 3 days. In some specific implementations, the dialysis is for a period of 2 days to 6 days, preferably 3 days.

In some specific embodiments, the triphosgene process is used to prepare L-methionine-N-ring anhydride. The flask was heated with a hot air gun to remove water, then 120.0mL anhydrous tetrahydrofuran, 15.0g L-methionine, and 23.0g triphosgene were added. The reaction was carried out in an oil bath at 60.0 ℃ and maintained in a nitrogen atmosphere. After about 1 hour, the bottle was taken out of the oil bath, the flow of nitrogen was increased, the flow of nitrogen was stopped when the remaining liquid was about 20.0mL, immediately precipitated with ice n-hexane, dissolved with a small amount of ethyl acetate, and washed three times with ice sodium chloride solution. Anhydrous magnesium sulfate is added for dewatering overnight, and then vacuum is carried out, thus obtaining solid L-methionine-N-cyclic anhydride.

According to the application, the temperature-sensitive hydrogel is dissolved in a buffer solution to obtain a temperature-sensitive hydrogel solution. In some specific embodiments, the buffers include, but are not limited to, PBS, and the application does not require special selection of buffers. In some specific implementations, the temperature of the mixing is from 0 ℃ to 8 ℃, preferably 4 ℃, and the time of the mixing is from 2 days to 6 days, preferably 3 days.

The application then mixes the temperature sensitive hydrogel solution, the second anti-tumor drug and the polyglycolide-lactide microsphere loaded with the first anti-tumor drug to obtain the hydrogel-microsphere composite drug-loaded material. In some specific implementations, the mixing time is 20min to 50min, preferably 30min. In some specific implementations, the mass concentration of the temperature-sensitive hydrogel in the temperature-sensitive hydrogel solution is 5wt% to 10wt%, preferably 7wt%.

The hydrogel-microsphere composite drug-carrying material provided by the application is capable of rapidly converting a liquid state into a gel state after being injected beside a tumor, rapidly releasing a second anti-tumor drug through diffusion, and slowly releasing a first anti-tumor drug along with degradation of the hydrogel and the polyglycolide-lactide microsphere, so that accurate space-time delivery of double drugs on local tumor is realized, the release rates of the two drugs can be effectively controlled, the release rates of the two drugs are about 15% different in 5 days, the release rates of the two drugs are about 30% different in 10 days, the polyamino acid and the polylactic acid can be degraded in vivo, degradation products can be directly discharged from the body through kidneys and are harmless to human bodies, and the hydrogel-microsphere composite drug-carrying material has good anti-tumor effect and biocompatibility and wide application prospect.

Drawings

FIG. 1 is a scanning electron microscope image of a first antitumor drug-loaded polyglycolide microsphere prepared in example 1 of the present application;

FIG. 2 is a graph showing the particle size distribution of the first antitumor drug-loaded polyglycolide-lactide microspheres prepared in example 1 of the present application;

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the temperature-sensitive hydrogel prepared in example 2 of the present application;

FIG. 4 is a graph showing the phase transition temperature of the temperature-sensitive hydrogel prepared in example 2 of the present application;

FIG. 5 is a circular dichroism spectrum of the temperature-sensitive hydrogel prepared in example 2 of the present application;

FIG. 6 is an in vitro drug release profile of the hydrogel-microsphere composite drug-loaded material prepared in example 3 of the present application;

FIG. 7 is a graph showing the tumor suppression obtained by in vivo tumor suppression experiments of the hydrogel-microsphere composite drug-loaded material prepared in example 3 of the present application;

FIG. 8 is a nuclear magnetic resonance hydrogen spectrum of the temperature-sensitive hydrogel prepared in comparative example 2 of the present application;

FIG. 9 is an in vitro drug release profile of the hydrogel-microsphere composite drug-loaded material prepared in comparative example 2 of the present application.

Detailed Description

It should be understood that one or more of the expressions ". The expressions" individually include each of the objects recited after the expressions and various 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 as having the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, should generally be construed as open-ended and non-limiting, e.g., not to exclude other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.

It should be understood that the order of steps or order of performing certain actions is not important so long as the application remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language, such as "e.g." or "comprising" herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

Furthermore, the numerical ranges and parameters setting forth the present application are approximations that may vary as precisely as possible in the exemplary embodiments. However, any numerical value inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless explicitly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used in this disclosure are modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.

The application provides a hydrogel-microsphere composite drug-carrying material which comprises, by mass, 5 to 10 parts of polyglycolide-lactide microspheres loaded with first antitumor drugs, 5 to 10 parts of second antitumor drugs and 0.1 to 50 parts of temperature-sensitive hydrogel.

