Controllable drug release microsphere as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a controllable medicine release microsphere as well as a preparation method and application thereof. Compared with the prior art, the drug and the gelatin are grafted on the thermal response polymer, so that the system is endowed with a thermal response and MMP-2 enzyme response hot top at the same time, the targeting effect of the controllable drug release microsphere on a tumor site is improved, and burst release of the drug can be remarkably reduced; the continuous delivery and the treatment effect of the medicine in the body are promoted; in addition, by adjusting the flow rate and the proportion of the dispersed phase solution and the continuous phase solution, the drug loading capacity and the particle size of the microspheres can be controlled, the particle size can be uniform and controllable, meanwhile, different drug loading effects can be achieved, and the release rate can be controlled; in addition, personalized adjustment of the drug release rate can be realized by adjusting the proportion of the two main substances of the dispersed phase.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to a controllable drug release microsphere, its preparation method and application. Background Technology

[0002] Cancer, also known as malignant tumor, is a chronic disease with a high mortality rate. It has become one of the major diseases seriously threatening human life and health worldwide. Cancer occurs due to the abnormal proliferation and differentiation of cells in the body, forming an uncontrolled mass or tumor. These tumors can invade surrounding normal tissues and even spread to other parts of the body through the blood or lymphatic system (i.e., metastasis).

[0003] Currently, there are various methods for treating cancer, such as surgery, radiotherapy, conventional chemotherapy, molecular targeted therapy, and immunotherapy, among which conventional chemotherapy remains one of the main methods. However, the efficacy of traditional systemic chemotherapy is often affected by the low drug concentration at the tumor site, resulting in poor treatment outcomes. Furthermore, chemotherapy drugs have poor targeting, exhibiting non-specific effects on both normal and cancerous cells. While killing tumors, they also damage normal tissues and cells, causing toxic side effects, increasing patient suffering, and reducing quality of life. This significantly limits the clinical application of chemotherapy drugs.

[0004] With the advancement of new technologies and concepts, further improving the efficacy of chemotherapy drugs, reducing toxic side effects, and enhancing the precision of tumor treatment are currently top priorities in clinical research. Microspheres are a promising option; they are particulate dispersion systems formed by adsorbing or dispersing drugs within polymeric materials. As an advanced delivery system, microspheres have been widely used in many fields such as food, pharmaceutical delivery, and cosmetics. Their functions and applications are constantly being actively explored, and the diversity of microsphere structures and material choices gives them advantages in controlling drug release rates and locations.

[0005] Drug-loaded microspheres, as a controlled-release drug system, offer advantages such as stable drug concentration, high utilization rate, release site closer to the pathogen, high efficacy, fewer side effects, and fewer dosing cycles. How to prepare uniform, controllable, and monodisperse microspheres and improve the targeting of delivery systems has become a current research hotspot. Microspheres have strict requirements for particle size and uniformity. The uniformity of microsphere size significantly affects the controllability of drug release location and rate. Furthermore, it is necessary to adjust the size of the microspheres to adapt to the dosing standards for different diseases. Traditional microsphere preparation methods mainly include ion crosslinking, spray drying, extrusion, and solvent evaporation. However, microspheres prepared by these methods typically have a wide particle size distribution, exhibiting drawbacks such as size inhomogeneity, poor structural uniformity, unstable drug release, and unsatisfactory sustained-release effects, severely impacting drug efficacy. Therefore, developing a technology capable of precisely controlling the particle size and distribution of microspheres is crucial. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a controllable drug release microsphere with uniform size distribution and controllable morphology, good drug release effect and good biocompatibility, as well as its preparation method and application.

[0007] This invention provides a method for preparing controllable drug release microspheres, comprising the following steps:

[0008] S1) Provides a dispersed phase solution and a continuous phase solution: the dispersed phase solution comprises polylactic acid, polyglycolic acid, and an organic solvent; the continuous phase solution comprises grafted gelatin, a thermoresponsive polymer of the drug, and water; the drug contains hydroxyl and / or carboxyl groups;

[0009] S2) The dispersed phase solution and the continuous phase solution are respectively introduced into the microfluidic chip to obtain controllable drug release microspheres.

[0010] Preferably, in step S2), the water-in-oil droplets formed by introducing the microfluidic chip are received by a solution containing a crosslinking agent and then crosslinked and fixed to obtain controllable drug release microspheres.

[0011] Preferably, the total mass concentration of polylactic acid and polyglycolic acid in the dispersed phase solution is 1% to 10%; the mass ratio of polylactic acid to polyglycolic acid is 10:90 to 90:10.

[0012] And / or, the drug is selected from anthracycline antibiotics;

[0013] And / or, the mass concentration of the grafted gelatin and the thermoresponsive polymer of the drug in the continuous phase solution is 1% to 5%;

[0014] And / or, the mass concentration of the crosslinking agent in the solution containing the crosslinking agent is 1% to 10%;

[0015] And / or, the crosslinking agent is selected from genipin and / or glutaraldehyde.

[0016] Preferably, the thermoresponsive polymer of the grafted gelatin and the drug is prepared according to the following method:

[0017] A1) The thermoresponsive polymer is activated with an activator and then mixed with gelatin under alkaline conditions to obtain a thermoresponsive polymer grafted with gelatin.

[0018] A2) The drug is reacted with a benzene ring derivative containing an aldehyde group, then a carboxyl activator is added to activate the carboxyl group, and then the drug is reacted with the thermo-responsive polymer of the grafted gelatin to obtain a thermo-responsive polymer of grafted gelatin and drug.

[0019] Preferably, the thermoresponsive polymer is selected from poloxamer;

[0020] And / or, the activator is selected from 4-nitrophenyl chloroformate;

[0021] And / or, the drug is selected from one or more of doxorubicin, daunorubicin, and pirarubicin;

[0022] And / or, the aldehyde-containing benzene ring derivative is selected from p-aldehyde benzoic acid;

[0023] And / or, the mass ratio of the thermoresponsive polymer to the activator is 10:(1-2);

[0024] And / or, the mass ratio of the thermoresponsive polymer to gelatin is (1-10):1;

[0025] And / or, the mass ratio of the drug to the thermoresponsive polymer is (0.1–5):5.

[0026] Preferably, step A1) specifically involves: mixing the thermoresponsive polymer with an organic solvent, adding it to an organic solution containing an activator for reaction, and precipitating it in cold diethyl ether after the reaction is complete to obtain the activated thermoresponsive polymer;

[0027] The activated thermoresponsive polymer was dissolved in an alcohol solvent and then mixed with gelatin and an alkaline solution to obtain a thermoresponsive polymer grafted with gelatin.

[0028] Preferably, the volume ratio of the dispersed phase solution to the continuous phase solution in S2) is 1:(4-6);

[0029] The flow rate of the dispersible solution is 1–5 μL / min;

[0030] The flow rate of the continuous phase solution is 10–20 μL / min.

[0031] The present invention also provides a controllable drug release microsphere prepared by the above preparation method.

[0032] The present invention also provides the application of the controllable drug release microspheres prepared by the above preparation method in the preparation of tumor treatment drugs.

