CN117159440A - A soluble microneedle co-loaded with immune adjuvants and immune checkpoint inhibitors and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a soluble microneedle for co-loading an immune adjuvant and an immune checkpoint inhibitor, and a preparation method and application thereof. The lipid nano-drug is obtained by encapsulating hydrophobic immunoadjuvant by liposome, and the chitosan nano-gel drug is obtained by loading protein immune checkpoint inhibitor by chitosan; the two obtained nano-drugs are further loaded on the needle tip part of the soluble microneedle, and the diffusion of the drugs to the microneedle substrate can be avoided; the prepared microneedle can act on shallow phenotype tumor, obviously improve the drug concentration and the availability of focus, and the co-delivery of the two nano drugs can synergistically enhance the killing function of the immune system of the organism on tumor cells. Meanwhile, the drug loaded on the soluble micro needle can reduce the dosage of the drug and avoid systemic toxic and side effects.

Description

A soluble microneedle co-loaded with immune adjuvants and immune checkpoint inhibitors and its Preparation methods and applications

Technical field

The invention belongs to the technical field of biomedicine. More specifically, it relates to a soluble microneedle co-loaded with an immune adjuvant and an immune checkpoint inhibitor and its preparation method and application.

Background technique

Cancer is the second leading cause of death in the world and poses a major threat to human life, health and social development. Since 1991, the cancer mortality rate in the United States has been declining year by year, which is partly due to the introduction of new treatments such as immune checkpoint blockade and targeted therapy for metastatic melanoma in the United States.

Commonly used methods to treat cancer mainly include chemotherapy, surgical resection and radiotherapy; in addition, immunotherapy, targeted therapy, and stem cell transplantation also play an important role. However, the above-mentioned treatment methods all have their own limitations. For example, surgical resection is only effective for solid tumors and is helpless for metastatic tumors, and there are risks such as poor prognosis and tumor recurrence; radiotherapy and chemotherapy not only kill cancer cells but also Normal cells attack indiscriminately; targeted therapy may cause cancer cells to develop drug resistance; stem cell transplantation is prone to graft-versus-host reaction. In order to achieve the goal of curing tumors while reducing side effects, it is imperative to develop more efficient and less toxic tumor treatment options.

The biggest difference between immunotherapy and other treatments is that immunotherapy can enhance the immune system's ability to recognize and kill tumors. That is to say, it targets the immune system that can resist cancerous lesions rather than tumor cells. Immunotherapy can promote the immune system to produce components that kill tumor cells, or counteract the signals produced by cancer cells that inhibit the immune response. Compared with traditional treatments such as chemotherapy, immunotherapy has fewer side effects, lower drug resistance, and lower off-target rates. Advantage. However, immunotherapy also has shortcomings in clinical application. For example, small molecule agonists or protein antibodies used in immunotherapy have problems such as poor water solubility and easy degradation when transported in the body. The introduction of nano-delivery systems can improve the bioavailability of immune drugs to a certain extent. However, nanomedicines are usually injected intravenously or intramuscularly, and usually only 2% to 8% of the drugs can eventually accumulate in tumors, resulting in insufficient drug delivery efficiency. In order to achieve the therapeutic window, intravenous or intramuscular injection of nanomedicines requires a large amount of drug, This not only increases the cost, but also increases the systemic toxicity of the drug. In addition, the tumor tissue is dense and the interstitial pressure is high, which not only limits the amount of in situ injection, but also makes it difficult for the drug to penetrate deeply into the tumor tissue.

