Protein drug-loaded GelMA microspheres, and preparation method and application thereof

Abstract

The invention discloses a protein drug loaded GelMA microsphere, a preparation method and application thereof, the protein drug loaded GelMA microsphere comprises a GelMA microsphere and a protein drug loaded on the GelMA microsphere, and the protein drug is composed of albumin paclitaxel and alphaPD-L1. The drug carrier is loaded with albumin paclitaxel and the alphaPD-L1 antibody, so that synergistic treatment can be realized, and the cancer treatment effect is improved; gelMA is used as a drug carrier, albumin paclitaxel and an alphaPD-L1 antibody are delivered, and the drug-loaded GelMA microspheres are injected into tumor tissues in situ, so that the targeting property and bioavailability of the drug can be effectively improved, side effects are reduced, the drug is enriched in a focus, and the circulating drug concentration is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a GelMA microsphere loaded with albumin-paclitaxel and αPD-L1, its preparation method, and its application. Background Technology

[0002] Currently, cancer treatment mainly relies on two methods: chemotherapy and immunotherapy.

[0003] In related technologies, systemic administration of traditional chemotherapy drugs is prone to toxicity problems such as bone marrow suppression, and the tumor targeting is low, resulting in low tumor tissue enrichment efficiency; in immunotherapy, the antibody half-life is short after intravenous injection, requiring high-dose administration in a short period of time, which poses certain safety risks.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing a GelMA microsphere loaded with a protein drug, its preparation method, and its application. This application achieves synergistic therapy and improves the efficacy of cancer treatment by loading albumin-bound paclitaxel and αPD-L1 antibody onto a drug carrier. By using GelMA as a drug carrier to deliver albumin-bound paclitaxel and αPD-L1 antibody, and by injecting the drug-loaded GelMA microspheres in situ into tumor tissue, the targeting and bioavailability of the drug can be effectively improved, side effects can be reduced, and the drug can be concentrated in the lesion, reducing the circulating drug concentration.

[0006] To achieve the above objectives, this application provides the following technical solution: According to one aspect of this application, a GelMA microsphere loaded with a protein drug is provided, the GelMA microsphere comprising a GelMA microsphere and a protein drug loaded on the GelMA microsphere, the protein drug being composed of albumin-bound paclitaxel and αPD-L1.

[0007] According to another aspect of this application, a method for preparing GelMA microspheres loaded with protein drugs is also provided, comprising: preparing GelMA microspheres using microfluidic technology; activating the protein drug using acrylate-polyethylene glycol-active ester, and covalently crosslinking the GelMA microspheres with the activated protein drug to obtain the GelMA microspheres loaded with protein drugs.

[0008] In some embodiments, the preparation of GelMA microspheres using microfluidic technology includes: dispersing methacrylamide gelatin and a photoinitiator in deionized water, stirring in the dark until completely dissolved to obtain a GelMA solution; converting the GelMA solution into GelMA microsphere droplets using microfluidic technology, wherein the aqueous phase in the microfluidic technology is the GelMA solution and the oil phase is the oil generated from the microdroplets; irradiating the GelMA microsphere droplets with a 365 nm, 90 W ultraviolet flashlight for 20 seconds to form solid GelMA microspheres; washing five times with 75% ethanol until no oil droplets remain under a microscope, centrifuging to obtain GelMA microspheres.

[0009] In some embodiments, the degree of amino substitution of the methacrylamide gelatin is 60%, the concentration of the methacrylamide gelatin in the GelMA solution is 80 mg / mL, the photoinitiator is LAP, and the concentration of LAP in the GelMA solution is 2.5 mg / mL.

[0010] In some embodiments, the activation of the protein drug using acrylate-polyethylene glycol-active ester includes: mixing acrylate-polyethylene glycol-active ester with albumin-paclitaxel and αPD-L1 in the dark, and incubating at 4°C for at least 18 hours to obtain the activated protein drug; the covalent cross-linking of the GelMA microspheres with the activated protein drug includes: mixing the activated protein drug with the GelMA microspheres and irradiating with 365 nm ultraviolet light for 10 seconds.

[0011] According to another aspect of this application, the use of the GelMA microspheres loaded with protein drugs described above, or the GelMA microspheres loaded with protein drugs prepared by the preparation method described above, in the preparation of cancer treatment drugs is also provided.

[0012] In some embodiments, the method of using the drug is to inject the drug in situ into the tumor tissue.

[0013] In some embodiments, the cancer includes one or more of breast cancer, non-small cell lung cancer, and pancreatic cancer.

[0014] In some embodiments, the cancer is breast cancer.