[0033] This invention also provides a method for preparing microspheres, comprising the following steps:

[0034] B1) Provides a dispersed phase solution and a continuous phase solution: the dispersed phase solution comprises polylactic acid, polyglycolic acid, and an organic solvent; the continuous phase solution comprises a thermoresponsive polymer grafted with gelatin and water;

[0035] B2) The dispersed phase solution and the continuous phase solution are respectively introduced into the microfluidic chip to obtain microspheres.

[0036] This invention provides a method for preparing controllable drug-release microspheres, comprising the following steps: S1) providing a dispersed phase solution and a continuous phase solution: the dispersed phase solution includes polylactic acid, polyglycolic acid, and an organic solvent; the continuous phase solution includes a thermoresponsive polymer grafted with gelatin and a drug, and water; the drug contains hydroxyl and / or carboxyl groups; S2) passing the dispersed phase solution and the continuous phase solution into a microfluidic chip respectively to obtain controllable drug-release microspheres. Compared with the prior art, this invention, by grafting a drug and gelatin onto a thermoresponsive polymer, endows the system with a thermally responsive and MMP-2 enzyme-responsive thermal top, which not only improves the targeting effect of the controllable drug-release microspheres on tumor sites, but also significantly reduces the burst release of the drug, promoting sustained drug delivery and therapeutic effect in vivo; in addition, by adjusting the flow rate and ratio of the dispersed phase solution and the continuous phase solution, the drug loading and particle size of the microspheres can be controlled, achieving different drug loading effects and controlling the release rate while achieving uniform and controllable particle size; furthermore, by adjusting the ratio of the two main substances in the dispersed phase, the drug release rate can be personalized. Attached Figure Description

[0037] Figure 1 This is a morphological diagram of the PLA / PGA / F127-g-Gelatin microspheres obtained in Example 1 of the present invention;

[0038] Figure 2 The figure shows the biocompatibility test results of PLA / PGA / F127-g-Gelatin microspheres obtained in Example 1 of this invention;

[0039] Figure 3 ADM standard curve diagram provided for this invention;

[0040] Figure 4This is an in vitro drug release curve of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres obtained in Example 2 of the present invention;

[0041] Figure 5 The image shows a comparison of the in vitro drug release assays of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres obtained in Example 2, PLA / PGA / F127-g / Gelatin / ADM drug-loaded microspheres ADM obtained in Comparative Example 1, blank microspheres (PLA / PGA / F127-g-Gelatin) obtained in Example 1, and ADM. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] This invention provides a method for preparing microspheres, comprising the following steps: B1) providing a dispersed phase solution and a continuous phase solution: the dispersed phase solution includes polylactic acid, polyglycolic acid and an organic solvent; the continuous phase solution includes a thermoresponsive polymer grafted with gelatin and water; B2) passing the dispersed phase solution and the continuous phase solution into a microfluidic chip respectively to obtain microspheres.

[0044] This invention utilizes microfluidic technology to prepare microspheres. This technology can control the flow and shearing of microfluidics within a microfluidic device to generate microdroplets with uniform and controllable particle size and monodispersity. Microfluidic technology has significant potential for development in the field of microspheres. Besides achieving uniform and controllable particle size, it also allows for the customization of the internal structure of microspheres to develop microspheres with ideal pharmacokinetic profiles and loading capacities.

[0045] In this invention, there are no special restrictions on the source of any raw materials; they can be commercially available.

[0046] In one specific embodiment of the present invention, the total mass concentration of polylactic acid (PLA) and polyglycolic acid (PGA) in the dispersed phase solution is preferably 1% to 10%; optionally, the total mass concentration of polylactic acid and polyglycolic acid in the dispersed phase solution is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or any two of the above values.

[0047] In one specific embodiment of the present invention, the mass ratio of polylactic acid to polyglycolic acid in the dispersed phase solution is preferably 10:90 to 90:10; optionally, the mass ratio of polylactic acid to polyglycolic acid is 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 or any two of the above ratios.

[0048] In a specific embodiment of the present invention, the total mass concentration of polylactic acid and polyglycolic acid in the dispersed phase solution is preferably 5%; the mass ratio of polylactic acid to polyglycolic acid is preferably 40:60 to 60:40.

[0049] In one specific embodiment of the present invention, the molecular weight of the polylactic acid (PLA) is preferably 10,000 to 100,000 g / mol; optionally, the molecular weight of the polylactic acid (PLA) is 10,000 g / mol, 20,000 g / mol, 30,000 g / mol, 40,000 g / mol, 50,000 g / mol, 60,000 g / mol, 70,000 g / mol, 80,000 g / mol, 90,000 g / mol, 100,000 g / mol, or a range between any two of the above values.

[0050] In one specific embodiment of the present invention, the molecular weight of the polyglycolic acid (PGA) is preferably 10,000 to 100,000 g / mol; optionally, the molecular weight of the polyglycolic acid (PGA) is 10,000 g / mol, 20,000 g / mol, 30,000 g / mol, 40,000 g / mol, 50,000 g / mol, 60,000 g / mol, 70,000 g / mol, 80,000 g / mol, 90,000 g / mol, 100,000 g / mol, or a range between any two of the above values.

[0051] In one specific embodiment of the present invention, the organic solvent in the dispersed phase solvent can be any organic solvent well known to those skilled in the art, and there are no special limitations, including but not limited to dichloromethane (DCM).

[0052] The dispersed phase solution can be prepared using methods well-known to those skilled in the art, without any special limitations. In a specific embodiment provided by the present invention, the dispersed phase solution is prepared by the following method: polylactic acid, polyvinyl alcohol, and an organic solvent are mixed to obtain a dispersed phase solution; the mixing method can be any method well-known to those skilled in the art, including but not limited to magnetic stirring, mechanical stirring, or ultrasound, etc., and ultrasound oscillation is preferred in the present invention; complete dissolution is ensured through mixing to form a homogeneous solution; after mixing, filtration is preferred to obtain the dispersed phase solution; the filtration method can be any method well-known to those skilled in the art, without any special limitations, and a fine filtration device is preferred in the present invention, more preferably a 0.22μm PTFE needle filter; filtration can not only remove insoluble impurities and obtain a clear and impurity-free solution, but also simultaneously achieve the purpose of sterilization.

[0053] In one specific embodiment of the present invention, the mass concentration of the thermoresponsive polymer grafted with gelatin in the continuous phase solution is preferably 1% to 5%; optionally, the mass concentration of the thermoresponsive polymer grafted with gelatin in the continuous phase solution is 1%, 2%, 3%, 4%, 5%, or any two of the above values. Gelatin is one of the substrates for matrix metalloproteinase-2 (MMP-2) overexpressed at tumor sites, which endows the system with MMP-2 enzyme-responsive properties and improves the targeting effect of microspheres on tumor sites.

[0054] In one specific embodiment of the present invention, the mass concentration of the thermoresponsive polymer grafted with gelatin in the continuous phase solution is 2%.