The hydrogel-microsphere composite drug-carrying material is characterized in that after the hydrogel-microsphere composite drug-carrying material is injected beside a tumor, the temperature-sensitive hydrogel is rapidly changed from a liquid state to a gel state, the second anti-tumor drug is rapidly released through diffusion, and the first anti-tumor drug is slowly released along with the degradation of the hydrogel and the polyglycolide microsphere, so that the accurate space-time delivery of double drugs on the part of the tumor is realized, the release rates of the two drugs can be effectively controlled, the release rates of the two drugs are about 15% different in 5 days, the release rates of the two drugs are about 30% different in 10 days, the polyamino acid and the polylactic acid can be degraded in vivo, degradation products can be directly discharged from the body through kidneys and are harmless to the human body, and the hydrogel-microsphere composite drug-carrying material has good anti-tumor effect and biocompatibility and wide application prospect.

The application is further illustrated by the following examples. The scope of the application is not limited by the following examples.

Example 1

The embodiment provides a polyglycolide-lactide microsphere loaded with a first antitumor drug, and the preparation method of the polyglycolide-lactide microsphere loaded with the first antitumor drug comprises the following steps:

100.0mg of polyglycolide and 20.0mg of SB525334 were dissolved in 10.0mL of methylene chloride, 20.0mg (about one drop mass) of Tween 80 was added, and the mixture was sonicated for 10 minutes to dissolve the same. The above solution was mixed with 10.0mL of PVA aqueous solution (1.5 wt%) to obtain a mixture. The ultrasonic uses a Y92-IIN ultrasonic emulsifying machine (Ningbo Xin Zhi Biotechnology Co., ltd., china) with working parameters of 50% of power, 2 minutes of total working time, 3.0 seconds of ultrasonic on time and 2.0 seconds of ultrasonic off time. The mixture was phacoemulsified and the resulting product was then poured into 45.0mL of aqueous polyvinyl alcohol (0.5 wt%) and magnetically stirred for 6 hours. Centrifuge at 3000r/min for 10 min, discard supernatant, add ultrapure water to resuspend product and wash 3 times. Freeze-drying to obtain the polyglycolide-lactide microsphere loaded with the first antitumor drug. The yield of the polyglycolide lactide microsphere loaded with the first antitumor drug is 89.5%, the drug loading rate is 5.0% and the drug loading efficiency is 30.1%.

Scanning electron microscope shooting and particle size testing are carried out on the polyglycolide-lactide microsphere loaded with the first antitumor drug:

The microspheres were resuspended in ultrapure water, dropped onto a silicon wafer and allowed to naturally dry, followed by metal spraying. The image of the microspheres under different multiplying powers is shot under JSM-7900F type thermal field emission scanning electron microscope, the scanning electron microscope of the polyglycolide-lactide microsphere loaded with the first antitumor drug prepared in the embodiment 1 of the application is shown in figure 1, the diameters of at least 100 microspheres are measured by imageJ software, the average particle size and variance are calculated, and the particle size distribution diagram of the polyglycolide-lactide microsphere loaded with the first antitumor drug prepared in the embodiment 1 of the application is shown in figure 2.

Example 2

The embodiment provides a temperature-sensitive polyamino acid hydrogel which is polyethylene glycol poly L-methionine-N-ring anhydride (structure shown in formula 2).

The preparation method of the temperature-sensitive polyamino acid hydrogel comprises the following steps:

Into the reaction flask, 1.0g of terminal monomethoxy polyethylene glycol was added and water was removed by azeotropic distillation with toluene. Adding 30.0mL of anhydrous dimethylformamide to dissolve terminal monomethoxy polyethylene glycol with terminal amination, adding 2.2g L-methionine-N-ring anhydride, reacting at room temperature for 3 days, dialyzing with ultrapure water for 3 days, and freeze-drying to obtain a powdery product, namely the temperature-sensitive polyamino acid hydrogel.

Nuclear magnetic testing is carried out on the obtained polyamino acid hydrogel polyethylene glycol poly L-methionine-N-cyclic anhydride, and a nuclear magnetic resonance hydrogen spectrum of the temperature-sensitive hydrogel prepared in the embodiment 2 of the application is shown in a figure 3.

