Multi-layer covered stent for unilateral controlled release of immune drugs and preparation method and application of multi-layer covered stent

Abstract

The invention discloses a multi-layer covered stent for unilateral controlled release of immune drugs and a preparation method and application thereof. The multi-layer covered stent comprises a substrate of a stent covered with an inner layer membrane, and the substrate is sequentially covered with a middle layer drug-loaded membrane and a blank pore membrane. The multi-layer covered stent is stable in structure and uniform in membrane layer distribution, the membrane layers and the stent are firmly combined, after the stent enters a tumor lesion site, medicine on the surface of the middle-layer medicine-carrying membrane is rapidly released to meet the treatment concentration, and after the outer-layer blank pore membrane absorbs lesion exudate, fiber network fusion occurs to generate small pores, so that the treatment effect is improved. The drug released by the middle-layer drug-loading membrane passes through pores of the outer-layer membrane at a relatively stable rate, and finally, a small amount of drug is slowly released at a stable rate along with degradation of the inner-layer membrane material, so that immune drugs and radiotherapy can form a continuous and stable synergistic treatment effect at a tumor part due to the zero-order release behavior; the tumor microenvironment immunity and the far-isolation effect are obviously improved, and the radiotherapy and immunity combined treatment of the malignant tumor accompanied by lumen obstruction is more efficiently realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a single-side controlled-release immune drug multilayer coated stent and a preparation method and application thereof. BACKGROUND

[0002] Radiotherapy is a tumor treatment method that kills or inhibits the growth and division of cancer cells through high-energy ionizing radiation, and is divided into internal irradiation and external irradiation, which can be used alone or in combination with other treatment methods.

[0003] Tumor immunotherapy drugs activate or enhance the ability of the human immune system to recognize and kill tumor cells, such as immune checkpoint inhibitors and cytokine drugs. Radiotherapy combined with local immunotherapy helps to produce immune regulation of the tumor microenvironment and systemic immunotherapy of non-irradiated lesions, and avoids the toxic side effects of systemic administration of tumor immunotherapy drugs.

[0004] Tumor obstruction refers to a pathological state in which the cavity organs or duct systems (such as the intestinal tract, bile duct, urethra, etc.) are blocked due to tumor growth or metastasis, causing dysfunction of the corresponding organs. Stent implantation is an important minimally invasive method for treating lumen obstruction, and drug-loaded covered stents cover a variety of polymer materials on the surface of bare stents to prevent tumor growth from penetrating into the stent interior to form new stenosis or occlusion, while achieving local drug release. If an immune drug-loaded covered stent is combined with external radiation therapy, or an immune drug-loaded covering is applied to the surface of an internal irradiation radioactive stent, multiple synergistic effects of relieving lumen obstruction, radiotherapy for malignant tumors, immune regulation of the tumor microenvironment, and systemic immunotherapy of non-irradiated lesions can be achieved.

[0005] The drug release time of the drug-loaded covered stent is short and unstable at present. For example, CN 107899093 A discloses a single-layer drug-containing nanofiber film coated on the surface of the stent by using electrospinning technology, which contains rapamycin and polylactic acid-glycolic acid copolymer (PLGA). The gradual degradation of the same molecular weight range PLGA in the body is used to achieve slow release of the drug. However, the electrospinning drug-containing film cannot avoid the adsorption of drug crystals on the surface of the nanofiber, which will cause obvious drug burst release after the stent is implanted in the body. CN 107496996 A discloses a multi-layer drug-containing film coated on the surface of the stent by using coating and impregnation technology, which contains PLGA of different molecular weights. The outer layer film carrier is low molecular weight PLGA 2000, and the middle layer film carrier is high molecular weight PLGA 95800. This multi-layer structure uses different molecular weight PLGA with different degradation times and release rates to achieve slow release of the drug by combining different release rates. However, the outer layer of low molecular weight PLGA has a rapid degradation behavior, which causes the loaded drug to be released quickly, and then the exposed middle layer of high molecular weight PLGA slowly degrades and releases the drug. This multi-phase release behavior of releasing the drug in front and back cannot meet the long-term stable blood drug concentration in the body. CN 113274557 A uses electrospinning technology to coat a multi-layer drug-containing film with one-sided drug release capability on the surface of the stent, wherein the drug-containing layer is the middle layer and the outer layer. The middle layer is a drug-loaded hydrophilic polyvinylpyrrolidone film, and the outer layer is a drug-loaded hydrophobic polycaprolactone / hydrophilic gelatin composite film. This multi-layer structure uses the hydrophilic and hydrophobic properties of the outer layer and the middle layer to regulate the drug release rate. Although it has a certain slow release capability, it also faces the phenomenon of burst release of the drug adsorbed on the surface of the outer layer (the cumulative release rate on the first day exceeds 50%), and the drug is released very slowly from the outer layer of the hydrophilic and hydrophobic composite film after the burst release ends (the cumulative release rate from the first day to the seventh day is less than 15%). The release curve after the superposition of the two release behaviors is poor in stability, and cannot guarantee the maintenance of effective therapeutic concentration.

[0006] Therefore, for the drug-loaded covered stent used for tumor treatment with lumen obstruction, the duration of the therapeutic window of the drug in the above-mentioned drug loading methods cannot match the half-life of the radioactive source, and a long-term and stable immune drug delivery strategy needs to be established to compensate for the decay of the late radiotherapy dose and improve the efficiency of immunogenicity utilization. SUMMARY

[0007] The purpose of the present application is to solve the problem of insufficient synergy between the instantaneous activity of immunotherapy drugs and radiotherapy during tumor interventional therapy. The present application provides a multi-layer covered stent for one-sided controlled release of immunotherapy drugs. The stent is a multi-layer nanofiber coating stent that can achieve the continuous half-month controlled release of immunotherapy drugs at a near constant speed. The stent can match the radiotherapy window, prolong immunogenicity stimulation, and enhance local and systemic anti-tumor response, which is used for efficient interventional therapy of malignant tumors with lumen obstruction combined with radiotherapy and immunotherapy.

[0008] The application further provides a preparation method of the single-side controlled-release immunodrug multilayer film covered stent and application thereof.