[0055] In a specific embodiment of this invention, the thermoresponsive polymer grafted with gelatin is prepared by the following method: the thermoresponsive polymer is activated with an activator, and then mixed and reacted with gelatin under alkaline conditions to obtain the thermoresponsive polymer grafted with gelatin; the thermoresponsive polymer can be any thermoresponsive polymer well known to those skilled in the art, and there are no special limitations. This invention includes, but is not limited to, one or more of poloxamer 124, poloxamer 127, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407; the activator can be any activator well known to those skilled in the art, and there are no special limitations. In this invention, 4-nitrophenylchloroformate is preferred; the thermoresponsive polymer... The preferred mass ratio of the thermoresponsive polymer to the activator is 10:(1-2); optionally, the mass ratio of the thermoresponsive polymer to the activator is 10:1, 10:1.2, 10:1.4, 10:1.6, 10:1.8, 10:2 or any two of the above values; the gelatin can be any gelatin known to those skilled in the art, and there are no special limitations. This invention includes, but is not limited to, type A gelatin; the preferred mass ratio of the thermoresponsive polymer to the gelatin is (1-10):1; optionally, the mass ratio of the thermoresponsive polymer to the gelatin is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or any two of the above values.

[0056] In a specific embodiment of the present invention, the thermoresponsive polymer grafted with gelatin is prepared by the following method: the thermoresponsive polymer is mixed with an organic solvent and then added to an organic solution containing an activator for reaction. After the reaction is completed, it is precipitated in cold diethyl ether to obtain an activated thermoresponsive polymer. The activated thermoresponsive polymer is dissolved in an alcohol solvent and then mixed with gelatin and an alkaline solution for reaction to obtain the thermoresponsive polymer grafted with gelatin.

[0057] In a specific embodiment of the present invention, a thermoresponsive polymer is mixed with an organic solvent and then added to an organic solution containing an activator for reaction. After the reaction is completed, the polymer is precipitated in cold diethyl ether to obtain the activated thermoresponsive polymer. The organic solvent can be any organic solvent well-known to those skilled in the art and is not particularly limited; dichloromethane (DCM) is preferred in this invention. The ratio of the thermoresponsive polymer to the organic solvent is preferably (10-100) g:100 mL. Optionally, the ratio of the thermoresponsive polymer to the organic solvent is 10 g:100 mL, 20 g:100 mL, 30 g:100 mL, 40 g:100 mL, 50 g:100 mL, 60 g:100 mL, 70 g:100 mL, 80 g:100 mL, 90 g:100 mL, 100 g:100 mL, or any range between two of the above values. The solvent in the organic solution containing the activator is preferably dichloromethane. The concentration of the activator in the solution is preferably 0.01–0.1 g / mL; optionally, the concentration of the activator in the organic solution containing the activator is 0.01 g / mL, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL, 0.05 g / mL, 0.06 g / mL, 0.07 g / mL, 0.08 g / mL, 0.09 g / mL, 0.1 g / mL, or any two of the above values; specifically, the thermoresponsive polymer is mixed with the organic solvent. The mixture is then added dropwise to an organic solvent containing an activator for reaction; the reaction is preferably carried out under vigorous stirring; the reaction is preferably carried out at room temperature; the reaction time is preferably 8-16 h, more preferably 10-12 h; the volume ratio of the organic solvent to cold diethyl ether is preferably 1:(20-100), more preferably 1:(40-80), even more preferably 1:(50-60), and most preferably 1:55; after precipitation, vacuum filtration is preferred to obtain the activated thermoresponsive polymer.

[0058] In one specific embodiment of the present invention, the activated thermoresponsive polymer is dissolved in an alcohol solvent, and then mixed and reacted with gelatin and an alkaline solution to obtain a gelatin-grafted thermoresponsive polymer. The alcohol solvent can be any solvent well-known to those skilled in the art and is not particularly limited; ethanol is preferred in this invention. The mass of the activated thermoresponsive polymer is 25%–45% of the total mass of the activated thermoresponsive polymer and the alcohol solvent, more preferably 28%–40%, even more preferably 30%–35%, and most preferably 33.3%. The alkaline solution can be any aqueous solution of an alkaline compound well-known to those skilled in the art and is not particularly limited. The alkaline compound is preferably an organic amine; the organic amine includes, but is not limited to, triethylamine, 2,4-diethylamine, etc. One or more of dimethylaminopyridine, etc.; the concentration of the alkaline compound in the alkaline solution is preferably 10-20 ml / L, more preferably 13-18 ml / L, even more preferably 14-16 ml / L, and most preferably 15 ml / L; the ratio of the thermoresponsive polymer to the alkaline solution is preferably (10-100) g: 1 L, more preferably (30-80) g: 1 L, even more preferably (40-60) g: 1 L, even more preferably (45-55) g: 1 L, and most preferably 50 g: 1 L; the mixing reaction is preferably carried out under vigorous stirring; the mixing reaction time is preferably 8-16 h, more preferably 10-12 h; after the mixing reaction, it is preferably dialyzed with deionized water and freeze-dried to obtain the thermoresponsive polymer grafted with gelatin.

[0059] In a specific embodiment of the present invention, the continuous phase solution can be prepared according to methods well known to those skilled in the art, without any special limitations. Specifically, it can be prepared by the following method: adding the thermoresponsive polymer of grafted gelatin to deionized water, heating and mixing to obtain a continuous phase solution; during the heating and mixing process, the appropriate temperature can be controlled to promote the complete dissolution and swelling process of the thermoresponsive polymer of grafted gelatin until a homogeneous and transparent solution is formed; the heating and mixing temperature is preferably 60℃~70℃, more preferably 65℃; during the heating and mixing process, it is necessary to ensure that no bubbles are generated to improve the stability of subsequent droplet formation; after heating and mixing, it is preferable to filter to obtain a continuous phase solution; the filtration method can be any method well known to those skilled in the art, without any special limitations. In the present invention, a fine filtration device is preferred, more preferably a 0.22μm PTFE needle filter; filtration can not only remove insoluble impurities and obtain a clear and impurity-free solution, but also achieve the purpose of sterilization.

[0060] The dispersed phase solution and the continuous phase solution are separately introduced into a microfluidic chip to obtain microspheres. Using a microfluidic device, the prepared dispersed phase solution and the continuous phase solution are injected into a microchannel at a precisely controlled flow rate, forming stable water-in-oil (W / O) droplets through interfacial tension. This step requires precise control of the two-phase flow rate ratio and the microchannel structure to ensure uniform droplet size and regular shape.

[0061] In one specific embodiment of the present invention, the volume ratio of the dispersed phase solution to the continuous phase solution is preferably 1:(4-6); optionally, the volume ratio of the dispersed phase solution to the continuous phase solution is preferably 1:4, 1:5, 1:6 or any two of the above ratios.

[0062] In one specific embodiment of the present invention, the volume ratio of the dispersed phase solution to the continuous phase solution is preferably 1:5.

[0063] In a specific embodiment of the present invention, the flow rate of the dispersed phase solution is preferably 1 to 5 μL / min; optionally, the flow rate of the dispersed phase solution is 1 μL / min, 2 μL / min, 3 μL / min, 4 μL / min, 5 μL / min or any two of the above values; the flow rate of the continuous phase solution is preferably 10 to 20 μL / min; optionally, the flow rate of the continuous phase solution is 10 μL / min, 11 μL / min, 12 μL / min, 13 μL / min, 14 μL / min, 15 μL / min, 16 μL / min, 17 μL / min, 18 μL / min, 19 μL / min, 20 μL / min or any two of the above values.

