CN117159440A

CN117159440A - Soluble microneedle with co-loaded immune adjuvant and immune checkpoint inhibitor as well as preparation method and application thereof

Info

Publication number: CN117159440A
Application number: CN202311052821.5A
Authority: CN
Inventors: 帅心涛; 黄思聪; 王家忱; 韦慧叶; 林敏钊
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2023-08-18
Filing date: 2023-08-18
Publication date: 2023-12-05

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

Soluble microneedle with co-loaded immune adjuvant and immune checkpoint inhibitor as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicine. More particularly, relates to a soluble microneedle for co-loading an immune adjuvant and an immune checkpoint inhibitor, a preparation method and application thereof.

Background

Cancer is the second leading cause of death worldwide, creating a major threat to human life health and social development. Since 1991, cancer mortality in the united states has declined year by year, due in part to new therapeutic approaches such as immune checkpoint blockade methods developed in the united states, targeted therapy of metastatic melanoma, and the like.

Common means for treating cancer mainly include chemotherapy, surgical excision and radiation therapy; in addition, immunotherapy, targeted therapy, and stem cell transplantation play an important role. However, the above treatments have respective limitations, such as: surgical excision is effective only on solid tumors, is not in charge of metastatic tumor bundles, and has risks of poor prognosis, tumor recurrence and the like; radiotherapy and chemotherapy kill cancer cells while they do no differential attack on normal cells; targeted therapy may lead to resistance in cancer cells; stem cell transplantation is prone to graft versus host reactions. In order to achieve the aim of reducing side effects while curing tumors, development of a more efficient and low-toxicity tumor treatment scheme is imperative.

Immunotherapy is most distinguished from other treatments in that it enhances the ability of the immune system to recognize and kill tumors, i.e. it aims at the immune system, not at tumor cells, which are resistant to cancerous lesions. The immunotherapy can promote the immune system to produce the component which kills the tumor cells, or offset the signals which inhibit the immune response and are produced by the cancer cells, and has the advantages of smaller toxic and side effect, low drug resistance, lower off-target rate and the like compared with the traditional treatment methods such as chemotherapy and the like. However, immunotherapy is also deficient in clinical applications. For example, small molecule agonists or protein antibodies used in immunotherapy have problems such as poor water solubility and susceptibility to degradation during in vivo delivery. The introduction of nano-drug delivery systems can improve the bioavailability of immune drugs to some extent. However, nano-drugs are usually injected intravenously or intramuscularly, and only 2% -8% of the drugs can be finally aggregated in tumors, so that the administration efficiency is insufficient; in order to achieve the therapeutic window, the dosage required by intravenous or intramuscular injection of the nano-drugs is large, which not only increases the cost, but also increases the systemic toxicity of the drugs. In addition, the tumor tissue is compact, the interstitial pressure is high, the dosage of in-situ injection is limited, and the medicine is difficult to penetrate into the tumor tissue deeply.