Compared to intravenous or intramuscular injections, microneedles can gently and painlessly deliver drugs through the stratum corneum of the skin in a minimally invasive manner. Microneedles have a micron-scale multi-array tip structure. Compared with traditional in situ single-point injection, microneedles can achieve dispersed multi-point injection, overcome the interstitial pressure of tumors, enhance the accumulation of drugs in lesions, and ultimately improve the bioavailability of drugs. Utilization. For example, Chinese patent application CN113694009A discloses a transdermal system that directly and locally co-delivers immune checkpoint inhibitors and chemotherapeutic agents through microneedles for collaborative immunochemotherapy. Although it has a certain effect on tumor cells, aPD-1 Co-delivery with CDDP is achieved using the same nanosystem loading. Based on hydrophobic and electrostatic non-specific interactions, aPD-1 is loaded into the phospholipid layer of liposomes. aPD-1 protein drugs are macromolecular substances that are limited by steric hindrance and are difficult to stably load into the phospholipid layer of liposomes. , resulting in low stability of the obtained nano drug-carrying system, and the drug easily leaks out of the nano system. When the above-mentioned lipid nanomedicine is loaded into microneedles, aPD-1 is easily released from the liposomes and diffuses to the microneedle base, resulting in a reduction in the microneedle drug loading capacity. In addition, this method uses the reverse phase microemulsion method to load CDDP and aPD-1 into liposomes. The reverse phase microemulsion method has two major problems: on the one hand, the protein antibody aPD-1 will come into contact with the remaining organic solvents; It will inevitably destroy the spatial structure of the protein antibody aPD-1, making it difficult to ensure the biological activity of such expensive antibodies; on the other hand, the preparation process of the reverse-phase microemulsion method is complex, the product stability is low, and the loading capacity is difficult under the same preparation conditions. It is guaranteed to be the same every time and is difficult to generalize to production practice. In addition, the use of lighter-density liposomes to encapsulate antibody drugs, although they can be deposited on the microneedle tips, is easier to diffuse to the back base of the microneedle and be scraped off during the preparation process, resulting in a reduction in the drug content in the needle tips. , and cause waste.

Contents of the invention

The technical problem to be solved by this invention is to overcome the defects and shortcomings in the existing microneedle drug delivery that the activity of protein drugs is easily destroyed and the nanomedicine loaded on the microneedle tip is easy to diffuse to the back base of the microneedle, resulting in a reduction in the drug content in the tip. A soluble microneedle co-loaded with an immune adjuvant and an immune checkpoint inhibitor is provided.

The object of the present invention is to provide a method for preparing the soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors.

Another object of the present invention is to provide the application of the soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors in the preparation of cancer treatment drugs or medical devices.

The above objects of the present invention are achieved through the following technical solutions:

The invention protects a soluble microneedle that is co-loaded with an immune adjuvant and an immune checkpoint inhibitor. The immune adjuvant is encapsulated in liposomes to obtain lipid nanomedicine, and the immune checkpoint inhibitor is loaded in chitosan. In the sugar nanogel, chitosan nanogel drug is obtained, and lipid nanomedicine and chitosan nanogel drug are jointly loaded on the tip of the soluble microneedle.

Immune adjuvants are usually hydrophobic small molecule drugs that can enhance the body's ability to respond to antigens. Liposomes have a phospholipid bilayer structure similar to that of a cell membrane, which can encapsulate hydrophobic drugs in the phospholipid layer to form lipid nanomedicines and improve their water solubility and bioavailability. Immune checkpoint blockers are usually protein antibodies, which are easily enzymatically inactivated in the body. Positively charged chitosan can load negatively charged immune checkpoint inhibitors through electrostatic interactions to form nanogel drugs. The obtained lipid nanomedicine and chitosan nanogel drug are jointly loaded on the needle tip of the soluble microneedle, which can perform immunotherapy on near-superficial tumors. It can increase the concentration of the drug in the lesion while reducing the dosage, which not only enhances the anti-tumor immune response, and reduce the systemic toxic and side effects of the drug, and the co-delivery of two nanomedicines can synergistically enhance the therapeutic effect.

Preferably, the immune adjuvant includes a Toll-like receptor 7 agonist (such as imiquimod R837), a Toll-like receptor 7/8 agonist (such as resiquimod R848) or a Toll-like receptor 9 agonist (such as unmethylated oligodeoxynucleotide CpG ODN).

Further, the immune checkpoint inhibitor is an IgG antibody.