[0064] In one specific embodiment of the present invention, the flow rate of the dispersed phase solution is preferably 3 μL / min; the flow rate of the continuous phase solution is preferably 14 μL / min.

[0065] In one specific embodiment of the present invention, an oil-in-water droplet formed by passing through a microfluidic chip is received by a solution containing a crosslinking agent and then crosslinked and fixed to obtain microspheres; the crosslinking effect of the crosslinking agent can fix the internal structure of the microspheres to prevent them from polymerizing or collapsing in subsequent processing.

[0066] In one specific embodiment of the present invention, the mass concentration of the crosslinking agent in the solution containing the crosslinking agent is preferably 1% to 10%; optionally, the mass concentration of the crosslinking agent in the solution containing the crosslinking agent is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or any two of the above values.

[0067] In one specific embodiment of the present invention, the mass concentration of the crosslinking agent in the solution containing the crosslinking agent is preferably 5%.

[0068] In one specific embodiment of the present invention, the crosslinking agent can be any crosslinking agent well known to those skilled in the art, and there are no special limitations. In the present invention, crosslinking agents such as genipin (GP) and / or glutaraldehyde are preferred.

[0069] In a specific embodiment of the present invention, the crosslinking fixation temperature is preferably room temperature; the crosslinking fixation time is preferably 10 to 60 minutes; optionally, the crosslinking fixation time is 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes or any two of the above values.

[0070] In one specific embodiment of the present invention, the crosslinking fixation time is 30 minutes.

[0071] In a specific embodiment of the present invention, after crosslinking and fixation, the microspheres are preferably cooled, centrifuged, washed, and freeze-dried to obtain microspheres. The crosslinked and fixed microspheres can be separated by centrifugation. The centrifugation speed is preferably 500-2000 rpm, more preferably 800-1500 rpm, and even more preferably 1000 rpm. The centrifugation time is preferably 10-60 s, more preferably 20-40 s, and even more preferably 30 s. The washing is preferably performed multiple times to thoroughly remove residual solvent and unreacted crosslinking agents and other impurities. The solvent used for washing is preferably petroleum ether and anhydrous ethanol. After washing, the moisture can be completely removed by freeze-drying in a low-temperature environment.

[0072] In one specific embodiment of the present invention, the microspheres are preferably sealed and stored at 4°C for later use.

[0073] The microspheres prepared by this invention can be used as a drug delivery system in the medical field.

[0074] The present invention also provides a method for preparing controllable drug release microspheres, comprising the following steps: S1) providing a dispersed phase solution and a continuous phase solution: the dispersed phase solution includes polylactic acid, polyglycolic acid and an organic solvent; the continuous phase solution includes grafted gelatin and a thermoresponsive polymer of the drug and water; the drug contains hydroxyl and / or carboxyl groups; S2) passing the dispersed phase solution and the continuous phase solution into a microfluidic chip respectively to obtain controllable drug release microspheres.

[0075] This invention grafts drugs and gelatin onto a thermoresponsive polymer, thereby endowing the system with a thermally responsive and MMP-2 enzyme-responsive thermal top. This not only improves the targeting effect of controllable drug-release microspheres on tumor sites but also significantly reduces drug burst release, promoting sustained drug delivery and therapeutic efficacy in vivo. Furthermore, by adjusting the flow rate and ratio of the dispersed phase solution and the continuous phase solution, the drug loading and particle size of the microspheres can be controlled, achieving different drug loading effects and controlling the release rate while maintaining uniform and controllable particle size.

[0076] The dispersed phase solution is the same as described above, and will not be repeated here.

[0077] In one specific embodiment of the present invention, the mass concentration of the grafted gelatin and the thermoresponsive polymer of the drug in the continuous phase solution is preferably 1% to 5%; optionally, the mass concentration of the grafted gelatin and the thermoresponsive polymer of the drug in the continuous phase solution is 1%, 2%, 3%, 4%, 5% or any two of the above values.

[0078] In one specific embodiment of the present invention, the mass concentration of the thermoresponsive polymer of grafted gelatin and drug in the continuous phase solution is 2%.

[0079] In a specific embodiment of this invention, the thermoresponsive polymer of grafted gelatin and drug is prepared by the following method: A1) The thermoresponsive polymer is activated with an activator, and then mixed and reacted with gelatin under alkaline conditions to obtain the thermoresponsive polymer of grafted gelatin; A2) The drug is reacted with a benzene ring derivative containing an aldehyde group, then a carboxyl activator is added to activate the carboxyl group, and then reacted with the thermoresponsive polymer of grafted gelatin to obtain the thermoresponsive polymer of grafted gelatin and drug.

[0080] The preparation method of the thermoresponsive polymer of the grafted gelatin is the same as described above, and will not be repeated here. However, after the reaction is completed, it is dialyzed with deionized water. It does not need to be lyophilized and can be directly used for the grafting of drugs in step A2).

[0081] In one specific embodiment of the present invention, the drug is any drug containing hydroxyl and / or carboxyl groups that is well known to those skilled in the art, and there are no special limitations. The drug is grafted onto the heat-responsive polymer via hydroxyl and / or carboxyl groups. In the present invention, an antitumor drug containing hydroxyl and / or carboxyl groups is preferred, an antibiotic antitumor drug is more preferred, an anthracycline antibiotic is even more preferred, and one or more of doxorubicin, daunorubicin, and pirarubicin are most preferred.

[0082] In a specific embodiment of the present invention, if the drug is a salt compound, it is preferred to first undergo desalting treatment before reacting with a benzene ring derivative containing an aldehyde group; the desalting treatment method can be any method well known to those skilled in the art and is not particularly limited, specifically: dissolving the drug salt compound in an organic solvent, adding an organic amine to react, and obtaining the desalted drug; the organic solvent can be any organic solvent well known to those skilled in the art and is not particularly limited, in the present invention, dimethyl sulfoxide (DMSO) is preferred; the molar ratio of the drug salt compound to the organic amine is preferably 1:(2-4), more preferably 1:(2.5-3.5), even more preferably 1:(3-3.5), and most preferably 1:3.2; the organic amine can be any organic amine well known to those skilled in the art and is not particularly limited, in the present invention, triethylamine is preferred; the reaction is preferably carried out under stirring conditions; the reaction time is preferably 5-15 h, more preferably 6-12 h, and even more preferably 8-10 h.

[0083] In a specific embodiment of the present invention, the molar ratio of the drug to the aldehyde-containing benzene ring derivative is preferably (1-1.5):1, more preferably (1.2-1.3):1, and even more preferably 1.25:1; the aldehyde-containing benzene ring derivative is selected from p-aldehyde benzoic acid.

[0084] In one specific embodiment of the present invention, the drug reacts with an aldehyde-containing benzene ring derivative in an organic solvent; the organic solvent is preferably dimethyl sulfoxide (DMSO); if the drug needs to be desalted, the aldehyde-containing benzene ring derivative can be added directly after the desalting treatment to carry out the reaction; the reaction time is preferably 20-30 h, more preferably 22-26 h, and even more preferably 24 h.