Compared to intravenous or intramuscular injection, the microneedles can deliver drugs through the stratum corneum in a gentle and painless manner in a minimally invasive manner. Compared with the traditional in-situ single-point injection mode, the micro-needle has a micron-scale multi-array needle point structure, can realize dispersive multi-point injection, overcomes the interstitial pressure of tumors, enhances the aggregation of medicines in focus, and finally improves the bioavailability of the medicines. For example, chinese patent application CN113694009a discloses a transdermal system for co-delivering immune checkpoint inhibitor and chemotherapeutic agent directly and locally via microneedles for synergistic immunochemistry, and while having a certain therapeutic effect on tumor cells, co-delivery of apc-1 and CDDP is achieved using the same nanosystem loading. Based on hydrophobic and electrostatic nonspecific interactions, the aPD-1 is loaded on a phospholipid layer of a liposome, the aPD-1 protein drugs belong to macromolecular substances, are limited by steric hindrance, and are difficult to stably load in the phospholipid layer of the liposome, so that the stability of the obtained nano drug-loading system is low, and the drugs are easy to leak out from the nano system. When the lipid nano-drug is loaded into the micro-needle, the aPD-1 is easily released from the liposome and is diffused to the micro-needle substrate, so that the drug loading capacity of the micro-needle is reduced. In addition, the method uses an inverse microemulsion method to load CDDP and aPD-1 into liposome, and the inverse microemulsion method has two major problems: on the one hand, the protein antibody aPD-1 can be contacted with the residual organic solvent, the space structure of the protein antibody aPD-1 can be inevitably destroyed, and the biological activity of the expensive antibody is difficult to ensure; on the other hand, the reverse microemulsion method has complex process and low product stability, and the load capacity is difficult to ensure the same each time under the same preparation conditions, so that the reverse microemulsion method is difficult to popularize in production practice. In addition, the use of the less dense liposome encapsulates the antibody drug, although capable of depositing onto the microneedle tips, is easier to diffuse towards the microneedle back substrate and be scraped off during the preparation process, resulting in reduced drug content of the tips and waste.

Disclosure of Invention

The invention aims to overcome the defects that the activity of protein drugs is easily damaged and the nano drugs loaded on the tip of the micro needle are easily diffused to the back substrate of the micro needle to reduce the content of the drugs on the tip in the existing micro needle drug administration, and provides a soluble micro needle with co-loaded immune adjuvant and immune checkpoint inhibitor.

The invention aims to provide a preparation method of the soluble microneedle for co-loading an immune adjuvant and an immune checkpoint inhibitor.

It is another object of the present invention to provide the use of the co-loaded immunoadjuvant and immune checkpoint inhibitor soluble microneedles in the manufacture of a medicament or medical device for treating cancer.

The above object of the present invention is achieved by the following technical scheme:

the invention provides a soluble microneedle for co-loading an immune adjuvant and an immune checkpoint inhibitor, wherein 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 the 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.

Immunoadjuvants are generally hydrophobic small molecule drugs capable of enhancing the body's ability to respond to antigens. The liposome has a phospholipid bilayer structure similar to a cell membrane, can encapsulate a hydrophobic drug in a phospholipid layer to form a lipid nano drug, and improves the water solubility and bioavailability of the lipid nano drug. The immune checkpoint blocker is usually a protein antibody, is easy to be enzymatically deactivated in vivo, and electropositive chitosan can load electronegative immune checkpoint inhibitors through electrostatic interaction to form a nanogel medicament. The obtained lipid nano-drug and chitosan nano-gel drug are loaded on the needle tip part of the soluble microneedle together, so that the near-superficial tumor can be treated by immunity, the drug administration dosage is reduced while the concentration of the focus drug is increased, the anti-tumor immune response is enhanced, the systemic toxic and side effects of the drug are reduced, and the co-delivery of the two nano-drugs can synergistically enhance the treatment effect.

Preferably, the immunoadjuvant comprises a Toll-like receptor 7 agonist (e.g., imiquimod R837), a Toll-like receptor 7/8 agonist (e.g., raschimod R848), or a Toll-like receptor 9 agonist (e.g., unmethylated oligodeoxynucleotide CpG ODN).

Further, the immune checkpoint inhibitor is an IgG antibody.

Preferably, the immune checkpoint inhibitor may be selected from one or more of IgG antibodies such as an anti-apoptotic receptor 1 antibody (i.e. agd-1) or an anti-apoptotic receptor-ligand 1 antibody (i.e. agd-L1).

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

More preferably, the neutral phospholipid comprises palmitoyl choline, distearoyl choline, dimyristoyl phosphatidylcholine, or phosphatidylcholine.

More preferably, the electronegative phospholipid comprises phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, or distearoyl phosphatidylethanolamine-polyethylene glycol.

More preferably, the electropositive phospholipid comprises a phospholipid stearamide cholesterol derivative. Generally positively charged phospholipids are substantially synthetic.