[0009] Technical scheme: In order to achieve the above-mentioned purpose, the single-side controlled-release immunodrug multilayer film covered stent provided by the application comprises a stent base covered with an inner layer film, and the stent base is sequentially covered with a middle layer drug-loaded film and an outermost blank porous film.

[0010] The material of the inner layer film is medical silicone rubber, polytetrafluoroethylene, polyurethane, polyester, or organic silicone polyurethane copolymer.

[0011] The material of the middle layer drug-loaded film and the outermost blank porous film is a mixed material composed of polymer A and polymer B, wherein the polymer A is any one or a mixture of multiple of polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), hydroxypropyl methyl cellulose (HPMC), and carboxymethyl cellulose (CMC), and the polymer B is poly (lactic-co-glycolic acid) (PLGA).

[0012] The stent is a net-shaped stent, a winding type stent, a tubular stent, a ring-shaped stent, or a tube-net-shaped stent, and the material of the stent is a metal material, a biodegradable material, or a mixture of the two.

[0013] The stent is radiated by one or more of radionuclide coating, neutron radiation activation, and loading of radioactive materials.

[0014] The middle layer drug-loaded film is loaded with an immunodrug, and the immunodrug comprises a mixture of one or more of an immune checkpoint inhibitor or a cytokine.

[0015] The immunodrug is any one or a mixture of multiple of an immune checkpoint inhibitor PD-1 / PD-L1 mAb, CTLA-4 mAb, an interferon, and an interleukin, the polymer A is any one or a mixture of multiple of a polymer with a molecular weight of 4×10-1.5×10 3 kDa, the PLGA has a molecular weight of 5×10-1×10 2 kDa, and the molecular ratio of lactic acid to glycolic acid is any one or a mixture of multiple of 75:25-50:50. The PLGA is a block copolymer composed of lactic acid and glycolic acid, the proportions of the two vary in different types of PLGA, and the proportions of the two have a great influence on the properties of the material.

[0016] As a preference, the polymer type A of the middle layer drug-loaded film is PEO, and the molecular weight is 1×10 2 -8×102 kDa; the PLGA has a molecular weight of 5 x 107.5 x 10 kDa, and the molecular ratio of lactic acid to glycolic acid is 50:50; preferably the outer layer of the porous membrane polymer of type A is PEO, and the molecular weight is 5 x 105 2 - 1 x 10 3 kDa; the PLGA has a molecular weight of 7.5 x 107.5 x 10 kDa, and the molecular ratio of lactic acid to glycolic acid is 75:25. 2

[0017] The preparation method of the single-side controlled-release immunodrug multilayer coated stent according to the present application comprises the following steps:

[0018] (1) dissolving polymer A and polymer B in a solvent, and adding an immunodrug in the middle layer drug-loaded membrane solution to prepare the middle layer drug-loaded membrane solution and the outer layer blank porous membrane solution, respectively;

[0019] (2) first electrospinning the middle layer drug-loaded membrane solution on the inner layer membrane stent to form a coating, and then electrospinning the outer layer blank porous membrane solution on the stent with the middle layer membrane to form a coating;

[0020] (3) vacuum drying the stent with the outer layer membrane to obtain the single-side controlled-release immunodrug multilayer coated stent.

[0021] In the present application, the mass ratio of polymer A to polymer B in the middle layer drug-loaded membrane solution is 8:1-2:1, and the mass fraction of polymer A and polymer B in the middle layer drug-loaded membrane solution is 3-8%; the mass ratio of polymer A to polymer B in the outer layer blank porous membrane solution is 1:1-1:8, and the mass fraction of polymer A and polymer B in the outer layer blank porous membrane solution is 5-10%; and the mass ratio of the middle layer drug-loaded membrane to the outer layer blank porous membrane is 1:2-4:1.

[0022] Further, the solvent is a mixture of dimethylformamide and N-methyl pyrrolidone, and the ratio is 1:5-5:1.

[0023] In the present application, the spinning parameters of the electrospinning of the middle layer drug-loaded membrane are as follows: the voltage is 15-18 kV, the needle type is 20-22G, the injection speed is 0.25-0.6 ml / h, the spinning distance is 5-10 cm, and the receiving roller speed is 80-120 rpm; the spinning parameters of the electrospinning of the outer layer blank porous membrane are as follows: the voltage is 18-20 kV, the needle type is 21-24G, the injection speed is 0.4-1.2 ml / h, the spinning distance is 5-10 cm, and the receiving roller speed is 80-120 rpm; and the electrospinning environment temperature is 18-25℃, and the humidity is 45-50%.

[0024] ​The application provides a single-side controlled-release immunodrug multilayer coated stent and a preparation method thereof.

[0025] As preferred, the single-side controlled-release immunodrug multilayer coated stent is prepared from a nickel-titanium memory alloy single wire in a diamond structure as a metal main body, and is prepared by means of radionuclide coating, neutron radiation activation or embedding radioactive materials in a groove, a sleeve or other structures.

[0026] Further, the specific operation steps are as follows:

[0027] Step (1) preparing a middle-layer drug-loaded membrane solution containing an immunodrug;

[0028] Step (2) preparing an outer-layer blank porous membrane solution;

[0029] Step (3) embedding the nickel-titanium alloy braided stent coated with a medical silicone rubber membrane into a receiving roller, and electrospinning the middle-layer drug-loaded membrane solution prepared in step (2) into a needle cylinder to obtain a stent coated with the middle-layer drug-loaded membrane;

[0030] Step (4) electrospinning the outer-layer blank porous membrane solution prepared in step (3) into the needle cylinder to continue electrospinning on the surface of the stent coated with the middle-layer drug-loaded membrane prepared in step (4), and finally obtaining the single-side controlled-release immunodrug multilayer coated stent.

[0031] Further, in the step (2), the preparation process of the middle-layer drug-loaded membrane solution is as follows: polymer A and PLGA are added into a mixed solvent of dimethylformamide and N-methylpyrrolidone (4:1 in volume ratio), then the immunodrug is added, and stirring is performed at room temperature for 12 h to obtain the middle-layer drug-loaded membrane solution.