[0085] In a specific embodiment of the present invention, the carboxyl activator can be any carboxyl activator well known to those skilled in the art, and there are no special limitations. In the present invention, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are preferred. The molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to N-hydroxysuccinimide (NHS) is preferably 1:2. The molar ratio of the drug to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is preferably 1:(1 to 1.5), more preferably 1:(1 to 1.2). The activation time of the carboxyl group is preferably 4 to 8 hours, more preferably 6 hours.

[0086] In a specific embodiment of the present invention, after activating the carboxyl groups, the mixture reacts with the thermoresponsive polymer grafted with gelatin. Preferably, the activated carboxyl group system is added dropwise to the thermoresponsive polymer grafted with gelatin under stirring. The mass ratio of the drug to the thermoresponsive polymer is preferably (0.1–5):5. Optionally, the mass ratio of the drug to the thermoresponsive polymer is 0.1:5, 0.5:5, 1:5, 2:5, 3:5, 4:5, 5:5, or any two of the above ratios. The reaction time is preferably 8–15 h, more preferably 10–12 h.

[0087] In one specific embodiment of the present invention, the mass ratio of the drug to the thermoresponsive polymer is preferably 3:5.

[0088] In one specific embodiment of the present invention, after the reaction is completed, the mixture is preferably dialyzed with deionized water and freeze-dried to obtain a thermoresponsive polymer of grafted gelatin and drug.

[0089] In a specific embodiment of the present invention, the continuous phase solution can be prepared according to methods well known to those skilled in the art, without any special limitations. Specifically, it can be prepared by the following method: adding grafted gelatin and the thermo-responsive polymer of the drug to deionized water, heating and mixing to obtain a continuous phase solution; during the heating and mixing process, the appropriate temperature can be controlled to promote the complete dissolution and swelling process of the grafted gelatin and the thermo-responsive polymer of the drug until a homogeneous and transparent solution is formed; the heating and mixing temperature is preferably 60℃~70℃, more preferably 65℃; during the heating and mixing process, it is necessary to ensure that no bubbles are generated to improve the stability of subsequent droplet formation; after heating and mixing, it is preferable to filter to obtain a continuous phase solution; the filtration method can be any method well known to those skilled in the art, without any special limitations. In the present invention, a fine filtration device is preferred, more preferably a 0.22μm PTFE needle filter; filtration can not only remove insoluble impurities and obtain a clear and impurity-free solution, but also achieve the purpose of sterilization.

[0090] The dispersed phase solution and the continuous phase solution are respectively introduced into a microfluidic chip to obtain controllable drug release microspheres. In a specific embodiment of the present invention, the volume ratio of the dispersed phase solution to the continuous phase solution is preferably 1:(4-6); optionally, the volume ratio of the dispersed phase solution to the continuous phase solution is preferably 1:4, 1:5, 1:6 or any two of the above ratios.

[0091] In one specific embodiment of the present invention, the volume ratio of the dispersed phase solution to the continuous phase solution is preferably 1:5.

[0092] In a specific embodiment of the present invention, the flow rate of the dispersed phase solution is preferably 1 to 5 μL / min; optionally, the flow rate of the dispersed phase solution is 1 μL / min, 2 μL / min, 3 μL / min, 4 μL / min, 5 μL / min or any two of the above values; the flow rate of the continuous phase solution is preferably 10 to 20 μL / min; optionally, the flow rate of the continuous phase solution is 10 μL / min, 11 μL / min, 12 μL / min, 13 μL / min, 14 μL / min, 15 μL / min, 16 μL / min, 17 μL / min, 18 μL / min, 19 μL / min, 20 μL / min or any two of the above values.

[0093] In one specific embodiment of the present invention, the flow rate of the dispersed phase solution is preferably 3 μL / min; the flow rate of the continuous phase solution is preferably 14 μL / min.

[0094] In one specific embodiment of the present invention, an oil-in-water droplet formed by passing through a microfluidic chip is received by a solution containing a crosslinking agent and then crosslinked and fixed to obtain a controllable drug release microsphere; the crosslinking effect of the crosslinking agent can fix the internal structure of the microsphere to prevent it from polymerizing or collapsing in subsequent processing.

[0095] In one specific embodiment of the present invention, the mass concentration of the crosslinking agent in the solution containing the crosslinking agent is preferably 1% to 10%; optionally, the mass concentration of the crosslinking agent in the solution containing the crosslinking agent is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or any two of the above values.

[0096] In one specific embodiment of the present invention, the mass concentration of the crosslinking agent in the solution containing the crosslinking agent is preferably 5%.

[0097] In one specific embodiment of the present invention, the crosslinking agent can be any crosslinking agent well known to those skilled in the art, and there are no special limitations. In the present invention, genipin (GP) and / or glutaraldehyde are preferred.

[0098] In a specific embodiment of the present invention, the crosslinking fixation temperature is preferably room temperature; the crosslinking fixation time is preferably 10 to 60 minutes; optionally, the crosslinking fixation time is 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes or any two of the above values.

[0099] In one specific embodiment of the present invention, the crosslinking fixation time is 30 minutes.

[0100] In a specific embodiment of the present invention, after cross-linking fixation, the microspheres are preferably cooled, centrifuged, washed, and freeze-dried to obtain controllable drug release microspheres. The cross-linked and fixed controllable drug release microspheres can be separated by centrifugation. The washing is preferably performed multiple times to thoroughly remove residual solvents and unreacted cross-linking agents and other impurities. The solvents used for washing are preferably petroleum ether and anhydrous ethanol. After washing, the moisture can be completely removed by freeze-drying in a low-temperature environment. The freeze-drying temperature is preferably -60℃ to -80℃. The freeze-drying time is preferably 24 to 60 hours, more preferably 36 to 48 hours.

[0101] In one specific embodiment of the present invention, the controllable drug release microspheres are preferably sealed and stored at 4°C for subsequent use.

[0102] The present invention also provides a controllable drug release microsphere prepared by the above preparation method.

[0103] The controllable drug release microspheres provided by this invention are a dual-response (thermal response and MMP-2 enzyme response) controllable release system prepared based on microfluidic technology. PLA and PGA, as biodegradable polymer substrates, possess both biocompatibility and controllable degradation characteristics, enabling long-term sustained drug release and reducing toxic side effects. However, their hydrophobicity can easily lead to non-specific binding of plasma proteins and the risk of thrombosis after intravenous injection. The introduction of gelatin effectively inhibits the interaction between the carrier and plasma proteins through its surface hydrophilic groups. Simultaneously, as a specific substrate for the MMP-2 enzyme, gelatin also endows the system with tumor microenvironment responsiveness, achieving targeted tumor enrichment.

[0104] Furthermore, a thermoresponsive polymer grafted with gelatin was constructed by chemically coupling a thermoresponsive polymer with gelatin, and then a drug such as doxorubicin (ADM) was covalently grafted onto it via an amidation reaction to prepare a thermoresponsive polymer composite system of grafted gelatin and drug. This design significantly inhibits burst release effects and improves the system's targeting by immobilizing drug molecules through covalent bonds, while also enhancing drug solubility, bioavailability, and chemical stability. Simultaneously, the integrated application of microfluidic technology enabled uniform microsphere size and controllable morphology, optimizing the controlled-release performance, drug loading capacity, and release stability of the drug-loaded microspheres, providing an efficient delivery platform for precision tumor treatment.