More preferably, the phospholipid-polymer conjugate comprises distearoyl phosphatidylethanolamine-polyethylene glycol, distearoyl phosphatidylcholine-polyethylene glycol, or dipalmitoyl phosphatidylcholine-polyethylene glycol.

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

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

Preferably, the base material of the needle portion and the base portion 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 immunoadjuvant to the liposome material is 1:10-20.

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

Further, the loading amount of the immunoadjuvant in the soluble micro needle is 5-15 mug per square centimeter.

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

The invention also provides a preparation method of the soluble microneedle for co-loading the immune adjuvant and the immune checkpoint inhibitor, which specifically comprises 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 microneedle step by step through a microneedle mould, wherein the loading sequence of the lipid nano-drug and the chitosan gel drug is not limited; if the chitosan nanogel drug is loaded first and then the lipid nanogel drug is loaded, the electronegative needle tip substrate can be adsorbed on the surface of the chitosan nanogel drug through electrostatic interaction, so that the density and the size of the substrate can be increased, the substrate can be gathered to the tip of the microneedle under the action of centrifugal force, the diffusion of the drug to the microneedle substrate is avoided, the drug loading rate of the microneedle is further improved, the waste of expensive drugs is reduced, and the treatment effect on tumors is exerted to the greatest extent; if the lipid nano-drug is loaded firstly and then the chitosan nano-gel drug is loaded, the substrate can also increase the density of the chitosan nano-gel drug when the substrate is injected to prepare a needle body, and if the substrate is an electronegative material, the substrate can be attached to the surface of the chitosan nano-gel drug through electrostatic interaction, so that the drug is prevented from diffusing to a microneedle substrate.

S3, sequentially preparing a needle body part and a base part on the needle point part obtained in the step S2.

The problem of low density exists in both small molecular medicines and antibodies, the medicines are difficult to gather to the needle tip through centrifugation in the preparation process of the micro-needle, and even the medicines reaching the needle tip are easy to diffuse to the back substrate of the micro-needle and scratch, so that the medicine carrying rate of the micro-needle is limited, the waste of expensive medicines is caused, and the treatment effect of tumors is finally affected. Through a great deal of research in the early stage, the applicant creatively discovers that in the process of preparing the soluble microneedle of the co-loaded immune adjuvant and the immune checkpoint inhibitor, the immune adjuvant is encapsulated in liposome to obtain lipid nano-medicament, the immune checkpoint inhibitor is loaded in chitosan nano-gel to obtain chitosan nano-gel medicament, and the chitosan nano-gel medicament is loaded on the needle point part of the soluble microneedle in a stepping way and the matrix material used subsequently can further increase the density of the liposome, so that the medicament is beneficial to being accumulated on the needle point of the microneedle and not easy to diffuse, thereby effectively improving the medicament carrying rate of the microneedle.

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

Preferably, the cross-linking agent adopted by the ionic gel method is sodium tripolyphosphate.

In step S2, the nano-drug and the base material of the needle point part are prepared into a solution, and then the solution is solidified to obtain the needle point part, wherein the concentration of the base material of the needle point part is 50-120 mg/mL, and the concentration of the nano-drug (the total concentration of the two drugs) is 1-4 mg/mL.

Preferably, the concentration of the nano-drug is 2mg/mL.

Preferably, when the chitosan nanogel drug is firstly loaded on the needle tip part of the soluble microneedle and then the lipid nanogel drug is loaded, the chitosan nanogel drug has higher mechanical strength and is beneficial to penetrating the skin. Compared with liposome, the chitosan nano gel has greatly increased density, and is more beneficial to being deposited at the bottom of the needle point; meanwhile, the invention also uses a multi-step centrifugation method, the lipid nano-drug mixed with the needle point substrate solution (negatively charged) is added after the chitosan nano-gel is solidified, the electronegative needle point substrate solution can be adsorbed on the surface of the chitosan through electrostatic interaction, the density and the size of the chitosan nano-gel drug are increased, the chitosan nano-gel drug is gathered to the needle point of the micro-needle under the action of centrifugal force, the drug is prevented from diffusing to the base of the micro-needle, and the availability of the expensive immune checkpoint inhibitor antibody is further ensured.