[0032] Further, in the step (3), the preparation process of the outer-layer blank porous membrane solution is as follows: PLGA and polymer A are added into a mixed solvent of dimethylformamide and N-methylpyrrolidone (4:1 in volume ratio), and stirring is performed at room temperature for 12 h to obtain the outer-layer blank porous membrane solution.

[0033] The mass ratio of the middle-layer drug-loaded membrane to the outer-layer blank porous membrane is 1:2-4:1; and the drug loading amount of the immunodrug in the coated membrane is 0.18-0.36 μg / mg.

[0034] Further, the single-side controlled-release immunodrug multilayer coated stent prepared by the preparation method is used in the radioimmunotherapy combined high-efficiency intervention treatment of malignant tumors with lumen obstruction.

[0035] The preparation and application mode of the single-side controlled release immunological drug multilayer coated stent of the application comprises: 1. coating the existing inner layer film stent with a drug-containing middle layer film and a blank outer layer film, implanting the stent into a tumor obstruction site, and cooperating with external irradiation to realize external radiotherapy and immunological combined treatment; 2. coating the existing inner layer film radiotherapy stent with a drug-containing middle layer film and a blank outer layer film, the radiotherapy stent can be endowed with radioactivity by one or more ways such as radionuclide coating, neutron radiation activation, and making recesses, sleeves and other structures to load radioactive materials, and the whole stent is implanted into a tumor obstruction site to realize internal radiotherapy and immunological combined treatment; 3. coating the existing inner layer film stent with a drug-containing middle layer film and a blank outer layer film, the radioactive materials are put into the recesses, sleeves and other structures, and then the whole stent is implanted into a tumor obstruction site to realize internal radiotherapy and immunological combined treatment.

[0036] The application discloses a single-side controlled release immunological drug multilayer coated stent and a preparation method and application thereof. The stent has radiotherapy activity and is coated with a medical silicone rubber film on the surface to realize single-side controlled release of the multilayer coated stent loaded with immunological drugs. In the application, the material used for the controlled release immunological drug multilayer coating is preferably a mixed material composed of polylactic acid-glycolic acid copolymer (PLGA) and polyethylene oxide (PEO), and the process used is electrospinning, wherein the middle layer is a drug-loaded film and the outer layer is a blank porous film. By adjusting the composition of the middle layer drug-loaded film and the blank porous film of the outer layer, the content ratio of different materials, and the mass ratio of the film layers, the single-side sustained near-zero release of the immunological drugs in the stent is realized. In the preparation of the single-side controlled release immunological drug multilayer coated stent, the cytokines and the PLGA / PEO mixed material are dissolved in a mixed solvent of dimethylformamide and N-methyl pyrrolidone to prepare solution A (middle layer drug-loaded film solution), and then the PLGA / PEO mixed material in different proportions is dissolved in the mixed solvent to prepare solution B (outer layer blank porous film solution); after the stent is fixed on a matching receiver, the stent surface is electrospun with solution A and solution B in sequence. In the application, the coated stent has stable structure, uniform film layer distribution, and firm combination of the film layer and the stent. When the multilayer coating loaded with immunological drugs enters a tumor lesion site along with the stent, the drugs on the surface of the middle layer drug-loaded film are first released rapidly to meet the treatment concentration, then the outer layer blank porous film absorbs the exudate of the lesion to cause fiber network fusion to generate small pores, the drugs released by the middle layer drug-loaded film pass through the pores of the outer layer film at a relatively stable rate, and finally a small amount of drugs are slowly released at a stable rate along with the degradation of the inner layer film material. The whole cycle of near-zero release behavior enables the immunological drugs and radiotherapy to form a sustained and stable synergistic treatment effect in the tumor site, significantly improves the tumor microenvironment immunity and the abscopal effect, and more efficiently realizes the radioimmunological combined treatment of malignant tumors with lumen obstruction.

[0037] The application prepares a single-side controlled-release immune drug multilayer coated stent, and the outer layer porous film of the stent forms pores in the medium to control drug release.

[0038] In the preparation process of the application, it is important to adjust the pores formed by the outer layer porous film in the medium, which can be controlled by controlling the proportion of different polymers in the film material, the thickness of the film, etc. A large number of experimental optimizations need to be carried out on the proportion of solvents, the solid content of the material in the solvent, the spinning parameters, etc. The drug dissolved from the inner layer film of the stent prepared in the application is released through the pores of the outer layer film, so the pores of the outer layer film are the key channels for controlling drug release. By adjusting the size of the pores of the outer layer film, the release behavior of the drug can be directly affected. The electron microscope images of the pores of the outer layer film at different time points in Example 2 of the application can directly show the changes in the pores of different formulations.

[0039] In addition, the effective treatment period of radioactive particles in radiotherapy is generally about half a month, and the immune drug prepared by the application is stably released in the treatment period of radiotherapy, which can enhance the immune treatment effect and induce sustained immune memory effect and distant effect.

[0040] Advantages: Compared with the prior art, the application has the following advantages:

[0041] 1. Unlike the strategy of adjusting the molecular weight and hydrophilic-hydrophobic ratio of the coating in the prior art, the application successfully realizes the sustained near-zero-order release of the immune drug in the stent by using the multilayer coating method of blank porous film and drug-loaded film. After the stent is implanted into the lumen, the lesion exudate in the lumen wall first infiltrates the outer layer blank porous film, and then infiltrates the middle layer drug-loaded film through the pores of the outer layer nanofiber. In the early stage of implantation, part of the drug adsorbed on the surface of the middle layer drug-loaded nanofiber needs to pass through the originally larger pores of the outer layer nanofiber network to infiltrate the lesion site, which avoids the direct burst release of the surface-adsorbed drug into the lesion; then under the action of the exudate, the outer layer nanofiber network fuses to form smaller pores, and the drug released from the middle layer drug-loaded nanofiber continuously and slowly passes through the smaller pores of the outer layer film, maintaining a stable release rate; finally, as the network structure of the outer layer porous film gradually degrades, although the pores of the outer layer film become larger again, a small amount of drug remaining in the middle layer drug-loaded film can pass through the outer layer porous film without obstacles, but the drug can also be stably released as the middle layer drug-loaded film slowly degrades, which supplements the defect of accelerated release caused by the increase in the size of the pores of the outer layer film in the later stage, and realizes the sustained near-zero-order release effect throughout the whole process.