[0105] The present invention also provides the application of the controllable drug release microspheres prepared by the above preparation method in the preparation of tumor treatment drugs.

[0106] The controllable drug release microspheres prepared by this invention can be used as a drug delivery system in the medical field.

[0107] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a controllable drug release microsphere, its preparation method, and its application.

[0108] All reagents used in the following examples are commercially available; the molecular weight of polylactic acid (PLA) is approximately 60,000 g / mol; the molecular weight of polyglycolic acid (PGA) is approximately 38,000–54,000 g / mol.

[0109] F127-g-Gelatin: 20g F127 was dissolved in 50 mL of DCM and added dropwise to a 50 mL DCM solution containing 3.2 g of 4-nitrophenyl chloroformate (p-NPC) while stirring vigorously. After reacting overnight, the resulting p-NPC-activated F127 was precipitated in 2750 mL of cold diethyl ether and then filtered under vacuum. 150 mL of p-NPC-activated F127 in 33.3% ethanol was added to 10 g of type A gelatin, followed by 1 L of deionized water containing 15 mL of triethylamine, and the mixture was stirred vigorously. After reacting overnight, the resulting F127-g-Gelatin was dialyzed against deionized water and then lyophilized for later use.

[0110] F127-g-Gelatin-ADM: Accurately weigh 12.0 g of doxorubicin hydrochloride (ADM·HCl), dissolve it in 100 mL of anhydrous DMSO, then add 64 mmol of triethylamine and stir for 10 h to obtain doxorubicin dehydrochloride; subsequently, add 2.48 g of p-aldehyde benzoic acid (p-CBA) and react for 24 h to form the p-CBA-ADM intermediate. Add 3.16 g of EDC and 4.74 g of NHS and react for 6 h to activate the para-carboxyl group of p-CBA. Under stirring, slowly add the activated p-CBA-ADM dropwise to the F127-g-Gelatin after dialyzing with deionized water, react for 10 h, stop the reaction, dialyze with deionized water, and then freeze-dry for later use.

[0111] Example 1

[0112] Preparation of PLA / PGA / F127-g-Gelatin microspheres

[0113] Continuous phase (preparation of 2% F127-g-Gelatin aqueous solution): Weigh 0.2g of F127-g-Gelatin powder, add 9.8g of ultrapure water, place in a 65℃ water bath and stir and heat to dissolve, then filter with a 0.22μm PTFE needle filter.

[0114] Dispersed phase (preparing 5% PLA and / or PGA dichloromethane solutions in mass ratios of 100% PGA, 20 / 80 PLA / PGA, 40 / 60 PLA / PGA, 60 / 40 PLA / PGA, 80 / 20 PLA / PGA, and 100% PLA): Weigh 0.1g of PLA and / or PGA powder according to the ratio, add 1.9g (1.43mL) of dichloromethane, sonicate to dissolve, and then filter through a 0.22μm PTFE needle filter to obtain dichloromethane solutions containing different proportions of PLA and / or PGA.

[0115] The dispersed and continuous phases were loaded into a microfluidic syringe: 5 mL of 2% F127-g-Gelatin aqueous solution was added to the channel 1 reservoir; 1 mL of 5% PLA and / or PGA dichloromethane solution was added to the channel 2 reservoir (black centrifuge tube); the air compressor and air source treatment switch were turned on; the pressure of channel 1 (aqueous phase) and channel 2 (oil phase) was set to purge air from the tubing and chip; after the air was purged (the tubing and chip were filled with liquid), the flow rates of the aqueous phase and oil phase were set to 14 μL / min and 3 μL / min, respectively. Water-in-oil droplets formed at the cross-shaped junction of the PDMS chip. Once the microdroplets were stably formed, they were collected into a glass culture dish containing 5% GP solution; collection was stopped after 60 min, and crosslinking was carried out at room temperature for 30 min. After the reaction was complete, the solution was removed, rapidly cooled to room temperature, centrifuged, and the microspheres were collected. Transfer the microspheres to centrifuge tubes, centrifuge, and collect the supernatant. Wash the precipitate with petroleum ether and anhydrous ethanol, respectively, and shake. Centrifuge at 1000 rpm for 30 seconds and collect the supernatant (this process can be repeated 1-2 times). Observe the solidified PLA / PGA microspheres under a microscope. Freeze-dry the collected microspheres (-80℃, 48h) and store a portion at 4℃ for later use.

[0116] Example 2

[0117] Preparation of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres

[0118] Drug-loaded microspheres were prepared using adriamycin (ADM) as a model drug, and the corresponding tests were performed.

[0119] Continuous phase (preparation of 2% F127-g-Gelatin-ADM aqueous solution): Weigh 0.2g of F127-g-Gelatin-ADM powder, add 9.8g of ultrapure water, place in a 65℃ water bath and stir and heat to dissolve, then filter with a 0.22μm PTFE needle filter.

[0120] Dispersed phase (prepared as a 5% PLA and / or PGA dichloromethane solution in mass ratios of 100% PGA, 20 / 80 PLA / PGA, 40 / 60 PLA / PGA, 60 / 40 PLA / PGA, 80 / 20 PLA / PGA, or 100% PLA): Weigh 0.1 g of PLA and / or PGA powder according to the ratio, add 1.9 g (1.43 mL) of dichloromethane, sonicate to dissolve, and then filter through a 0.22 μm PTFE needle filter to obtain dichloromethane solutions containing different proportions of PLA and / or PGA.

[0121] The dispersed and continuous phases were loaded into a microfluidic injector: 5 mL of 2% F127-g-Gelatin-ADM aqueous solution was added to the channel 1 reservoir; 1 mL of 5% PLA and / or PGA dichloromethane solution was added to the channel 2 reservoir (black centrifuge tube); the air compressor and air source treatment switch were turned on; the pressure of channel 1 (aqueous phase) and channel 2 (oil phase) was set to purge air from the tubing and chip; after the air was purged (the tubing and chip were filled with liquid), the flow rates of the aqueous and oil phases were set to 14 μL / min and 3 μL / min, respectively. The flow rates were adjusted to increase the feedback value to quickly reach the predetermined settings; water-in-oil droplets were formed at the cross-shaped junction of the PDMS chip. Once the microdroplets were stably formed, they could be collected into a glass culture dish containing 5% GP solution; collection was stopped after 60 min, crosslinking was performed at room temperature for 30 min, and after the reaction was completed, the solution was removed, rapidly cooled to room temperature, centrifuged, and the microspheres were collected. Transfer the microspheres to centrifuge tubes, centrifuge, and collect the supernatant. Wash the precipitate with petroleum ether and anhydrous ethanol, respectively, and shake. Centrifuge at 1000 rpm for 30 seconds and collect the supernatant (this process can be repeated 1-2 times). Transfer the microspheres to centrifuge tubes again; centrifuge and collect the supernatant. Add ultrapure water at a ratio of V(supernatant):V*(water) = 1:4 and shake. Centrifuge at 1000 rpm for 30 seconds and collect the supernatant (this process can be repeated 1-2 times). Observe the solidified microspheres under a microscope. Freeze-dry the collected microspheres (-80℃, 48h) and store a portion at 4℃ for later use.