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

Further, in step S3, the substrate concentration of the needle portion is 300 to 400mg/mL, and the substrate concentration of the base portion is 300 to 400mg/mL. The preparation method specifically comprises the following steps: a needle portion is prepared on the needle tip portion and a base portion is prepared on the needle portion.

Further, the needle tip, the needle body and the base are respectively arranged in a microneedle mould, and are obtained by centrifugation and solidification; wherein the rotational speed of the centrifugation is 3000-10000 rpm, and the time is 5-30 min.

The invention aims to solve the problems that the drug delivery load of the micro-needle is small and the drug cannot be concentrated on the tip of the micro-needle, and the micro-needle tip and the needle body are prepared by a two-step method.

The invention also protects the application of the soluble microneedle of the co-loaded immune adjuvant and immune checkpoint inhibitor in preparing medicines or medical appliances for treating cancers.

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

The invention has the following beneficial effects:

the invention provides a soluble microneedle for co-loading an immune adjuvant and an immune checkpoint agonist, which is characterized in that liposome is used for encapsulating the immune adjuvant to form a lipid nano-drug, and chitosan is used for loading an immune checkpoint inhibitor to form a chitosan nano-gel drug; the obtained two nano-drugs are further loaded on the tip part of the soluble microneedle step by step, 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 of focus, and the co-delivery of two nano drugs can synergistically enhance the killing function of the immune system of the organism on tumor cells; meanwhile, the dosage of the medicine can be reduced, and systemic toxic and side effects are avoided.

Drawings

FIG. 1 is a data statistical graph of particle sizes of RNPs and αNPs nano-drugs in examples 1 and 2.

FIG. 2 is a data statistical graph of surface potential of RNPs and αNPs nano-drugs in examples 1 and 2.

FIG. 3 is a transmission electron microscope scan of RNPs nano-drug according to example 1.

Fig. 4 is a transmission electron microscope scan of an αnps nano-drug of example 2.

FIG. 5 is a scanning electron microscope image of the soluble microneedle of example 3.

Fig. 6 is a hand-held microscope image of the soluble microneedle of example 3.

Fig. 7 is a data statistical chart of drug loading test results of the soluble microneedles of example 4.

FIG. 8 is a statistical plot of the survival of different concentrations of R848, RNPs and RNP@DMN micro-target DC2.4 cells loaded with RNPs in example 5.

Fig. 9 is a solid view of tumor volume recordings in example 6.

Fig. 10 is a statistical plot of the data for tumor volume change in example 6 (three asterisks) representing p < 0.001, indicating that there is a very significant statistical difference).

FIG. 11 is a statistical graph of the weight change of mice in example 6.

FIG. 12 is a graph of HE staining of the heart, liver, spleen, lung and kidney of the mice in example 6.

FIG. 13 is a data statistics of liver and kidney function parameters (ALT, BUN, TBIL, CREA) of the mice in example 6.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

PC-98T, high purity egg yolk lecithin, available from Ai Weita (Shanghai) pharmaceutical technologies Co., ltd;

chol, cholesterol, available from Ai Weita (Shanghai) pharmaceutical technologies Co., ltd;

DSPE-mPEG2000: namely phosphatidylethanolamine distearate-polyethylene glycol 2000, available from Ai Weita (Shanghai) pharmaceutical technologies.

EXAMPLE 1 Synthesis of lipid nanodrug RNPs encapsulating immunoadjuvants (exemplified by R848)

The preparation method of the lipid nano-drug RNPs encapsulating the immune adjuvant specifically comprises the following steps:

SI-1, 20mg of liposome membrane material (PC-98T: chol: DSPE-mPE2000=10:5:1, molar ratio) and 2mg of R848 were dissolved in 15mL of chloroform, poured into a 250mL eggplant-shaped bottle, and chloroform was removed by rotary evaporation (35 ℃,135rpm,20 min) to obtain a uniform lipid membrane;

SI-2, adding 4mL of pure water into an eggplant-shaped bottle, and peeling the lipid membrane under ultrasound;

SI-3, obtaining the lipid nano-drug RNPs by probe ultrasound (130 w,20kHz,20 min).