[0042] 2. The drug-loaded coated stent of the application can release the drug at a sustained zero-order rate for half a month, the release is stable, the continuous release time matches the effective time of radiotherapy well, and the immune and distant effects of the tumor microenvironment can be greatly improved, so that the radioimmunotherapy for malignant tumors with lumen obstruction can be more efficiently realized.

[0043] 3、The prepared multilayer covered stent can fully utilize the curative effect advantages of tumor local drug delivery and combined treatment of radioimmunity, and can inhibit the lumen obstruction caused by the malignant growth of the lumen tumor, the preparation method of the covered stent is simple, quality control and industrial production are convenient, and the covered stent is convenient for clinical treatment and operation treatment of the clinician. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Scanning electron microscope images of in vitro degradation of outer blank porous membranes with different ratios (PEO:PLGA).

[0045] Figure 2 In vitro release curves of middle drug-loaded membranes with different ratios (PEO:PLGA).

[0046] Figure 3 In vitro release curves of drug-loaded multilayer covered stents prepared by outer blank porous membranes with different ratios (PEO:PLGA).

[0047] Figure 4 In vitro release curves of drug-loaded multilayer covered stents prepared by different mass ratios (middle drug-loaded membrane: outer blank porous membrane).

[0048] Figure 5 In vitro release curves of drug-loaded multilayer covered stents prepared by different mass ratios (middle drug-loaded membrane: outer blank porous membrane).

[0049] Figure 6 In vivo pharmacodynamic evaluation of the anti-tumor effect of mice and tumor immune microenvironment treatment.

[0050] Figure 7 In vivo pharmacodynamic evaluation of the anti-tumor distant effect of mice.

[0051] Figure 8 Results of the in vitro release of IL-12 of the comparative multilayer covered stent. DETAILED DESCRIPTION

[0052] The present application is further illustrated below in conjunction with the embodiments and the accompanying drawings.

[0053] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified. The experimental methods not specifically indicated in the examples are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

[0054] Since the immunological drugs are all water-soluble biological macromolecules, have similar physicochemical properties, and have similar solubility and release behavior in liquid environment, in the following specific examples 1-8, interleukin 12 (IL-12, novoprotein, CM39) is taken as a model drug for research.

[0055] The esophageal stent adopted in the embodiment of the present application is made of nickel-titanium alloy, and the film material is medical silicone rubber film. The specification model is MTN-SKE-G-S-20 / 80-B-8 / 650, the registration certificate number is Guojianzhunqing 20173130120, and the manufacturer is Nanwei Medical Science and Technology Co., Ltd. In the present application, the esophageal stent is used as a metal radiotherapy stent, the inner surface of the metal radiotherapy stent is covered with a medical silicone rubber film, and the outer surface is provided with a metal capsule (nickel-titanium alloy) for loading I 125 particles.

[0056] 125 I Particles, CIAE-6711, China Institute of Atomic Energy.

[0057] PLGA is purchased from Shandong Jinan Daigang Biological Engineering Co., Ltd., PEO is purchased from IFF Company in the United States, IL-12 is purchased from Proteintech in the United States, and DMF and NMP are purchased from Shanghai Maikelin Reagent Co., Ltd.

[0058] Electrospinning machine, Foshan Weimaikai Technology Co., Ltd., model MN60.

[0059] Example 1

[0060] Preparation of outer blank porous membrane with different proportions (PEO:PLGA)

[0061] An appropriate amount of PEO (molecular weight 1×10 3 kDa) and PLGA (molecular weight 7.5×10 kDa, molecular ratio of lactic acid to glycolic acid 75:25) with different proportions were added into a mixed solvent of dimethylformamide and N-methyl pyrrolidone (volume ratio 4:1) (solid content is shown in Table 1, the solution density in the example is calculated according to 1 g / cm 3 After stirring at room temperature for 12 h, electrospinning was performed, the whole preparation system was controlled at a temperature of 18-25℃ and a humidity of 45-50%, the receiving roller speed was 100 rpm, the spinning distance was 7 cm, and the prepared outer blank porous membrane was placed in a desiccator for storage. The specific prescription amount proportion, solid-liquid dosage and spinning process parameters are shown in Table 1.

[0062] Table 1. Prescription and process parameters for investigating the amount proportion of PEO and PLGA in the outer blank porous membrane

[0063]

[0064]

[0065] The four outer blank porous membranes prepared by the above method all have smooth surfaces without obvious granular protrusions.

[0066] Example 2

[0067] In-vitro degradation morphology observation of outer blank porous membranes with different proportions (PEO: PLGA)

[0068] To simulate the in-vivo degradation environment of the multilayer coated stent with unilateral controlled-release immunological drugs after intervention on the inner wall of the lumen, the outer blank porous membranes with prescriptions 1, 2 and 3 prepared in Example 1 were cut into appropriate sizes and placed on the membrane of a Fanz diffusion cell (completely covering the membrane), 6.5 mL of phosphate buffer solution (PBS, pH = 7.4) and 0.1% sodium azide were added into the bottom chamber, then the top chamber was sealed with a sealing film to prevent liquid evaporation, the upper and lower chambers were clamped with a clamp, and the diffusion cell was placed in a 37°C water bath with a magnetic stirring speed of 500 rpm. A small amount of sample was taken at 2h, 12h, 7d, 12d and 14d, dried and observed for surface morphology using a scanning electron microscope to observe the changes in porosity at different time points. The results are shown in Figure 1

[0069] As can be seen from Figure 1 , the nanofibers of prescription 1 remained independent for the first 7 days, and the fiber diameter decreased significantly at 12d. No obvious fiber network fusion occurred during the entire degradation period, and no effective physical barrier could be established for the inner layer drug. The nanofiber network of prescription 2 began to fuse at 2h, and at 12h, a large area of nanofiber network fusion was observed, the porosity was significantly reduced, a physical barrier was produced and maintained for 12 days. At 14d, it was found that the nanofiber network fusion occurred obvious erosion and fracture, the porosity increased significantly, and the physical barrier was lost, and the performance was the best. The nanofiber network of prescription 3 also began to fuse at 2h, but at 12h, the nanofiber network began to fracture, then the porosity gradually increased with the increase of fiber fracture degree, and the physical barrier was lost.