[0015] Compared with the prior art, the advantages and positive effects of this application are as follows: This application loads albumin-bound paclitaxel and αPD-L1 antibody onto a drug carrier. Albumin-bound paclitaxel can exert its effect by enhancing the sensitivity of tumor cells to the immune system, while αPD-L1 antibody enhances the anti-tumor activity of T cells by relieving the inhibition of the immune system by tumor cells. Combining these two drugs can achieve synergistic treatment and improve the effect of cancer treatment.

[0016] Furthermore, by using GelMA as a drug carrier to deliver albumin-bound paclitaxel and αPD-L1 antibody, and by injecting drug-loaded GelMA microspheres in situ into tumor tissue, the targeting and bioavailability of the drug can be effectively improved, side effects reduced, and the drug concentrated in the lesion, thereby reducing the circulating drug concentration.

[0017] Furthermore, stable drug loading was achieved by covalently cross-linking AC-PEG-NHS activating protein drugs with GelMA microspheres. Compared with traditional physical adsorption or encapsulation methods, covalent cross-linking can more effectively control drug release, reduce drug leakage, and improve the stability and efficiency of drug delivery. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The illustration shows the luminescence of various tissues in the mammary tissue of healthy mice after injection of GelMA microspheres in Example 3 of this application.

[0020] Figure 2 The illustration shows the luminescence of various tissues in the liver tissue of healthy mice after injection of GelMA microspheres in Example 3 of this application.

[0021] Figure 3 The illustration shows the luminescence of various tissues in the lung tissue of healthy mice after injection of GelMA microspheres in Example 3 of this application.

[0022] Figure 4 The illustration shows the luminescence of various tissues after injecting GelMA microspheres into the tumor tissue of 4T1 mice in Example 3 of this application.

[0023] Figure 5 The images show fluorescence micrographs of sections of mammary tissue from healthy mice injected with GelMA microspheres at 12 h and 24 h after application in Example 3 of this application.

[0024] Figure 6 The images show fluorescence micrographs of liver tissue sections from healthy mice injected with GelMA microspheres at 6 h and 24 h after the injection in Example 3 of this application.

[0025] Figure 7 This image shows a fluorescence microscope photograph of a section of tumor tissue from 4T1 mice 24 hours after injection of GelMA microspheres in Example 3 of this application.

[0026] Figure 8The tumor volume of different treatment groups in Example 4 of this application is shown.

[0027] Figure 9 The image shows the body weight of mice in different treatment groups in Example 4 of this application. Detailed Implementation

[0028] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0029] Albumin-bound paclitaxel (commonly known as Abraxane) is an anticancer drug primarily used to treat certain types of cancer, such as breast cancer, non-small cell lung cancer, and pancreatic cancer.

[0030] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] Experimental equipment and materials: Microfluidic equipment: FluidicLab, Droplet / Microsphere Preparation System - Droplet Microfluidic System (DG-01); Microfluidic chip: FluidicLab, PDMS standard microdroplet generation microfluidic chip; Methacrylamide gelatin: Engineering ForLife, EFL-GM-60; Lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP): Engineering ForLife, EFL-GM-60 compatible LAP; Microdroplet generating oil: FluidicLab, Drop-Sruf microdroplet generating oil; Albumin-bound paclitaxel: Qilu Pharmaceutical, Paclitaxel for Injection (albumin-bound); αPD-L1: Invivo anti-mouse PD-L1 Recombinant mAb (S0B0038); Mice: Vital River, BALB / c mice; Acrylate-polyethylene glycol-active ester: Engineering For Life, AC-PEG-NHS.

[0032] Example 1: Preparation of drug-loaded GelMA microspheres loaded with albumin-bound paclitaxel and αPD-L1.

[0033] S100. Disperse methacrylamide gelatin (GelMA) and photoinitiator in deionized water and stir in the dark until completely dissolved to obtain a GelMA solution.

[0034] The amino substitution degree of the methacrylamide gelatin (GelMA) was 60%, the concentration of GelMA in the GelMA solution was 80 mg / mL, and the photoinitiator was lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), with a concentration of 2.5 mg / mL in the GelMA solution.

[0035] S200: GelMA solution is converted into GelMA microsphere droplets using microfluidic technology.

[0036] In this embodiment, a microfluidic chip is used to prepare microspheres. The GelMA solution is the aqueous phase, and the oil generated from the microdroplets is the oil phase. A pressure (200 mbar) is applied to both phases using a microfluidic device to make them flow stably through the microfluidic chip. At the cross structure of the chip, water-in-oil droplets are formed by shearing and collected to obtain GelMA microsphere droplets.

[0037] S300, microsphere curing, washing.