Preferably, the immune checkpoint inhibitor can be selected from IgG antibodies such as anti-programmed death receptor 1 antibody (i.e., aPD-1) or anti-programmed death receptor-ligand 1 antibody (i.e., aPD-L1). one or more.

Preferably, the liposome material includes one or more of neutral phospholipids, electronegative phospholipids, electropositive phospholipids, phospholipid-polymer conjugates or cholesterol.

More preferably, the neutral phospholipid includes palmitoylcholine, distearoylcholine, dimyristoylphosphatidylcholine or phosphatidylcholine.

More preferably, the electronegative phospholipid includes phosphatidic acid, phosphatidylglycerol, phosphatidylinositol or distearoylphosphatidylethanolamine-polyethylene glycol.

More preferably, the electropositive phospholipid includes a phospholipid stearylamide cholesterol derivative. Generally, positively charged phospholipids are basically artificially synthesized.

More preferably, the phospholipid-polymer conjugate includes distearoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylcholine-polyethylene glycol or dipalmitoylphosphatidylcholine-polyethylene glycol. diol.

Preferably, the base material of the tip portion of the soluble microneedle is selected from one or more of hyaluronic acid, cellulose, sodium alginate, trehalose, and the like.

Preferably, the molecular weight of the hyaluronic acid is less than 10 kDa.

Preferably, the base material of the needle body part and the base part of the soluble microneedle is selected from one or more of polyvinylpyrrolidone K90, hyaluronic acid, dextran, cellulose and gelatin.

Preferably, the mass ratio of the immune adjuvant to the liposome material is 1:10-20.

Preferably, the mass ratio of the immune checkpoint inhibitor to chitosan is 1:0.5-2.

Further, the loading amount of the immune adjuvant in the soluble microneedles is 5-15 μg per square centimeter.

Further, the loading amount of the immune checkpoint inhibitor in the soluble microneedles is 2-10 μg per square centimeter.

The present invention also protects the preparation method of the soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors, which specifically includes the following steps:

S1. Encapsulate immune adjuvants in liposomes to obtain lipid nanomedicines; load immune checkpoint inhibitors into chitosan nanogels to obtain chitosan nanogel drugs;

S2. Mix the lipid nanomedicine obtained in step S1 with the base material solution at the needle tip evenly, and then load the chitosan nanogel drug on the needle tip of the soluble microneedle through the microneedle mold step by step, and is not limited to lipids. The loading sequence of nanomedicine and chitosan gel medication; if the chitosan nanogel medication is loaded first and then the lipid nanomedicine is loaded, the negatively charged tip base material can be adsorbed to the surface of the chitosan nanogel medication through electrostatic interaction , increase its density and size, causing it to gather at the tip of the microneedle under the action of centrifugal force, avoiding the diffusion of drugs to the base of the microneedle, further improving the drug loading rate of the microneedle, reducing the waste of expensive drugs, and maximizing the effect on tumors. The therapeutic effect; if the lipid nanomedicine is loaded first and then the chitosan nanogel drug is loaded, when the base material is injected to prepare the needle, the base material can also increase the density of the chitosan nanogel drug. If the base material is Negative materials can be attached to the drug surface of chitosan nanogel through electrostatic interaction to prevent the drug from diffusing to the microneedle base.

S3. Prepare the needle body part and the base part in sequence on the needle tip obtained in step S2, and that is the result.

Whether it is small molecule drugs or antibodies, there is a problem of low density. During the preparation process of microneedles, it is difficult to gather to the needle tip through centrifugation. Even if the drug reaches the needle tip, it is easy to diffuse to the back base of the microneedle and be scraped off, which not only limits the The drug loading rate of microneedles is reduced, which also leads to the waste of expensive drugs and ultimately affects the therapeutic effect of tumors. After extensive preliminary research, the applicant creatively discovered that in the process of preparing soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors, the immune adjuvants are encapsulated in liposomes to obtain lipid nanomedicines, which combine immune The checkpoint inhibitor is loaded into the chitosan nanogel to obtain the chitosan nanogel drug, which is then loaded step by step on the tip of the soluble microneedle, and the subsequent matrix material used can further increase the density of the liposome, with It is conducive to the accumulation of drugs on the microneedle tips and is not easy to spread, thereby effectively improving the drug loading rate of the microneedle.