The particle size, potential, surface morphology, etc. of RNPs nanoparticles were measured and the results are shown in fig. 1 to 3. As can be seen from the graph, the hydrated particle size of the RNPs nano-particles is about 85nm, the zeta potential is about-27 mV, and the transmission electron microscope image shows that the RNPs have a membrane structure with a cavity, and the particle size after drying and collapsing is about 60 nm.

EXAMPLE 2 Synthesis of immune checkpoint inhibitor loaded Chitosan nanogel drug alpha NPs (exemplified by aPD-1)

The preparation method of the chitosan nanogel drug alpha NPs loaded with the immune checkpoint inhibitor specifically comprises the following steps:

SII-1, dissolving chitosan in 2% acetic acid water solution, wherein the mass concentration is 2.5mg/mL; the aPD-1 was dissolved in 1 XPBS at a mass concentration of 2mg/mL, and sodium Tripolyphosphate (TPP) was dissolved in pure water at a mass concentration of 0.25mg/mL.

Under the conditions of SII-2 and 4 ℃, the aPD-1 solution and the TPP solution are uniformly mixed, and are dropwise added into the chitosan solution under the condition of high-speed vortex (1500-3000 rpm) for reaction for 2 hours. The mass ratio of the aPD-1 to the chitosan is 1:1.

SII-3, centrifuging the solution obtained in SII-2 at high speed (10000 rpm) for 15min, discarding supernatant, and dispersing the lower solid with equal amount of pure water to obtain chitosan nanogel drug alpha NPs loaded with immune checkpoint inhibitor.

The characteristics of the alpha NPs nanoparticle such as particle diameter, potential, surface morphology and the like were measured, and the results are shown in FIGS. 1 to 2 and FIG. 4. As can be seen from the graph, the hydrated particle size of the alpha NPs nano particles is about 94nm, the zeta potential is about 46mV, and the transmission electron microscope graph photographed after the alpha NPs are dyed by uranium acetate shows that the particle size of the alpha NPs nano particles after the alpha NPs nano particles are dried and collapsed is about 60 nm.

EXAMPLE 3 preparation of soluble microneedles co-loaded with immune adjuvant and immune checkpoint blocker

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

SIII-1, pouring 100 mug of the alpha NPs nano particles obtained in the embodiment 2 into a microneedle mould, centrifuging for 5min at a rotating speed of 4000rpm, centrifuging for 5min again at a rotating speed of 180 ℃, scraping off redundant liquid, centrifuging for 30min, and drying until the solution is solidified;

SIII-2, 2.5mg of RNPs nanoparticle obtained in example 1 was mixed with 70mg/mL aqueous Hyaluronic Acid (HA) solution; pouring the HA solution containing RNPs into a microneedle mould with the needle tip part solidified with alpha NPs, centrifuging for 5min at 4000rpm, centrifuging for 5min again at 180 DEG, scraping off excessive liquid, centrifuging for 30min, drying until the solution is solidified, and taking the solution and the alpha NPs as the needle tip part;

SIII-3, preparing 300mg/mL HA solution, pouring the prepared HA solution into a microneedle mould, centrifuging for 5min at a rotating speed of 4000rpm, centrifuging for 5min again at a rotating speed of 180 ℃, scraping off redundant liquid, centrifuging for 30min, and drying until the solution is solidified to obtain a needle body part;

SIII-4, preparing 300mg/mL PVP K90 aqueous solution, pouring the prepared PVP K90 aqueous solution into a microneedle mould, maintaining vacuum negative pressure for 15min, extracting bubbles, scraping out the bubble-containing solution, adding a new PVP K90 aqueous solution, filling the whole mould, drying until the solution is solidified, and taking the solution as a base part to obtain the soluble microneedle carrying the immune adjuvant and the immune checkpoint inhibitor together;

wherein, the adopted microneedle mould is a PDMS microneedle mould, the morphological parameters are needle height 1200 mu m, base diameter 300 mu m, needle tip diameter less than 10 mu m, and array number more than 12×12.