And (3) performing a solution-gel transition experiment on the obtained polyamino acid type hydrogel polyethylene glycol poly L-methionine-N-cyclic anhydride, namely determining the solution-gel transition temperature of the hydrogel by using a test tube inversion method. Four different hydrogel solutions with mass concentrations of 6wt%, 7wt%, 8wt% and 9wt%, respectively, were prepared with phosphate buffered saline at ph=7.4 and stirred at 4 ℃ for 3 days. 300.0. Mu.L of each hydrogel was placed in a glass bottle having a diameter of 11mm, and placed in a water bath set to heat from 0 ℃. After 5 minutes of stabilization at each temperature rise of 2 ℃, an attempt was made to invert the vial. If the hydrogel does not flow within 30 seconds, it is considered to have transitioned from the liquid state to the gel state. The solution-gel phase transition temperature diagram of the temperature-sensitive hydrogel prepared in example 2 of the present application is shown in fig. 4.

The polyethylene glycol poly L-methionine-N-cyclic anhydride obtained was subjected to a round dichromatic chromatography test, and the test results are shown in FIG. 5. With the increase of temperature, the secondary structure of the polymer is continuously changed, physical crosslinking is provided for the polymer network, and the transformation of sol-gel is promoted.

Example 3

The embodiment provides a hydrogel-microsphere composite drug-carrying material, and the preparation method of the hydrogel-microsphere composite drug-carrying material comprises the following steps:

a solution of the temperature-sensitive hydrogel provided in example 2 was prepared with PBS at a mass concentration of 7wt% and stirred for 3 days at 4 ℃. Subsequently, 9.0mg Anlotinib and 9.0mg of the first antitumor drug-loaded polyglycolide lactide microsphere provided in example 1 were added to each 1.0mL of the temperature-sensitive hydrogel solution, and the mixture was mixed by stirring for 30 minutes.

In vitro drug release experiment of hydrogel-microsphere composite drug-carrying material for space-time delivery of antitumor drug, 300.0 μl of mixed solution of hydrogel-microsphere composite material is placed in a glass bottle with diameter of 16mm, and placed in 37 deg.C water bath for 30min to form stable gel state. 1.0mL of PBS and 1.0mL of PBS containing 0.2mg/mL of elastase were then added to the vials, respectively, and the top solution was collected at the preset time point, followed by the addition of the corresponding new solution. The concentration of the drug contained in the removing liquid is measured by a high performance liquid chromatography, the accumulated release rate of the drug is calculated, an in-vitro drug release diagram of the hydrogel-microsphere composite drug-carrying material prepared in the embodiment 3 of the application is shown in fig. 6, the release speed of the second anti-tumor drug is high, the first anti-tumor drug is slowly released along with the degradation of the hydrogel and the polyglycolide-lactide microsphere, so that the accurate space-time delivery of the two drugs in local tumor is realized, the release rates of the two drugs are about 15% different in 5 days, and the release rates are about 30% different in 10 days. Within 30 days, the cumulative release amounts of the second and first antitumor drugs in PBS reached 70.4% and 42.7%, respectively, while the cumulative release amounts in PBS plus elastase (simulating tumor tissue microenvironment) were 92.4% and 58.5%, respectively. The slow release ensures the effective concentration of the medicine at the tumor part and reduces the toxic and side effects. The release speed of the second anti-tumor drug is always faster than that of the first anti-tumor drug, so that the hydrogel-microsphere composite system achieves the expected aim of sequentially releasing the two drugs, and can be used for subsequent animal experiments.

The in-vivo tumor inhibition experiment of the hydrogel-microsphere composite drug-loaded material comprises the steps of selecting a male C57 mouse (6-8 weeks old) and constructing a lung cancer bone metastasis model for the male C57 mouse, wherein when LLC cells are cultured to a logarithmic phase, the LLC cells are digested and configured into a cell suspension with the concentration of 1.0X10 ⁷ cells/mL. We aspirate the cell suspension with a 1.0mL syringe and place it on ice for use. Mice were anesthetized with isoflurane and then skin preparation was performed on the right hind limbs of C57 mice. We used a syringe to break through the right hindlimb tibial plateau, into the bone marrow cavity, injecting 50.0 μl of cell suspension. After 6 days, the tumor breaks through the tibial plateau, and can be treated in situ when the tumor volume is about 100mm ³.

The mice were grouped according to different treatment methods, PBS group (control), dual free drug group (second antitumor drug+first antitumor drug), hydrogel-loaded Anlotinib group (hydrogel/second antitumor drug), hydrogel-loaded Anlotinib +sb525334 group (hydrogel/(second antitumor drug+first antitumor drug), and hydrogel-loaded Anlotinib +sb 525334-loaded polyglycolide microsphere group (hydrogel/(second antitumor drug+polyglycolide microsphere/second antitumor drug),. Final dose was Anlotinib:9.0 mg/mouse, SB525334:9.0 mg/mouse. The treatments were both intratumoral injection and single administration.