Further, in step S1, the chitosan nanogel drug is prepared using an ion gel method.

Preferably, the cross-linking agent used in the ion gel method is sodium tripolyphosphate.

Further, in step S2, the nanomedicine and the base material at the needle tip part are made into a solution and then solidified to obtain the needle tip part. The concentration of the base material at the needle tip part is 50-120 mg/mL, and the nanomedicine (total concentration of the two drugs) The concentration is 1~4mg/mL.

Preferably, the concentration of the nanomedicine is 2 mg/mL.

Preferably, when the chitosan nanogel drug is first loaded on the needle tip of the soluble microneedle, and then the lipid nanomedicine is loaded, it has higher mechanical strength and is easier to penetrate the skin. Compared with liposomes, chitosan nanogel has a significantly higher density and is more conducive to deposition at the bottom of the needle tip; at the same time, the present invention also uses a multi-step centrifugation method to add and mix the chitosan nanogel after solidification The needle tip base material solution (negatively charged) lipid nanomedicine, the negatively charged needle tip base material solution can be adsorbed to the chitosan surface through electrostatic interaction, increasing the density and size of the chitosan nanogel drug, making it Under the action of centrifugal force, it gathers on the tip of the microneedle, preventing the drug from diffusing to the base of the microneedle, further ensuring the utilization of expensive immune checkpoint inhibitor antibodies.

Further, in step S2, the microneedle mold is a PDMS microneedle mold, the morphological parameters are needle height 400-1500 μm, base diameter 300-500 μm, needle tip diameter less than 10 μm, and the number of arrays is greater than 8×8.

Further, in step S3, the base material concentration of the needle body part is 300-400 mg/mL, and the base material concentration of the base part is 300-400 mg/mL. The preparation process specifically includes: preparing a needle body part on the needle tip part, and preparing a base part on the needle body part.

Furthermore, the needle tip, needle body and base part are obtained by placing the base material solution in a microneedle mold respectively, centrifuging and solidifying; wherein the centrifugal speed is 3000-10000 rpm and the time is 5-30 min.

In order to solve the problem that the microneedle drug delivery load is small and the drug cannot be concentrated on the microneedle tip, the present invention prepares the microneedle tip and needle body through a two-step method.

The present invention also protects the application of the soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors in the preparation of cancer treatment drugs or medical devices.

Further, the cancer is superficial cancer, including breast cancer, melanoma, and skin cancer.

The invention has the following beneficial effects:

The invention provides a soluble microneedle that is co-loaded with an immune adjuvant and an immune checkpoint agonist. The immune adjuvant is encapsulated in liposomes to form lipid nanomedicine, and chitosan is used to load immune checkpoint inhibitors to form chitosan. Sugar nanogel drug; the two obtained nanomedicines are further loaded on the tip of the soluble microneedle step by step, and can prevent the drug from diffusing to the microneedle base; the prepared microneedle can act on superficial tumors and significantly improve the focus of the disease. The drug concentration is high, and the co-delivery of the two nanomedicines can synergistically enhance the killing function of the body's immune system against tumor cells; at the same time, the dosage can also be reduced to avoid systemic toxic side effects.

Description of drawings

Figure 1 is a statistical graph showing the particle size of RNPs and αNPs nanomedicines in Examples 1 and 2.

Figure 2 is a statistical graph showing the surface potential of RNPs and αNPs nanomedicines in Examples 1 and 2.

Figure 3 is a transmission electron microscope scanning image of RNPs nanomedicine in Example 1.

Figure 4 is a transmission electron microscope scanning image of αNPs nanomedicine in Example 2.

Figure 5 is a scanning electron microscope image of the soluble microneedles in Example 3.

Figure 6 is a handheld microscope image of the soluble microneedles in Example 3.