[0122] Comparative Example 1

[0123] Preparation of PLA / PGA / F127-g / Gelatin / ADM microspheres (simple mixing)

[0124] Continuous phase: Prepare a 2% F127-g / Gelatin aqueous solution. Weigh 0.2g of F127-g / Gelatin (2:1) powder, add 9.8g of ultrapure water, place in a 65℃ water bath and stir and heat to dissolve. Then filter with a 0.22μm PTFE needle filter.

[0125] Dispersed phase: Prepare a 5% PLA and / or PGA-dichloromethane solution containing doxorubicin (mass ratios of 100% PGA, 20 / 80 PLA / PGA, 40 / 60 PLA / PGA, 60 / 40 PLA / PGA, 80 / 20 PLA / PGA, 100% PLA). Weigh 0.1g of PLA and / or PGA powder and an appropriate amount of doxorubicin, add 1.9g (1.43mL) of dichloromethane, sonicate to dissolve, and then filter through a 0.22μm PTFE needle filter.

[0126] The dispersed and continuous phases were loaded into a microfluidic syringe: 5 mL of 2% F127-g-Gelatin aqueous solution was added to the channel 1 reservoir; 1 mL of a 5% PLA and / or PGA dichloromethane solution containing doxorubicin was added to the channel 2 reservoir (black centrifuge tube); the air compressor and air source treatment switch were turned on; the pressure of channel 1 (aqueous phase) and channel 2 (oil phase) was set to purge air from the tubing and chip; after the air was purged (the tubing and chip were filled with liquid), the flow rates of the aqueous and oil phases were set to 14 μL / min and 3 μL / min, respectively. Water-in-oil droplets formed at the cross-shaped junction of the PDMS chip. Once the microdroplets were stably formed, they were collected into a glass culture dish containing 5% GP solution; collection was stopped after 60 min, and crosslinking was performed at room temperature for 30 min. After the reaction was complete, the solution was removed, rapidly cooled to room temperature, and centrifuged. The microspheres were collected. Transfer the microspheres to centrifuge tubes, centrifuge, and collect the supernatant. Wash the precipitate with petroleum ether and anhydrous ethanol, and shake. Centrifuge at 1000 rpm for 30 seconds and collect the supernatant (this process can be repeated 1-2 times). Transfer the microspheres to centrifuge tubes again; centrifuge and collect the supernatant. Add ultrapure water at a ratio of V(supernatant):V*(water) = 1:4, and shake. Centrifuge at 1000 rpm for 30 seconds and collect the supernatant (this process can be repeated 1-2 times). Observe the solidified microspheres under a microscope. Freeze-dry the collected microspheres (-80℃, 48h) and store a portion at 4℃ for later use.

[0127] The performance of the microspheres obtained in Examples 1-2 and Comparative Example 1 was tested, as follows:

[0128] Microsphere appearance

[0129] PLA / PGA / F127-g-Gelatin microspheres were successfully prepared using a microfluidic device. Precise control of the microsphere size and shape is crucial for achieving the drug effect. The microspheres were uniformly dispersed in physiological saline and stained with methylene blue for 15 min. Subsequently, the morphology of the microspheres with a PLA to PGA mass ratio of 2:3 was observed using a conventional optical microscope, and their appearance is shown in the image below. Figure 1 As shown.

[0130] Biocompatibility of PLA / PGA / F127-g-Gelatin microspheres

[0131] 1 g of sterilized microspheres were cultured in 10 mL of complete culture medium at 37 °C for 24 hours. The extract was collected by centrifugation, filtered through a membrane, and sterilized for later use. Mouse fibroblast L-929 cells were counted using a cell counting chamber at a value of 5 × 10⁻⁶. 4 Cells / well were seeded into 96-well plates and incubated for 24 h. Then, 100 μL of extraction medium was added to each well, with 6 replicates per group, and incubated for another 24 h. Next, 10 μL of CCK8 solution (5 mg / mL) was added to each well, and the plates were incubated for 4 h, then shaken well in the dark. Untreated cells were used as a control group. Absorbance was measured at 570 nm to calculate cell viability, and the biocompatibility of PLA / PGA / F127-g-Gelatin microspheres was tested. To assess the potential cytotoxicity of the control and microspheres, different ratios of microspheres were co-cultured with mouse fibroblast L-929 cells for 24 h, and the relative cell viability was compared using the CCK8 assay. According to ISO 10993-5, a decrease in cell viability of more than 30% is considered cytotoxic. Figure 2 As shown, with changes in microsphere concentration and culture, the relative cell viability of cells co-cultured with microspheres of different ratios remained above 85% after 24 hours, indicating that the microspheres not only did not inhibit cell growth but actually promoted cell survival. These experimental results demonstrate that the prepared microspheres possess excellent biocompatibility, thus showing great promise for drug delivery.

[0132] ADM Standard Curve

[0133] First, accurately weigh 10.0 mg of doxorubicin, dissolve it in PBS, and dilute to volume in a 10 mL volumetric flask to obtain a 1.0 mg / mL doxorubicin solution. Place an appropriate amount of this solution in a UV spectrophotometer and perform a wavelength scan using standard procedures to obtain the maximum absorption wavelength of doxorubicin. Then, pipette different volumes (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mL) of the prepared doxorubicin solution into 10 mL volumetric flasks, and dilute to the mark with an appropriate amount of PBS solution to obtain different concentrations (10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0 μg / mL) of doxorubicin solution. Measure the absorbance (OD value) at 480 nm using UV spectrophotometry. Finally, analyze the data to obtain the ADM standard curve, as shown below. Figure 3 As shown.

[0134] In vitro drug release assay of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres

[0135] Accurately weigh 20 mg of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres, uniformly disperse them in 10 mL of PBS (pH 7.4), place them in a sealed dialysis bag, and then place the bag in 75 mL of PBS. Simulate the physiological environment in vivo by dialysis at a constant temperature (37±1℃) and constant speed (100 r / min) in an air shaker. Collect 10 mL of dialysate at intervals of 4 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 96 h, and 120 h. Detect the OD480 of the dialysate by ultraviolet spectrophotometry. Calculate the drug release at each time point based on the known standard curve, and then calculate the cumulative release. After each 10 mL of dialysate was removed, 10 mL of fresh PBS was added to the container to maintain a total volume of 75 mL. The experiment was repeated three times. In vitro drug release curves of the drug-loaded microspheres were plotted using the mean ± standard deviation of the cumulative drug release as the parameter. The in vitro drug release curves of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres are shown in the figure below. Figure 4 As shown. By Figure 4It is evident that the drug release process of drug-loaded microspheres exhibits a typical two-stage characteristic: a rapid release phase in the initial stage (0-24 hours) where the drug is released at a relatively fast rate; after 24 hours, a sustained-release phase begins, with the release rate significantly slowing down. This release pattern offers clinical therapeutic advantages—rapid initial release effectively addresses the peak drug demand during acute disease flare-ups, while the subsequent stable sustained release maintains an effective therapeutic concentration. Further analysis of release curves with different carrier ratios revealed that a higher proportion of polylactic acid (PLA) in the carrier resulted in a slower overall release rate. This is because the biodegradation rate of PLA is significantly slower than that of polyglycolic acid (PGA). As the PLA proportion increases, the degradation of the carrier structure slows down, leading to a decrease in drug release rate. Therefore, by precisely controlling the PLA to PGA ratio, personalized adjustments to the release rate can be achieved: for patients requiring long-term micro-dose administration, using drug-loaded microspheres with a high PLA proportion can achieve a more ideal sustained-release effect; while for acute disease treatment, increasing the PGA proportion can accelerate drug release. This carrier design strategy provides customized solutions for different clinical needs.