[0070] Example 3

[0071] Preparation of middle layer drug-loaded membranes with different proportions (PEO: PLGA)

[0072] An appropriate amount of PEO (molecular weight 5 x 10 2 ​The PEO (molecular weight 4.0 x 10 kDa) and PLGA (molecular weight 7.5 x 10 kDa, the molecular ratio of lactic acid and glycolic acid is 50:50) were added into the mixed solvent of dimethylformamide and N-methylpyrrolidone (volume ratio is 4:1), and an appropriate amount of IL-12 (the drug loading in the membrane is 0.2 ug / mg) was added, and after stirring at room temperature for 12 h, electrospinning was performed, the whole preparation system was controlled at a temperature of 18-25°C and a humidity of 45-50%, the receiving roller speed was 100 rpm, the spinning distance was 7 cm, and the prepared middle layer drug-loaded membrane was placed in a desiccator for storage. The specific prescription amount ratio and solid-liquid dosage and spinning process parameters are shown in Table 2.

[0073] Table 2. Prescription and process parameters for investigating the amount ratio of PEO and PLGA in the middle layer drug-loaded membrane

[0074]

[0075] The middle layer drug-loaded membranes of prescriptions 5, 6 and 7 prepared by the above method have smooth and flat surfaces, and the middle layer drug-loaded membrane of prescription 4 has small granular protrusions on the surface, which may be due to incomplete evaporation of the solution. Therefore, the middle layer drug-loaded membranes of prescriptions 5, 6 and 7 are selected for subsequent experiments.

[0076] Example 4

[0077] In vitro release of middle layer drug-loaded membranes with different ratios (PEO:PLGA)

[0078] In order to simulate the in vivo drug release environment of the single-sided controlled-release immunodrug multilayer coated stent after intervention on the lumen wall, the middle layer drug-loaded membranes of prescriptions 5, 6 and 7 were placed in the Fanz diffusion cell according to the method described in Example 2 (completely covering the entire membrane), 1.0 mL of sample solution was taken at a certain time interval, and then 1.0 mL of fresh medium was added. The concentration of IL-12 in the sample was determined using an Elisa kit, and the cumulative release rate was calculated by bringing in the standard curve, and the results are shown in Figure 2

[0079] Figure 2 ​The results show that the three kinds of middle layer drug-loaded films all present obvious burst release (>30%) in the initial release period (3h), and the burst release of the middle layer drug-loaded film of prescription 5 is the most obvious (>50%), which is due to the rapid dissolution of IL-12 on the surface of the fiber. Subsequently, the middle layer drug-loaded film of prescription 6 continuously releases drugs at a relatively fast rate, but the release rate of the middle layer drug-loaded film of prescription 7 is slow. After 120h, the small amount of drugs contained in the middle layer drug-loaded film of prescription 6 present a slow release behavior with a relatively stable rate, while the release rate of the middle layer drug-loaded film of prescription 7 obviously accelerates, which may be due to the accumulation of acidic fragments in the high content of PLGA molecules in the film material, which automatically catalyzes the degradation of the film material, thereby causing the release rate to accelerate. From the results of example 2, it can be known that the outer layer blank porous film will lose the physical barrier effect in the later period, thereby reducing the controlled release ability. In order to meet the requirement that the drug-loaded multilayer coated film maintains a stable release rate throughout the release period, the middle layer drug-loaded film of prescription 6 is selected for subsequent experiments.

[0080] Example 5

[0081] Preparation of drug-loaded multilayer coated films with different proportions (PEO:PLGA) of outer layer blank porous films and in vitro release investigation.

[0082] The metal radiotherapy stent is coated with a medical silicone rubber film on the inner surface and is provided with a metal capsule shell on the outer surface, which is used to load I 125 particles of the esophageal stent. First, the metal radiotherapy stent is fixed on the receiver of the electrospinning machine, and the composition and spinning conditions of the middle layer drug-loaded film of prescription 6 are prepared according to the description in example 3. The middle layer drug-loaded film solution is configured, and the middle layer drug-loaded film is formed by electrospinning the middle layer drug-loaded film solution on the surface of the stent. Then, the composition and spinning conditions of the outer layer drug-loaded film of prescriptions 1-3 are prepared according to the description in example 1. The outer layer drug-loaded film solution is configured, and the outer layer blank porous film is formed by electrospinning the outer layer drug-loaded film solution on the surface of the middle layer drug-loaded film. The middle layer film and the outer layer film are spun until they can cover the entire stent. The finally prepared drug-loaded multilayer coated film has the composition shown in table 3 (the solid mass ratio in the middle layer drug-loaded film solution and the outer layer blank porous film solution is 1:1). The prepared drug-loaded multilayer coated film is placed in a desiccator for storage.

[0083] Table 3 Composition of drug-loaded multilayer coated films prepared from middle layer drug-loaded films with different proportions (PEO:PLGA)

[0084]

[0085] The surface of the drug-loaded multilayer coated film of prescription 8, 9, 10 prepared according to the above method is smooth and flat, and has no obvious granular protrusions.