Figure 7 is a statistical graph showing the drug loading test results of the soluble microneedles in Example 4.

Figure 8 is a statistical graph showing the survival rate of DC2.4 cells using different concentrations of R848, RNPs and RNP@DMN microneedles loaded with RNPs in Example 5.

Figure 9 is a physical diagram of tumor volume recording in Example 6.

Figure 10 is a statistical graph of tumor volume changes in Example 6 (three asterisks (***) represent p<0.001, indicating an extremely significant statistical difference).

Figure 11 is a statistical graph showing body weight changes of mice in Example 6.

Figure 12 is a HE staining picture of the heart, liver, spleen, lung and kidney of mice in Example 6.

Figure 13 is a statistical graph of liver and kidney function parameters (ALT, BUN, TBIL, CREA) of mice in Example 6.

Detailed ways

The invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.

PC-98T: High-purity egg yolk lecithin, purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.;

Chol: Cholesterol, purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.;

DSPE-mPEG2000: distearic acid phosphatidylethanolamine-polyethylene glycol 2000, purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

Example 1 Synthesis of lipid nanomedicine RNPs encapsulating immune adjuvants (taking R848 as an example)

The preparation method of the lipid nanomedicine RNPs encapsulating immune adjuvants specifically includes the following steps:

SI-1. Dissolve 20 mg of liposome membrane material (PC-98T:Chol:DSPE-mPEG2000=10:5:1, molar ratio) and 2 mg of R848 in 15 mL of chloroform, pour it into a 250 mL eggplant-shaped bottle, and pass Rotate evaporate (35°C, 135rpm, 20min) to remove chloroform and obtain a uniform lipid film;

SI-2. Add 4 mL of pure water to the eggplant-shaped bottle and peel off the lipid film under ultrasound;

SI-3. Obtain lipid nanomedicine RNPs through probe ultrasound (130w, 20kHz, 20min).

The particle size, potential, surface morphology and other characteristics of RNPs nanoparticles were measured. The results are shown in Figures 1 to 3. As can be seen from the figure, the hydrated particle size of RNPs nanoparticles is about 85nm, and the zeta potential is about -27mV. The transmission electron microscope image shows that RNPs exhibit a membrane structure with cavities, and the particle size after drying and collapse is about 60nm.

Example 2 Synthesis of chitosan nanogel drug αNPs loaded with immune checkpoint inhibitors (taking aPD-1 as an example)

The preparation method of the chitosan nanogel drug αNPs loaded with immune checkpoint inhibitors specifically includes the following steps:

SII-1. Dissolve chitosan in 2% acetic acid aqueous solution with a mass concentration of 2.5 mg/mL; aPD-1 is dissolved in 1×PBS with a mass concentration of 2 mg/mL. Sodium tripolyphosphate (TPP) is dissolved in pure In water, the mass concentration is 0.25mg/mL.

Under the conditions of SII-2 and 4°C, mix the aPD-1 solution and the TPP solution evenly, add it drop by drop to the chitosan solution under high-speed vortex conditions (1500~3000rpm), and react for 2 hours. The mass ratio of aPD-1 to chitosan is 1:1.

SII-3. Centrifuge the solution obtained in SII-2 at high speed (10000 rpm) for 15 minutes, discard the supernatant, and disperse the lower solid with an equal amount of pure water to obtain chitosan nanogel drug αNPs loaded with immune checkpoint inhibitors. .

The particle size, potential, surface morphology and other characteristics of αNPs nanoparticles were measured. The results are shown in Figures 1 to 2 and Figure 4. As can be seen from the figure, the hydrated particle size of αNPs nanoparticles is about 94nm, and the zeta potential is about 46mV. The transmission electron microscope image taken after αNPs was dyed with uranyl acetate shows that its particle size after drying and collapse is about 60nm.