[0038] In this embodiment, the GelMA microsphere droplets were irradiated with a 365 nm UV flashlight (90 W) for 20 seconds to form solid GelMA microspheres. The microspheres were then washed five times with 75% ethanol until no oil droplets remained as observed under a microscope, and collected by centrifugation.

[0039] S400, drug load.

[0040] (1) Drug activation: Acrylate-polyethylene glycol-active ester (AC-PEG-NHS) was mixed with albumin-bound paclitaxel and anti-PD-L1 monoclonal antibody (αPD-L1) and incubated at 4°C for at least 18 hours in the dark. AC-PEG-NHS was used to activate protein drugs (albumin-bound paclitaxel and αPD-L1).

[0041] αPD-L1 is a blocking antibody that targets programmed death-ligand-1 (PD-L1).

[0042] (2) Covalent cross-linking: The activated protein drug was mixed with the GelMA microspheres prepared in step S300 and irradiated with 365 nm ultraviolet light for 10 seconds to form drug-loaded GelMA microspheres. The average particle size of the drug-loaded GelMA microspheres was 90 μM and the CV was 3.9%.

[0043] Example 2: Construction of a 4T1 mouse model.

[0044] The experimental mice were purchased from Vital River, SPF grade, and numbered 35. 4T1 mouse breast cancer cells were subcutaneously inoculated into the mice to construct a 4T1 mouse model.

[0045] 4T1 breast cancer cells were digested, washed with PBS, and resuspended in a cell suspension. 5 x 10-1 cells were injected subaxillarily into each mouse. 6 Four T1 cells were collected. The major axis (L) and minor axis (W) of the tumor were measured using calipers. The volume was calculated according to the formula V = L x W. 2 / 2 calculation, the tumor volume reaches 100 mm. 3 The model was successfully built.

[0046] Example 3: Permeability verification of GelMA microspheres.

[0047] (1) Preparation of fluorescently labeled GelMA microspheres (without drug loading).

[0048] The fluorescent molecules were mixed with the GelMA solution obtained in step S100 of Example 1, and then prepared into droplets in step S200. The droplets were irradiated with 365 nm ultraviolet light for 10 seconds, and the free fluorescent molecules were washed away with 75% ethanol solution. The fluorescently labeled GelMA microspheres were collected by centrifugation.

[0049] (2) Validation using mouse models.

[0050] Fluorescently labeled GelMA microspheres were injected orally into the mammary glands, liver, lungs of healthy mice and tumors of 4T1 mice. Mice were sacrificed at different times (6 h, 12 h, and 24 h) and tissues were collected. The permeability of GelMA microspheres was determined by detecting fluorescence in the tissues using a small animal in vivo imaging system.

[0051] Figure 1 The illustration shows the luminescence of various tissues in the mammary tissue of healthy mice after injection of GelMA microspheres in Example 3 of this application. Figure 2 The illustration shows the luminescence of various tissues in the liver tissue of healthy mice after injection of GelMA microspheres in Example 3 of this application. Figure 3 The illustration shows the luminescence of various tissues in the lung tissue of healthy mice after injection of GelMA microspheres in Example 3 of this application. Figure 4 This illustrates the luminescence of various tissues in 4T1 mouse tumor tissue after injection of GelMA microspheres in Example 3 of this application. For example... Figure 1-4 As shown, the injected fluorescently labeled GelMA microspheres mainly aggregated in the injection area and did not spread widely or appear in other organs.

[0052] Tissue was fixed using paraformaldehyde, dehydrated using a sucrose gradient, embedded using OCT embedding agent, and finally prepared into frozen sections. The frozen sections were stained with DAPI dye, and fluorescence was observed under a fluorescence microscope.

[0053] Figure 5The images show fluorescence micrographs of sections of mammary tissue from healthy mice injected with GelMA microspheres at 12 h and 24 h after application in Example 3 of this application. Figure 6 The images show fluorescence micrographs of liver tissue sections from healthy mice injected with GelMA microspheres at 6 h and 24 h after the injection in Example 3 of this application. Figure 7 This image shows a fluorescence microscope photograph of a section of tumor tissue from 4T1 mice 24 hours after injection of GelMA microspheres, as described in Example 3 of this application. Figure 5-7 As shown, the injected fluorescent molecular microspheres mainly aggregate in the injection area and do not diffuse over a wide area.

[0054] Example 4: Evaluation of the efficacy of drug-loaded GelMA microspheres.

[0055] Drug-loaded GelMA microspheres and free drug with different drug addition amounts were prepared according to the method in Example 1. 4T1 mice were constructed according to the method for constructing the 4T1 mouse model in Example 2. The drug (albumin-bound paclitaxel) was added at 2.5 mg / kg mouse body weight, 5 mg / kg mouse body weight, and 10 mg / kg mouse body weight, and αPD-L1 was added at 10 mg / kg mouse body weight.