Characterization is carried out on the morphology of the obtained soluble micro-needles loaded with the immune adjuvant and the immune checkpoint inhibitor, and the characterization results are shown in figures 5-6.

Example 4 drug load testing of soluble microneedles

(1) Preparation of soluble microneedles (RNP@DMN) carrying lipid nanodrug RNPs only

Taking 2.5mg of RNPs nano-drug obtained in the example 1, and uniformly mixing with 70mg/mL of Hyaluronic Acid (HA) aqueous solution; pouring the HA solution containing the RNPs into a microneedle mould, centrifuging for 5min at 4000rpm, rotating for 180 DEG, centrifuging for 5min again, scraping off redundant liquid, centrifuging for 30min, and drying until the solution is solidified to be used as a needle point part;

and preparing the soluble microneedle RNP@DMN by the rest steps and parameters according to reference example 3.

(2) Preparation of soluble microneedle (alpha NP@DMN) only loaded with chitosan nanogel drug alpha NPs

Taking 100 mug of the alpha NPs nano particles obtained in the example 2, pouring the alpha NPs nano particles into a microneedle mould, centrifuging for 5min at the rotating speed of 4000rpm, centrifuging for 5min again at the rotating speed of 180 DEG, scraping off redundant liquid, centrifuging for 30min, and drying until the solution is solidified to be used as a needle body part;

and preparing the rest steps and parameters in reference example 3 to obtain the soluble micro-needle alpha NP@DMN.

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

Ensuring that the addition amount of R848 is the same as the R848 loaded by RNP weighed in the preparation process of the RNP@DMN in the preparation process of the R848@DMN, dissolving a proper amount of R848 in 1mL of chloroform through conversion, and uniformly mixing with 70mg/mL of Hyaluronic Acid (HA) aqueous solution; pouring the HA solution containing R848 into a microneedle mould, centrifuging for 5min at 4000rpm, rotating for 180 degrees, centrifuging for 5min again, scraping off redundant liquid, centrifuging for 30min, and drying until the solution is solidified to be used as a needle point part;

and the soluble microneedle R848@DMN can be obtained by preparing the rest steps and parameters in reference example 3.

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

Ensuring that the addition amount of the aPD-1 in the preparation process of the aPD-1@DMN is the same as that of the aPD-1 loaded by the alpha NPs weighed in the preparation process of the RNP@DMN, dissolving a proper amount of the aPD-1 in 1mL of 1 xPBS through conversion, pouring into a microneedle mould, centrifuging for 5min at the rotating speed of 4000rpm, centrifuging for 5min again at the rotating speed of 180 ℃, scraping off redundant liquid, centrifuging for 30min, and drying until the solution is solidified to be used as a needle body part;

and preparing the rest steps and parameters in reference example 3 to obtain the soluble microneedle aPD-1@DMN.

(5) Drug loading test of soluble microneedles:

scraping the soluble microneedles obtained in the steps (1) - (4) from the microneedle patch by using a surgical blade, collecting, weighing, dissolving with a proper amount of ultrapure water, diluting the dissolved solution to a proper concentration by using DMSO, and measuring the concentration of R848 in the microneedles by using a high performance liquid chromatography-ultraviolet detector; the same method is adopted to collect the aPD-1, and the drug loading amount of the aPD-1 is measured by an IgG ELISA kit.