Example 3 Preparation of soluble microneedles co-loaded with immune adjuvants and immune checkpoint blockers

The preparation method of co-loading immune adjuvant and immune checkpoint blocker specifically includes the following steps:

SIII-1. Take 100 μg of αNPs nanoparticles obtained in Example 2, pour it into a microneedle mold, centrifuge at 4000 rpm for 5 min, rotate 180° and centrifuge again for 5 min, scrape off excess liquid, centrifuge for 30 min, and dry until the solution solidifies;

SIII-2. Take 2.5 mg of the RNPs nanoparticles obtained in Example 1 and mix them evenly with a 70 mg/mL hyaluronic acid (HA) aqueous solution; pour the HA solution containing RNPs into the needle tip obtained in SIII-1 and the needle tip has solidified. In the microneedle mold of αNPs, centrifuge at 4000 rpm for 5 minutes, rotate 180° and centrifuge again for 5 minutes, scrape off excess liquid, centrifuge for 30 minutes, dry until the solution solidifies, and serve as the needle tip together with αNPs;

SIII-3. Prepare 300 mg/mL HA solution, pour the prepared HA solution into the microneedle mold, centrifuge at 4000 rpm for 5 min, rotate 180° and centrifuge again for 5 min, scrape off excess liquid, centrifuge for 30 min, and dry to The solution solidifies as part of the needle body;

SIII-4. Prepare a 300 mg/mL PVP K90 aqueous solution, pour the prepared PVP K90 aqueous solution into the microneedle mold, maintain the vacuum negative pressure for 15 minutes, extract the bubbles, scrape off the bubble-containing solution, and add a new PVP K90 aqueous solution. Fill the entire mold, dry until the solution solidifies, and use it as the base part to obtain soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors;

Among them, the microneedle mold used is a PDMS microneedle mold, whose morphological parameters are needle height 1200 μm, base diameter 300 μm, needle tip diameter less than 10 μm, and the number of arrays is greater than 12×12.

The morphology of the obtained soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors was characterized, and the characterization results are shown in Figures 5-6.

Example 4 Drug Loading Test of Soluble Microneedles

(1) Preparation of soluble microneedles (RNP@DMN) loaded only with lipid nanomedicine RNPs

Take 2.5 mg of the RNPs nanomedicine obtained in Example 1 and mix it evenly with a 70 mg/mL hyaluronic acid (HA) aqueous solution; pour the HA solution containing RNPs into the microneedle mold, centrifuge at 4000 rpm for 5 min, and rotate 180° Centrifuge again for 5 minutes, scrape off excess liquid, centrifuge for 30 minutes, dry until the solution solidifies, and use it as the needle tip;

The remaining steps and parameters are prepared with reference to Example 3, and the soluble microneedle RNP@DMN is obtained.

(2) Preparation of soluble microneedles (αNP@DMN) loaded only with chitosan nanogel drug αNPs

Take 100 μg of the αNPs nanoparticles obtained in Example 2, pour them into a microneedle mold, centrifuge at 4000 rpm for 5 min, rotate 180° and centrifuge again for 5 min, scrape off excess liquid, centrifuge for 30 min, dry until the solution solidifies, and serve as the needle body;

The remaining steps and parameters are prepared with reference to Example 3, and the soluble microneedle αNP@DMN is obtained.

(3) Preparation of R848 soluble microneedles (R848@DMN)

Ensure that during the preparation process of R848@DMN, the added amount of R848 is the same as the RNP-loaded R848 weighed during the preparation process of RNP@DMN. After conversion, an appropriate amount of R848 is dissolved in 1mL of chloroform and mixed with 70mg/mL of hyaluronic acid (HA). ) Mix the aqueous solution evenly; pour the HA solution containing R848 into the microneedle mold, centrifuge at 4000 rpm for 5 minutes, rotate 180° and centrifuge again for 5 minutes, scrape off excess liquid, centrifuge for 30 minutes, dry until the solution solidifies, and serve as the needle tip;

The remaining steps and parameters can be prepared by referring to Example 3 to obtain soluble microneedles R848@DMN.