[0136] A comparison of in vitro drug release experiments between simple mixed microspheres (i.e., PLA / PGA / F127-g / Gelatin / ADM microspheres prepared in Comparative Example 1, i.e., control group) and PLA / PGA / F127-gGelatin-ADM drug-loaded microspheres prepared in Example 2, i.e., experimental group).

[0137] Accurately weigh 20 mg each of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres (PLA to PGA mass ratio 3:2), PLA / PGA / F127-g / Gelatin / ADM drug-loaded microspheres (PLA to PGA mass ratio 3:2), blank microspheres (PLA / PGA / F127-g-Gelatin, PLA to PGA mass ratio 3:2), and ADM. Disperse each microsphere evenly in 10 mL of PBS (pH 7.4), place in a sealed dialysis bag, and incubate in a 75 mL PBS solution. Dialysis was performed in PBS at a constant temperature (37±1℃) and constant speed (100 r / min) in an air shaker, simulating the physiological environment in vivo. Ten mL of dialysate was collected at intervals of 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. The OD480 of the dialysate was measured by UV spectrophotometry. The drug release at each time point was calculated based on a known standard curve, and the cumulative release was then calculated. After each collection of 10 mL of dialysate, 10 mL of fresh PBS was added to the container to maintain a total volume of 75 mL. The experiment was repeated three times. The in vitro drug release curve of the drug-loaded microspheres was plotted using the mean ± standard deviation of the cumulative drug release as the parameter. The comparison graph of the in vitro drug release test is shown below. Figure 5 As shown. Figure 5The in vitro drug release rates of blank microspheres, ADM, experimental group, and control group were measured. Figure 5 It was found that the blank group showed no drug release; the ADM group released the drug completely within a short time without any sustained-release effect; the release efficiency of PLA / PGA / F127-g / Gelatin / ADM microspheres (control group) was significantly higher than that of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres (experimental group), but it could not achieve the ideal drug release effect; the drug release process of PLA / PGA / F127-g-Gelatin-ADM drug-loaded microspheres (experimental group) exhibited a typical two-stage characteristic: the initial release stage (0-24 hours) was a burst release stage, in which the drug was released at a relatively rapid rate; after 24 hours, it entered a sustained-release stage, and the release rate slowed down significantly. This release mode has clinical therapeutic advantages—the initial rapid drug release can effectively cope with the peak drug demand during the acute phase of the disease, while the later stable sustained release can maintain an effective therapeutic concentration.

[0138] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a controllable drug release microsphere, characterized by, The method comprises the following steps: S1) providing a dispersed phase solution and a continuous phase solution; the dispersed phase solution comprises polylactic acid, polyglycolic acid and an organic solvent; the continuous phase solution comprises grafted gelatin, a drug containing hydroxyl and / or carboxyl, a thermoresponsive polymer and water; S2) passing the dispersed phase solution and the continuous phase solution into a microfluidic chip respectively to obtain the controllable drug release microspheres. The water-in-oil droplets formed by passing the dispersed phase solution and the continuous phase solution into the microfluidic chip in the step S2) are received into a solution containing a crosslinking agent to be crosslinked and fixed to obtain the controllable drug release microspheres.

2. The production method according to claim 1, characterized by, The total mass concentration of the polylactic acid and the polyglycolic acid in the dispersed phase solution is 1% to 10%; the mass ratio of the polylactic acid to the polyglycolic acid is 10:90 to 90:10; 3. The production method according to claim 2, characterized by, The drug is selected from anthracycline antibiotics; The mass concentration of the grafted gelatin and the thermoresponsive polymer of the drug in the continuous phase solution is 1% to 5%; The mass concentration of the crosslinking agent in the solution containing the crosslinking agent is 1% to 10%; The crosslinking agent is selected from genipin and / or glutaraldehyde. The grafted gelatin and the thermoresponsive polymer of the drug are prepared by the following method:

4. The method of claim 1, wherein, A1) activating the thermoresponsive polymer with an activating agent, and then mixing and reacting the thermoresponsive polymer with gelatin under alkaline conditions to obtain the thermoresponsive polymer grafted with gelatin; A2) reacting the drug with an aldehyde group-containing benzene ring derivative, then activating the carboxyl group by adding a carboxyl activating agent, and then reacting the carboxyl group with the thermoresponsive polymer grafted with gelatin to obtain the thermoresponsive polymer grafted with gelatin and the drug. The thermoresponsive polymer is selected from poloxamer; 5. The preparation method according to claim 4, characterized in that, The activating agent is selected from 4-nitrophenyl chloroformate; The drug is selected from one or more of adriamycin, daunorubicin and pirarubicin; The aldehyde group-containing benzene ring derivative is selected from p-aldehyde benzoic acid; The mass ratio of the thermoresponsive polymer to the activating agent is 10:(1-2); The mass ratio of the thermoresponsive polymer to gelatin is (1-10):1; The mass ratio of the drug to the thermoresponsive polymer is (0.1-5):

5. The step A1) specifically comprises: mixing the thermoresponsive polymer with an organic solvent, then adding an organic solution containing the activating agent to react, and then precipitating in cold diethyl ether after the reaction to obtain the activated thermoresponsive polymer; 6. The preparation method according to claim 4, characterized in that, The activated thermoresponsive polymer is dissolved in an alcohol solvent, and then mixed and reacted with gelatin and an alkaline solution to obtain the thermoresponsive polymer grafted with gelatin. The volume ratio of the dispersed phase solution to the continuous phase solution in the step S2) is 1:(4-6); 7. The preparation method according to claim 1, characterized in that, The flow rate of the dispersed phase solution is 1-5 μL / min; The flow rate of the continuous phase solution is 10-20 μL / min.

8. The controllable drug release microspheres prepared by the preparation method in any one of claims 1-7.

9. The use of the controllable drug release microspheres prepared by the preparation method in any one of claims 1-7 in the preparation of a tumor treatment drug. The method comprises the following steps:

10. A method of preparing microspheres, characterized by, B1) providing a dispersed phase solution and a continuous phase solution; the dispersed phase solution comprises polylactic acid, polyglycolic acid and an organic solvent; ​ The continuous phase solution comprises a thermally responsive polymer grafted gelatin and water; B2) separately passing the dispersed phase solution and the continuous phase solution into the microfluidic chip to obtain the microspheres.

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