[0056] Finally, drug-loaded GelMA microspheres and free drug groups were obtained. The drug-loaded GelMA microsphere groups were MS@ (2.5 mg / kg PTX + 10 mg / kg αPD-L1), MS@ (5 mg / kg PTX + 10 mg / kg αPD-L1), and MS@ (10 mg / kg PTX + 10 mg / kg αPD-L1), respectively. The free drug groups were 2.5 mg / kg PTX + 10 mg / kg αPD-L1, 5 mg / kg PTX + 10 mg / kg αPD-L1, and 10 mg / kg PTX + 10 mg / kg αPD-L1, respectively.

[0057] The size of subcutaneous tumors in mice was measured using vernier calipers. Tumor volume was calculated as: tumor length * tumor width * tumor width / 2. When the tumor volume increased to 100 mm... 3 During treatment, mice were injected orally into the tumor with drug-loaded GelMA microspheres, free drug, GelMA microspheres alone (MS), or an equal volume of saline (Control). Tumor size and mouse weight were continuously monitored. The Control group consisted of 5 mice, while the other groups each consisted of 4 mice.

[0058] Figure 8 The tumor volume of different treatment groups in Example 4 of this application is shown. Figure 9 The image shows the body weight of mice in different treatment groups in Example 4 of this application. Figure 8-9As shown, the drug delivery method using drug-loaded GelMA microspheres can significantly improve the inhibitory effect of the drug on tumor growth; the normal weight gain of mice indicates that the GelMA drug-loaded microspheres have good safety.

[0059] This application describes a method for achieving sustained drug release by injecting GelMA microspheres loaded with albumin-bound paclitaxel and αPD-L1 into 4T1 tumor mice in situ. By limiting the drug release range (1-2 cm), the method prolongs the drug's duration of action in vivo, thereby enhancing the therapeutic effect on tumors and reducing adverse reactions and damage to normal tissues.

[0060] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A GelMA microsphere loaded with a protein drug, characterized in that, The GelMA microspheres loaded with the protein drug comprise GelMA microspheres and a protein drug loaded on the GelMA microspheres, the protein drug being composed of albumin-bound paclitaxel and αPD-L1.

2. The method for preparing GelMA microspheres loaded with protein drugs according to claim 1, characterized in that, include: GelMA microspheres were prepared using microfluidic technology; The protein drug was activated using acrylate-polyethylene glycol-active ester, and the GelMA microspheres were covalently cross-linked with the activated protein drug to obtain the GelMA microspheres loaded with the protein drug.

3. The preparation method according to claim 2, characterized in that, The fabrication of GelMA microspheres using microfluidic technology includes: Methacrylamide gelatin and photoinitiator were dispersed in deionized water and stirred in the dark until completely dissolved to obtain a GelMA solution. The GelMA solution is converted into GelMA microsphere droplets using microfluidic technology, wherein the aqueous phase is the GelMA solution and the oil phase is the oil generated from the microdroplets. The GelMA microsphere droplets were irradiated with a 365 nm, 90 W UV flashlight for 20 seconds to form solid GelMA microspheres. They were then washed five times with 75% ethanol until no oil droplets remained under a microscope. After centrifugation, the GelMA microspheres were obtained.

4. The preparation method according to claim 3, characterized in that, The degree of amino substitution of the methacrylamide gelatin is 60%, the concentration of the methacrylamide gelatin in the GelMA solution is 80 mg / mL, the photoinitiator is LAP, and the concentration of LAP in the GelMA solution is 2.5 mg / mL.

5. The preparation method according to claim 2, characterized in that, The method of activating protein drugs using acrylate-polyethylene glycol-active esters includes: mixing acrylate-polyethylene glycol-active esters with albumin-paclitaxel and αPD-L1 in the dark, and incubating at 4°C for at least 18 hours to obtain activated protein drugs. The step of covalently crosslinking the GelMA microspheres with the activated protein drug includes: mixing the activated protein drug with the GelMA microspheres and irradiating with 365 nm ultraviolet light for 10 seconds.

6. The use of the GelMA microspheres loaded with protein drugs according to claim 1 or the GelMA microspheres loaded with protein drugs prepared by any one of claims 2-5 in the preparation of cancer treatment drugs.

7. The application according to claim 6, characterized in that, The method of using the drug is to inject it in situ into the tumor tissue.

8. The application according to claim 7, characterized in that, The cancers include one or more of breast cancer, non-small cell lung cancer, and pancreatic cancer.

9. The application according to claim 8, characterized in that, The cancer in question is breast cancer.

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