As shown in fig. 7, it is clear from the graph that the loading rate of the soluble microneedle with the co-supported immune adjuvant and immune checkpoint inhibitor, which were obtained by encapsulating R848 in a liposome, loading the agd-1 in a chitosan nanogel, and then loading the soluble microneedle, was significantly improved compared with R848 and agd-1, which were directly loaded on the soluble microneedle without encapsulation.

EXAMPLE 5 Effect of varying concentrations of R848, RNPs and soluble microneedle αNP-RNP@DMN loaded with RNPs on DC2.4 cell viability

Soluble microneedles loaded with RNPs (rnp@dmn) were prepared by the method of reference example 4, and the effect of different concentrations of R848, RNP, and soluble microneedles loaded with RNPs (rnp@dmn) on DC2.4 cell viability was tested using the MTT method, respectively.

As shown in FIG. 8, it is clear from FIG. 8 that the unencapsulated R848 has increased toxicity to DC2.4 cells (target cells of R848) with increasing concentration, but has an effect of promoting proliferation of DC2.4 cells when encapsulated in liposomes or loaded into microneedles. Encapsulation of the liposomes demonstrated reduced toxicity of R848 to DC2.4 cells.

EXAMPLE 6 evaluation of the therapeutic Effect and safety of soluble microneedles co-loaded with immune adjuvant and immune checkpoint inhibitor on tumors

1. Experimental materials:

a murine triple negative breast cancer 4T1 cell line; BALB/c mice of 4-6 weeks old were purchased from animal experiment center in Guangdong province. All animal procedures were conducted following the university of Zhongshan "laboratory animal care and use guidelines". All experiments involving animals strictly followed the national animal management regulations (revised in 1988, 2017) and the national guidelines for laboratory animal humane treatment (MOST 2006).

2. The experimental method comprises the following steps:

modeling a tumor animal model: the back hair of the mice was removed, and 100. Mu.L (1X 10) of 4T1 breast cancer cell suspension was used ⁶ Individual) were injected into the back of mice, and the mice were divided into 5 groups, which were 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 i.t.), and co-loaded RNPs and αnps microneedle group (αnp-rnp@dmn), respectively. Building constructionThe mice body weight and tumor volume were recorded every two days after 7 days in the model with different dosing treatments according to the different groups. The results are shown in FIGS. 9 to 11.

The toxic and side effects of the drug are clarified by HE staining of mice heart, liver, spleen, lung and kidney, and the staining results are shown in FIG. 12; meanwhile, liver and kidney functions of the mice were detected by a mouse biochemical analyzer, and the results are shown in fig. 13.

3. Experimental results:

as can be seen from fig. 9 to 13, the cancer inhibiting effect of the combined administration group of the immune adjuvant and the immune checkpoint inhibitor is obviously better than that of the control group and the single administration group, and the αnp-rnp@dmn group administered by the microneedle has better curative effect than that of the αnp+rnp i.t. group injected with the mixed drug solution in the tumor; the αnp-rnp@dmn group tumors began to show a tendency to become smaller at the tenth day of dosing compared to the other groups with continuously rising tumor sizes. In addition, the body weight of each group of mice is not obviously reduced, the biochemical indexes of the liver and the kidney are in the normal range, no obvious difference exists among the groups, and the H & E sections of the heart, the liver, the spleen and the kidney of each group do not show obvious damage, so that no obvious toxic or side effect is proved by the administration of the micro-needle. The H & E section of the lung in fig. 12 shows that the triple negative breast cancer of the mouse causes fibrosis and thickening of the lung interstitial mass, which threatens the life and health of the mouse, and the treatment of the αnp+rnp i.t. group and the αnp-rnp@dmn group can effectively inhibit the pulmonary fibrosis of the mouse and maintain the normal functions of the pulmonary organs of the mouse.

According to the above results, it can be demonstrated that the soluble microneedle co-loaded with the immune adjuvant and the immune checkpoint inhibitor has a remarkable tumor inhibition effect, shows no toxicity to normal organs, and can maintain the normal morphology of the mouse lung.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

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:

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;

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.