(4) Preparation of aPD-1 soluble microneedles (aPD-1@DMN)

Ensure that the amount of aPD-1 added during the preparation of aPD-1@DMN is the same as the αNPs-loaded aPD-1 weighed during the preparation of RNP@DMN. After conversion, dissolve an appropriate amount of aPD-1 in 1mL 1×PBS and pour into the microneedle mold, centrifuge at 4000 rpm for 5 minutes, rotate 180° and centrifuge again for 5 minutes, scrape off excess liquid, centrifuge for 30 minutes, dry until the solution solidifies, and serve as the needle body;

The remaining steps and parameters are prepared with reference to Example 3, and the soluble microneedle aPD-1@DMN is obtained.

(5) Drug loading test of soluble microneedles:

Use a scalpel blade to scrape the soluble microneedles obtained in (1) to (4) above from the microneedle patch, collect and weigh them, dissolve them with an appropriate amount of ultrapure water, and then dilute the above dissolved solution with DMSO to a suitable The concentration of R848 in the microneedles was determined by high-performance liquid chromatography-UV detector; aPD-1 was collected using the same method, and the drug loading capacity of aPD-1 was determined by IgG ELISA kit.

The measurement results are shown in Figure 7. It can be seen from the figure that compared to R848 and aPD-1, which are directly loaded on soluble microneedles without encapsulation, R848 is encapsulated in liposomes and aPD-1 is loaded on chitosan. After loading the nanogel into soluble microneedles, the loading rate of the soluble microneedles obtained by co-loading immune adjuvants and immune checkpoint inhibitors has been significantly improved.

Example 5 Effects of different concentrations of R848, RNPs and soluble microneedles αNP-RNP@DMN loaded with RNPs on the survival rate of DC2.4 cells

Refer to the method of Example 4 to prepare soluble microneedles loaded with RNPs (RNP@DMN), and use the MTT method to test the effects of different concentrations of R848, RNP, and soluble microneedles loaded with RNPs (RNP@DMN) on the viability of DC2.4 cells. Influence.

The test results are shown in Figure 8. It can be seen from Figure 8 that the toxicity of unencapsulated R848 to DC2.4 cells (the target cells of R848) increases as the concentration increases, but when it is encapsulated in liposomes or loaded into microspheres, After injection, it promotes the proliferation of DC2.4 cells. It was proved that liposome encapsulation reduced the toxicity of R848 to DC2.4 cells.

Example 6 Therapeutic effect and safety evaluation of soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors on tumors

1. Experimental materials:

Mouse-derived triple-negative breast cancer 4T1 cell line; BALB/c mice aged 4 to 6 weeks were purchased from the Guangdong Provincial Animal Experiment Center. All animal operating procedures were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" of Sun Yat-sen University. All experiments involving animals strictly abide by the "China Animal Management Regulations" (1988, revised in 2017) and the "China Guidelines for the Humane Treatment of Laboratory Animals" (MOST 2006).

2. Experimental method:

Tumor animal model modeling: Remove the hair from the back of mice, inject 100 μL (1×10 ⁶ cells) of 4T1 breast cancer cell suspension into the back of mice, divide the mice into 5 groups, namely control group (Control), RNPs single drug microneedle group (RNP@DMN), αNPs single drug microneedle group (αNP@DMN), RNPs and αNPs mixed solution intratumoral injection group (αNP+RNP it), and co-loaded RNPs and αNPs microneedle group (αNP -RNP@DMN). After 7 days of modeling, different administration treatments were performed according to different groups, and the mouse body weight and tumor volume were recorded every two days. The results are shown in Figures 9 to 11.

The toxic and side effects of the drug were clarified by HE staining of the heart, liver, spleen, lung and kidney of mice. The staining results are shown in Figure 12. At the same time, the liver and kidney functions of the mice were detected by a mouse biochemical instrument, and the results are shown in Figure 13.