Tumor length and diameter were measured every other day, and tumor volume was calculated using the following formula:

Tumor volume = long diameter x short diameter/2.

Mice were sacrificed after 10 days of measurement and tumor inhibition curves were drawn, and the tumor inhibition curves obtained by the in vivo tumor inhibition experiments of the hydrogel-microsphere composite drug-loaded material prepared in example 3 of the present application are shown in fig. 7. On treatment day 10, the hydrogel-microsphere composite material group (397.1 +/-116.5 mm ³) has the best anti-tumor effect, which is smaller than one third of the average tumor volume of the control group, and the tumor volume of the hydrogel-microsphere composite material group (397.1 +/-116.5 mm ³) is obviously smaller than the hydrogel/(second anti-tumor drug+polyglycolide microsphere/second anti-tumor drug) group (667.7 +/-85.7 mm ³), which proves that the composite material realizes better anti-tumor effect through the sequential release of drugs.

Example 4

The present example provides a first antitumor drug loaded polyglycolide microsphere, which differs from example 1 in that the drug loaded polyglycolide microsphere has a centrifugal rotational speed of 9000r/min.

The preparation method comprises the following steps:

100.0mg of polyglycolide and 20.0mg of SB525334 were dissolved in 10.0mL of methylene chloride, 20.0mg (about one drop mass) of Tween 80 was added, and the mixture was sonicated for 10 minutes to dissolve the same. The above solution was mixed with 10.0mL of PVA aqueous solution (1.5 wt%) to obtain a mixture. The ultrasonic uses a Y92-IIN ultrasonic emulsifying machine (Ningbo Xin Zhi Biotechnology Co., ltd., china) with working parameters of 50% of power, 2 minutes of total working time, 3.0 seconds of ultrasonic on time and 2.0 seconds of ultrasonic off time. The mixture was phacoemulsified and the resulting product was then poured into 45.0mL of aqueous polyvinyl alcohol (0.5 wt%) and magnetically stirred for 6 hours. Centrifuge at 9000r/min for 10 min, discard supernatant, add ultrapure water to resuspend product and wash 3 times. Freeze-drying to obtain the polyglycolide-lactide microsphere loaded with the first antitumor drug. The yield of the first antitumor drug loaded polyglycolide lactide microsphere is 79.1%, the drug loading rate is 3.8% and the drug loading efficiency is 41.8%.

Comparative example 1

The comparative example provides a hydrogel-microsphere composite drug-carrying material, which is different from example 3 in that nanoparticles carrying a first antitumor drug are used.

The preparation method comprises the following steps:

The adopted nano particles are polymers (the polymerization degree is 20:10) of glutamic acid and phenylalanine initiated by terminal monomethoxy polyethylene glycol with terminal amination. 17.6mg of nanoparticle polymer powder was dissolved in 1.0mL of dimethylformamide, and 0.6mg of SB525334 was dissolved in 0.6mL of dimethyl sulfoxide. The two were then mixed and slowly added dropwise to 5.0mLPBS with a micropulsor. Stirring for 3 hours, then dialyzing with primary water for 3 hours, and freeze-drying to obtain the nano particles loaded with the first antitumor drug. The yield of the nanoparticle loaded with the first antitumor drug was 54.9%, the drug loading rate was 0.62% and the drug loading efficiency was 18.7%. The nanoparticle is too low in drug loading rate and drug loading efficiency, so that the nanoparticle is not considered for a subsequent composite system.

Comparative example 2

The comparative example provides a hydrogel-microsphere composite drug-carrying material, which is different from the example 3 in that the hydrogel is an L-norleucine-N-cyclic anhydride polymer hydrogel initiated by terminal amino terminal monomethoxy polyethylene glycol (polyethylene glycol poly L-norleucine-N-cyclic anhydride, formula 5).

into the reaction flask, 1.0g of terminal monomethoxy polyethylene glycol was added and water was removed by azeotropic distillation with toluene. Adding 30.0mL of anhydrous dimethylformamide to dissolve terminal monomethoxy polyethylene glycol with amination, adding 1.96g L-norleucine-N-ring anhydride, reacting at room temperature for 3 days, dialyzing with ultrapure water for 3 days, and lyophilizing to obtain a powdery product, namely the temperature-sensitive polyamino acid hydrogel.

Nuclear magnetic testing is carried out on the obtained polyamino acid hydrogel polyethylene glycol poly L-norleucine-N-cyclic anhydride, and a nuclear magnetic resonance hydrogen spectrogram of the temperature-sensitive hydrogel prepared in comparative example 2 is shown in figure 8.