[0086] Then, to simulate the in-vivo drug release environment of the single-side controlled release immunotherapeutic drug-coated multilayer stent after intervention on the luminal wall, the drug-loaded multilayer coatings of Formulations 8, 9, and 10 were placed in Franz diffusion cells, with the outer blank porous membrane contacting the release medium on one side. Then, 1.0 mL of sample solution was taken at specific time intervals and 1.0 mL of fresh medium was added. The concentration of IL-12 in the sample was determined using an Elisa kit and a standard curve was used to calculate the cumulative release rate, and the results are shown in Table 2. Figure 3

[0087] Figure 3 The results show that the burst release phenomenon of the drug-loaded multilayer coatings of Formulations 8, 9, and 10 is significantly reduced (about 20%) compared to that of Formulation 2 at the initial stage (3 h). Subsequently, the drug-loaded multilayer coating of Formulation 10 shows approximately first-order release behavior, with almost complete release at 240 h, which is due to the fact that the nanofiber network of the outer blank porous membrane (Formulation 3) in Formulation 10 does not effectively fuse during the release process, and the loose nanofiber network allows the dissolved IL-12 in the middle layer to quickly pass through and be released. The drug-loaded multilayer coatings of Formulations 8 and 9 show approximately zero-order release behavior, and the release rate of Formulation 10 is significantly lower than that of Formulation 9, because although the drug-loaded multilayer coatings of Formulations 8 and 9 can quickly fuse the fiber network at the initial stage of release and form a physical barrier effect, the outer blank porous membrane (Formulation 1) in Formulation 8 has a faster degradation rate and poorer physical barrier stability, which accelerates the diffusion of the dissolved IL-12 in the middle drug-loaded membrane. In addition, the drug-loaded multilayer coatings of Formulations 8, 9, and 10 all slow down the release rate of the drug compared to the middle drug-loaded membrane of Formulation 6, indicating that the outer porous membrane in this multilayer membrane structure can effectively form a physical barrier to the drug released from the middle drug-loaded membrane, thereby enabling stable drug-controlled release on the surface of the lesion tissue.

[0088] Example 6

[0089] Preparation of drug-loaded multilayer coatings with different mass ratios (middle drug-loaded membrane: outer blank porous membrane) and in-vitro release investigation

[0090] According to the method in Example 5, the metal radiotherapy stent was fixed on the receiver of the electrospinning machine, and the middle drug-loaded membrane of Formulation 6 and the outer blank porous membrane of Formulation 2 were sequentially prepared on the surface of the stent according to the methods described in Example 3 and Example 1 (the mass ratio of the solid content in the middle drug-loaded membrane solution to the outer blank porous membrane solution used for spinning). The composition of the finally prepared drug-loaded multilayer coating is shown in Table 4. The prepared drug-loaded multilayer coating was placed in a desiccator for storage.

[0091] Table 4 Composition of drug-loaded multilayer coatings prepared with different mass ratios (middle drug-loaded membrane: outer blank porous membrane) ​

[0092]

[0093] The drug-loaded multilayer coatings of prescriptions 11, 12, and 13 prepared according to the above method have smooth and flat surfaces without obvious granular protrusions.

[0094] Then, as described in Example 6, the drug-loaded multilayer films of formulations 11, 12, and 13 were placed in a Fanz diffusion cell. 1.0 mL of sample solution was added at specific time intervals, followed by the addition of 1.0 mL of fresh medium. The concentration of IL-12 in the samples was determined using an ELISA kit, and the cumulative release rate was calculated by subtracting the values ​​from the standard curve. The results are as follows: Figure 4 As shown.

[0095] Figure 4 The results showed that drug-loaded multilayer membranes of formulations 11, 12, and 13 all exhibited a certain burst release phenomenon in the initial stage (3 hours), with formulation 13 showing a more pronounced burst release. Subsequently, formulation 13 exhibited near-first-order release behavior, achieving complete release at 240 hours. In contrast, formulations 11 and 12 exhibited near-zero-order release behavior, both achieving complete release at 336 hours. This may be because the thinner outer blank porous membrane is more easily wetted by the medium in the initial stage, and the shorter pore length of the fused fiber network allows the drug dissolved in the middle drug-loaded membrane to pass through the pores more easily and be released. When the outer blank porous membrane reaches a certain thickness, the fine pore structure of the fused longer fiber network can create a stable physical barrier for the drug dissolved in the middle drug-loaded membrane. At this point, the drug dissolved in the middle drug-loaded membrane passes through the outer porous membrane at a stable rate and is released. Therefore, formulation 12 was selected as the optimal drug-loaded multilayer membrane.

[0096] Example 7

[0097] Drug-loaded multilayer coatings prepared from different polymers A and their in vitro release study

[0098] Weigh out an appropriate amount of PVP (polyvinylpyrrolidone, molecular weight 4×10⁻⁶). 2 kDa), HPMC (hydroxypropyl methylcellulose, molecular weight 8×10⁻⁶ kDa), 2 kDa) and CMC (carboxymethyl cellulose, molecular weight 5×10 2The solids were mixed with PLGA (molecular weight 5 × 10 kDa, lactic acid to glycolic acid molecular ratio 50:50) in a mixed solvent of dimethylformamide and N-methylpyrrolidone (volume ratio 4:1) (solid content shown in Table 5). After adding IL-12 (IL-12 loading in the coating was 0.2 μg / mg), the mixture was stirred at room temperature for 12 h and then electrospun. The electrospinning environment was 18-25℃, humidity 45-50%, spinning distance 7 cm, and receiving roller speed 100 rpm. Then, an appropriate amount of PVP (molecular weight 1 × 10 kDa) was added. 3 kDa), HPMC (molecular weight 1.5 × 10⁻⁶) 3 kDa) and CMC (molecular weight of 1×10 3 The drug-loaded multilayer membranes (Mcglycine to Glycolic acid) were mixed with PLGA (molecular weight 7.5 × 10 kDa, molecular ratio of lactic acid to glycolic acid 75:25) in a mixed solvent of dimethylformamide and N-methylpyrrolidone (volume ratio 4:1). After stirring at room temperature for 12 h, electrospinning was performed. The electrospinning environment was 18-25℃, humidity 45-50%, spinning distance 7 cm, and receiver roller speed 100 rpm. Following the method in Example 5, the metal radiotherapy stent was fixed to the receiver of the electrospinning machine. Specific formulations of drug-loaded multilayer membranes prepared from different polymers A on the stent surface are shown in Table 5, and specific process parameters are shown in Table 6. The final drug-loaded multilayer membrane had a middle drug-loaded membrane to an outer blank porous membrane mass ratio of 2:1.

[0099] Table 5. Specific formulations for drug-loaded multilayer coatings prepared from different polymers A.