3. Experimental results:

As can be seen from Figures 9 to 13, the tumor suppressor effect of the combined administration of immune adjuvants and immune checkpoint inhibitors was significantly better than that of the control group and single drug administration group, while the αNP-RNP@DMN group administered through microneedles Compared with the αNP+RNP i.t. group injected with mixed drug solution into the tumor, it has better efficacy; compared with the continued increase in tumor size in other groups, the tumors in the αNP-RNP@DMN group began to shrink on the tenth day of administration. the trend of. In addition, the weight of mice in each group did not decrease significantly, the biochemical indicators of the liver and kidneys were within the normal range, and there was no significant difference between the groups. The H&E sections of the heart, liver, spleen, and kidneys in each group showed no obvious damage, proving that microorganisms Injection administration has no obvious side effects. Among them, the H&E sections of the lungs in Figure 12 show that triple-negative breast cancer in mice will cause fibrosis and thickening of the lung interstitium, threatening the life and health of the mice. αNP+RNPi.t. group and αNP-RNP Treatment in the @DMN group can effectively inhibit pulmonary fibrosis in mice and maintain the normal function of the mouse lung organs.

Based on the above results, it can be proved that the soluble microneedles co-loaded with immune adjuvants and immune checkpoint inhibitors have significant tumor inhibition effect, show no toxicity to normal organs, and can maintain the normal morphology of mouse lungs.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (10)

1. The soluble microneedle for co-loading the immune adjuvant and the immune checkpoint inhibitor is characterized in that the immune adjuvant is encapsulated in liposome to obtain a lipid nano-drug, the immune checkpoint inhibitor is loaded in chitosan nano-gel to obtain a chitosan nano-gel drug, and the lipid nano-drug and the chitosan nano-gel drug are jointly loaded at the needle tip part of the soluble microneedle.

2. The soluble microneedle co-loaded with an immune adjuvant and an immune checkpoint inhibitor according to claim 1, wherein the immune adjuvant comprises a Toll-like receptor 7 agonist, a Toll-like receptor 7/8 agonist or a Toll-like receptor 9 agonist.

3. The soluble microneedle co-loaded with an immune adjuvant and an immune checkpoint inhibitor according to claim 1, wherein the immune checkpoint inhibitor is an IgG antibody.

4. The soluble microneedle co-loaded with an immune adjuvant and an immune checkpoint inhibitor according to claim 1, wherein the substrate of the tip site of the soluble microneedle is selected from one or more of hyaluronic acid, cellulose, sodium alginate and trehalose.

5. The soluble microneedle co-loaded with an immune adjuvant and an immune checkpoint inhibitor according to claim 1, wherein the loading of the immune adjuvant in the soluble microneedle is 5-15 μg per square centimeter.

6. The soluble microneedle co-loaded with an immune adjuvant and an immune checkpoint inhibitor according to claim 1, wherein the loading amount of the immune checkpoint inhibitor in the soluble microneedle is 2-10 μg per square centimeter.

7. The co-loaded immune adjuvant and immune checkpoint inhibitor soluble microneedle according to claim 1, wherein the liposome material comprises one or more of neutral phospholipids, electronegative phospholipids, electropositive phospholipids, phospholipid-polymer conjugates or cholesterol.

8. The method for preparing the soluble microneedle of the co-supported immune adjuvant and the immune checkpoint inhibitor according to any one of claims 1 to 7, which is characterized by comprising the following steps:

s1, encapsulating an immune adjuvant in a liposome to obtain a lipid nano-drug; loading an immune checkpoint inhibitor into chitosan nanogel to obtain a chitosan nanogel drug;

s2, uniformly mixing the lipid nano-drug obtained in the step S1 with the substrate solution at the needle point part, and loading the mixture with the chitosan nano-gel drug at the needle point part of the soluble micro-needle step by step through a micro-needle die;

s3, sequentially preparing a needle body part and a base part on the needle point part obtained in the step S2.

9. Use of the co-loaded immunoadjuvant and immune checkpoint inhibitor soluble microneedle according to any one of claims 1 to 7 for the preparation of a medicament or medical device for the treatment of cancer.

10. The use according to claim 9, wherein the cancer is a superficial cancer, including breast cancer, melanoma, skin cancer.