This comparative example provides a hydrogel-microsphere composite drug-loaded material, which is different from example 3 in that the hydrogel used is provided in comparative example 2.

A solution of the temperature-sensitive hydrogel provided in comparative example 2 having a mass concentration of 7wt% was prepared with PBS and stirred at 4℃for 3 days. Subsequently, 9.0mg Anlotinib and 9.0mg of the first antitumor drug-loaded polyglycolide lactide microsphere provided in example 4 were added to each 1.0mL of the temperature-sensitive hydrogel solution, and the mixture was mixed by stirring for 30 minutes.

In vitro drug release experiment of hydrogel-microsphere composite drug-carrying material for space-time delivery of antitumor drug, 300.0 μl of mixed solution of hydrogel-microsphere composite material is placed in a glass bottle with diameter of 16mm, and placed in 37 deg.C water bath for 30min to form stable gel state. 1.0mL of PBS was then added to the vial, and the top solution was collected at the preset time point, followed by the corresponding new solution. The concentration of the drug contained in the removal solution was measured by high performance liquid chromatography, and the cumulative release rate of the drug was calculated, and the in vitro drug release profile of the hydrogel-microsphere composite drug-loaded material prepared in comparative example 2 of the present application is shown in fig. 9. The cumulative release rate of the second antitumor drug on day 10 was 33.4%, and the cumulative release rate of the first antitumor drug on day 10 was 15.1% which differ by 18.3%. Under the same conditions, the release rate of the hydrogel-microsphere composite system of example 3 was 21.3% different than that of comparative example 2. And the release rates of both antitumor drugs in comparative example 2 were lower than those in example 3. This is probably because norleucine has a side chain of a linear alkyl group and has high hydrophobicity. This hydrophobicity makes norleucine more prone to tight hydrophobic interactions in hydrogels, which may lead to denser network structures, slowing drug release rates. The hydrogel-microsphere composite system provided in example 3 is a better choice in combination with the difference in drug release rates and the rapid growth of bone metastasis and tumor in lung cancer (which requires rapid release of the antitumor drug).

The foregoing is only a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any person skilled in the art, who is within the scope of the present application, shall cover the scope of the present application by equivalent substitution or modification according to the technical scheme of the present application and the application concept thereof.

Claims

1. A hydrogel-microsphere composite drug-carrying material, characterized in that it comprises, by mass: 5 to 10 parts of poly(lactide-glycolide) 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.

2. The hydrogel-microsphere composite drug-loaded material according to claim 1 is characterized in that the particle size of the poly(lactide-glycol) microspheres loaded with the first anti-tumor drug is 0.2-2.0 μm; the first anti-tumor drug comprises one or more of SB525334, LY2157299, LY2109761, EPZ005687, Tazemetostat, GSK343, Lirametostat, Dactolisib or LY294002; the mass ratio of the poly(lactide-glycol) to the first anti-tumor drug is 5:1 to 20:1.

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

4. The hydrogel-microsphere composite drug-carrying material according to claim 1, characterized in that the thermosensitive hydrogel has a structure of Formula 1;

n is 20 to 30.

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

6. A method for preparing a hydrogel-microsphere composite drug-carrying material, comprising:

The thermosensitive hydrogel, the second anti-tumor drug and the poly(lactide-glycolide) microspheres loaded with the first anti-tumor drug are mixed to obtain a hydrogel-microsphere composite drug-loaded material.

7. The preparation method according to claim 6, characterized in that the preparation method of the poly(lactide-glycol) microspheres loaded with the first anti-tumor drug comprises:

The poly(lactide-glycol)-dextrose, the first anti-tumor drug, a surfactant and an organic solvent are mixed, centrifuged and freeze-dried to obtain poly(lactide-glycol)-dextrose microspheres loaded with the first anti-tumor drug.

8. The preparation method according to claim 7, characterized in that the centrifugal speed is 2000r/min to 9000r/min;

The mass ratio of the poly(lactide-glycolide) 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, characterized in that the preparation method of the temperature-sensitive hydrogel comprises:

The thermosensitive hydrogel is obtained by reacting L-methionine-N-cyclic anhydride, an initiator and a solvent.

10. The preparation method according to claim 9, characterized in that the molar ratio of the initiator to L-methionine-N-intracyclic anhydride is 1:20 to 1:30;

The solvent includes dimethylformamide;

The initiator includes terminally amino-terminated monomethoxy polyethylene glycol.