[0100]

[0101] Table 6. Specific process parameters for drug-loaded multilayer coatings prepared from different polymers A

[0102]

[0103]

[0104] The drug-loaded multilayer coatings of prescriptions 14, 15, and 16 prepared according to the above method have smooth and flat surfaces without obvious granular protrusions.

[0105] The in vitro release of drug-loaded multilayer films prepared from different polymers A was investigated according to the method described in Example 6. The concentration of IL-12 in the samples was determined using an ELISA kit, and the cumulative release rate was calculated by subtracting the values ​​from the standard curve. The results are as follows: Figure 5 As shown.

[0106] The results show that the drug-loaded multilayered films prepared from three different kinds of polymer A (PVP, HPMC, CMC) and PLGA all have good control release ability for IL-12, and can release continuously for half a month. However, compared with the drug-loaded multilayered film prepared from PEO / PLGA of prescription 12, the control release ability is poorer, so the drug-loaded multilayered film with polymer A as PEO is preferred for subsequent in vivo pharmacodynamic evaluation.

[0107] Example 8

[0108] Preparation of the multilayered film stent of the optimal prescription 12

[0109] The metal radiotherapy stent is coated with a medical silicone rubber film on the inner surface and is provided with a metal capsule shell on the outer surface, which is used to load I 125 particles of the esophageal stent.

[0110] According to the method in Example 6, the multilayered film stent was prepared according to prescription 12, and the specific process was as follows:

[0111] The PLGA in the middle layer drug-loaded film solution has a molecular weight of 5x10 kDa, a molecular ratio of lactic acid to glycolic acid of 50:50, and a PEO molecular weight of 3x10 2 kDa. The PEO and PLGA were dissolved in the DMF / NMP mixed solution in a ratio of 4:1, and the total solid content was 6%. Then IL-12 was added (the drug loading amount in the film was 0.2 μg / mg).

[0112] The PLGA in the outer layer blank porous film has a molecular weight of 7.5x10 kDa, a molecular ratio of lactic acid to glycolic acid of 75:25, and a PEO molecular weight of 5x10 2 kDa. The PEO and PLGA were dissolved in the DMF / NMP mixed solution in a ratio of 1:2, and the total solid content was 9%.

[0113] After the intermediate drug-loaded membrane solution and the outer blank porous membrane solution are prepared, the metal radiotherapy stent is fixed to the receiver of the electrospinning machine. First, the intermediate drug-loaded membrane solution is electrospinned on the stent surface: the ambient temperature for electrospinning is 18-25℃, the humidity is 45-50%, the spinning voltage is 18kV, the injection rate is 0.5mL / h, the needle type is 21G, the receiver rotation speed is 100rpm, and the spinning distance is 7cm. After the intermediate drug-loaded membrane is prepared, the outer blank porous membrane solution is further electrospinned: the ambient temperature for electrospinning is 18-25℃, the humidity is 45-50%, the spinning voltage is 20kV, the injection rate is 0.95mL / h, the needle type is 24G, the receiver rotation speed is 100rpm, and the spinning distance is 7cm. The middle and outer membranes are spun until they can cover the entire scaffold. The final fabricated multilayer membrane radiotherapy scaffold with unilateral controlled-release immunodrug has a middle drug-loaded membrane to outer blank porous membrane mass ratio of 2:1, and the immunodrug (IL-12) loading in the membrane is 0.25 ug / mg. It is then stored at room temperature in a desiccant oven.

[0114] Before clinical use, the doctor gently punctures the membrane covering the capsule surface using a medical needle (>26 gauge) to insert... I 125 radioactive particles were placed into the capsule, and then interventional drug delivery was performed. Example 9 used a method involving the placement of... I The amount of 125 radioactive particles loaded into the scaffold is selected based on the tumor size, with a viability of 0.8-1.0. I One 125-particle device was surgically implanted into the tumor site. The cumulative radiation dose was calculated to be 4-6 Gy based on the experimentally set time points.

[0115] Example 9

[0116] The difference between stable (drug-loaded stent (Prescription 12)) and unstable (drug-loaded stent (Prescription 6), drug-loaded stent (Prescription 11)) release leads to differences in tumor microenvironment immunotherapy and its remote effects.

[0117] To elucidate the effects of drug-loaded scaffolds with different release behaviors on immunotherapy of the tumor immune microenvironment, a Hepa1-6 tumor-bearing mouse model was established and in vivo pharmacodynamic evaluation was performed. Five groups were established: a blank control group (blank), a group without an outer porous membrane (drug-loaded scaffold (Formula 6), prepared and spun on the scaffold according to the method in Example 3), a multilayer coated group with unstable release (drug-loaded scaffold (Formula 11), prepared and spun on the scaffold according to the method in Example 6), and a multilayer coated group with stable release (drug-loaded scaffold (Formula 12), prepared and spun on the scaffold according to the method in Example 8). After implantation at the tumor site, tumor volume was measured every 3 days and calculated using the following formula:

[0118]

[0119] wherein V(mm 3 ) represents the tumor volume, a(mm) and b(mm) represent the length and width of the tumor, respectively.

[0120] On the 15th day of treatment, 3 mice were randomly selected from each group for euthanasia, and then the expression levels of immune cells in the tumor tissue were quantitatively detected by flow cytometry, and the differences between multiple groups were compared by one-way ANOVA * p<0.05, ** p<0.01, *** p<0.001), and the results are shown in Figure 6 .

[0121] Figure 6 The results show that the tumor volume of the drug-loaded stent group is significantly reduced compared with the stent group, indicating that the immunotherapy and radiotherapy have a synergistic therapeutic effect. The immune cell level of the tumor microenvironment in the stent group has no obvious change compared with the blank group, while the immune cell level of the tumor microenvironment in the drug-loaded stent group is significantly increased, indicating that the immune drugs released in the drug-loaded coating can produce a synergistic therapeutic effect with radiotherapy by activating the tumor immune microenvironment. In addition, by comparing different drug-loaded stent groups, it can be found that the drug-loaded stent group (prescription 12) significantly improves the immune cell level of the tumor microenvironment and produces the best anti-tumor effect, which may be because the smoothly released immune drugs continuously act on the immune cells within the half-life period of the rays, and the matching action time of the two will produce the best tumor immune microenvironment.

[0122] Further, to clarify the influence of drug-loaded stents with different release behaviors on the abscopal effect of radioimmunotherapy, the above-mentioned 5 groups of experimental model mice were implanted with a second tumor lesion at a position away from the initial tumor at 60 days after drug administration, and then the volume of the second tumor was detected every 4 days, and the calculation formula is as above. On the 76th day of treatment, 3 mice were randomly selected from each group for euthanasia, and then the expression levels of immune cells in the spleen were quantitatively detected by flow cytometry, and the differences between multiple groups were compared by one-way ANOVA * p<0.05, ** p<0.01, *** p<0.001), and the results are shown in Figure 7 .

[0123] Figure 7The results show that the stent group has no obvious change in the second tumor volume compared with the blank group, while the drug-loaded stent group is significantly reduced, which indicates that the combination of radiotherapy and immunotherapy can produce a significant distant effect in vivo. The immune drugs released by the drug-loaded coated stent combined with radiotherapy can stimulate the expression of immune cells in the spleen tissue and activate the adaptive immune system in vivo, thereby producing anti-tumor activity. In addition, by comparing different drug-loaded stent groups, it can be found that the drug-loaded stent group (prescription 12) significantly improves the level of immune cells in the spleen tissue and produces the best distant effect, which may be because the steady release of immune drugs can maximize the immune stimulation within the half-life of the rays, and the immune cells produced can act on the spleen and activate the adaptive immune system in vivo, thereby producing a sustained distant effect.

[0124] Comparative Example 1

[0125] Preparation of IL-12-loaded multilayer coated stent and in vitro release investigation

[0126] The multilayer coated stent prepared according to Example 2 in the reference patent CN113274557 A was used as the control, except that the drug loaded was replaced by an equal amount of IL-12. Then the in vitro release investigation was carried out according to the in vitro experimental scheme described in Example 3. The in vitro release curve is shown in Figure 8 .

[0127] Figure 8 The results show that the IL-12-loaded multilayer coated stent exhibits a significant burst release behavior at the initial stage of release, which causes a large amount of IL-12 to be released rapidly and easily degraded and inactivated by various proteases in vivo. Subsequently, although it exhibits a slow release behavior, the cumulative release rate is low, making it difficult to maintain a long-term drug concentration locally.

Claims

1. A multilayer covered scaffold for unilateral controlled-release of immunotherapeutic drugs, characterized in that, The multilayer membrane-coated stent includes a base of a stent covered with an inner membrane, on which a middle drug-loaded membrane and an outermost blank porous membrane are sequentially coated.

2. The multilayer covered scaffold for unilateral controlled-release immunodrugs according to claim 1, characterized in that, The inner membrane is made of medical-grade silicone rubber, polytetrafluoroethylene, polyurethane, polyester, or silicone polyurethane copolymer.

3. The multilayer covered scaffold for unilateral controlled-release immunodrugs according to claim 1, characterized in that, The materials of the middle drug-loaded membrane and the outermost blank porous membrane are a mixture of polymer A and polymer B, wherein polymer A is any one or more of polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), and carboxymethyl cellulose (CMC), and polymer B is preferably polylactic-co-glycolic acid copolymer (PLGA).

4. The multilayer covered scaffold for unilateral controlled-release immunodrugs according to claim 1, characterized in that, The stent is a mesh stent, a spiral stent, a tubular stent, a ring stent, or a tubular mesh stent, and the stent material is a metal material, a biodegradable material, or a mixture of two materials.

5. The multilayer covered scaffold for unilateral controlled-release immunodrugs according to claim 1, characterized in that, The stent is made radioactive through one or more methods, such as coating with radionuclides, activating with neutron radiation, or loading with radioactive materials.

6. The multilayer covered scaffold for unilateral controlled-release immunodrugs according to claim 1, characterized in that, The middle drug-loaded membrane is loaded with an immunomodulatory drug, which includes one or more of immune checkpoint inhibitors or cytokines.

7. A method for preparing a multilayer covered scaffold for unilateral controlled-release immunodrugs as described in claim 1, characterized in that, Includes the following steps: (1) Polymer A and polymer B are dissolved in a solvent, and an immunomodulator is added to the middle drug-loaded membrane solution to prepare the middle drug-loaded membrane solution and the outer blank pore membrane solution, respectively. (2) First, electrospin the middle layer drug-loaded membrane solution onto the inner layer membrane support, and then continue electrospin the outer layer blank porous membrane solution onto the support with the middle layer membrane already coated. (3) The scaffold covered with the outer membrane is vacuum dried to finally obtain a multilayer membrane scaffold for unilateral controlled release of immunotherapeutic drugs.

8. The method for preparing a multilayer covered scaffold for unilateral controlled-release immunodrugs according to claim 7, characterized in that, The mass ratio of polymer A to polymer B in the intermediate drug-loaded membrane solution is 8:1-2:1, and the mass fraction of polymer A and polymer B in the intermediate drug-loaded membrane solution is 3-8%; the mass ratio of polymer A to polymer B in the outer blank pore membrane solution is 1:1-1:8, and the mass fraction of polymer A and polymer B in the outer blank pore membrane solution is 5-10%; the mass ratio of the intermediate drug-loaded membrane to the outer blank pore membrane is 1:2-4:

1.

9. The method for preparing a multilayer covered scaffold for unilateral controlled-release immunodrugs according to claim 7, characterized in that, The electrospinning parameters for the middle drug-loaded membrane are as follows: voltage 15-18kV, needle type 20-22G, injection speed 0.25-0.6ml / h, spinning distance 5-10cm, and receiving roller speed 80-120rpm; the electrospinning parameters for the outer blank porous membrane are as follows: voltage 18-20kV, needle type 21-24G, injection speed 0.4-1.2ml / h, spinning distance 5-10cm, and receiving roller speed 80-120rpm; the ambient temperature for electrospinning is 18-25℃, and the humidity is 45-50%.

10. The use of a multilayer covered stent of unilateral controlled-release immunotherapy as described in claim 1 in the preparation of tools or materials for radioimmunoassay combined with interventional therapy for malignant tumors with luminal obstruction.

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