Construction method and application of biomimetic membrane coated drug-loaded nano system

Abstract

The invention belongs to the field of polymer chemistry and biomedical engineering, and discloses a construction method and application of a biomimetic membrane coated drug-loaded nano system. According to the system disclosed by the invention, small-molecule traditional Chinese medicine (sinomenine) is loaded in mesopores, and homologous cell membranes wrap drug-loaded nanoparticles to obtain a final nano-drug system which can effectively and passively target nasopharyngeal carcinoma cells. The sinomenine can cooperate with photothermal therapy to jointly induce mitochondrial injury, promote cell apoptosis and cause ICD immunoreaction, the sinomenine inhibits PD-L1 expression to relieve immunosuppression of T cells, meanwhile, infiltration of cytotoxic T cells in tumor sites is increased, distant metastasis of nasopharyngeal carcinoma can be inhibited by inhibiting MMPs and PD-L1, and the nasopharyngeal carcinoma can be inhibited by inhibiting MMPs and PD-L1. Therefore, the effects of enhancing nasopharynx cancer immunotherapy and inhibiting metastasis are achieved.

Description

Technical Field

[0001] This invention relates to the fields of polymer chemistry and biomedical engineering, specifically to a method for constructing and applying a biomimetic membrane-coated drug-loaded nanosystem. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is one of the most common head and neck tumors, characterized by high rates of metastasis and recurrence. Over the past three decades, radiotherapy and chemotherapy have evolved and become fundamental treatments for NPC. Although the 5-year survival rate for localized disease is 70%-90%, adjuvant chemotherapy in advanced stages leads to poor prognostic outcomes for many patients, with a high proportion experiencing recurrence and metastasis in the later stages of treatment. This underscores the urgent need to find new adjuvant therapies.

[0003] In recent decades, the tumor microenvironment (TME) has been recognized as playing a crucial role in promoting tumor progression, metastasis, and mediating drug resistance. Immunotherapy delivered via vectors designed based on specific tumor microenvironments has provided a range of effective treatment options for nasopharyngeal carcinoma (NPC). NPC is characterized by abundant immune infiltration, including T cells, B cells, dendritic cells (DCs), monocytes, and eosinophils. Furthermore, NPC exhibits a high expression rate of programmed death-ligand-1 (PD-L1) at 89-95%. The immunosuppressive tumor microenvironment in NPC contributes to the development of a dysfunctional and depleted CD8+ tumor microenvironment. + T cells and effectors are constructed by overexpressing inhibitory immune checkpoint signaling molecules, such as programmed cell death protein-1 (PD-1) or its ligand PD-L1, as well as various chemokines and cytokines. The exhaustion and weakness of immune cells allow NPC cells to evade host immune clearance. Many PD-1 / PD-L1 inhibitors have progressed from preclinical studies to clinical trials for the treatment of recurrent / metastatic (R / M) nasopharyngeal carcinoma, including nivolumab, pembrolizumab, camrelizumab, toripalimab, and tislelizumab. PD-L1 has been shown to enhance tumor cell migration and invasion, and promote distant metastasis, by activating signaling pathways such as PI3K / AKT and Wnt / β-catenin, thereby inducing EMT.

[0004] Cancer metastasis is a complex process involving multiple factors and processes, including cell detachment from the primary tumor, extracellular matrix (ECM) degradation, cell invasion into surrounding tissues, entry into blood vessels or lymphatic vessels, and ultimately, exudation into new tissues. Matrix metalloproteinases (MMPs) are important enzymes that promote tumor cell migration, invasion, and metastasis. Among these MMPs, MMP-2 and MMP-9 are the main proteases involved in the metastasis process. Current literature demonstrates that sinomenine can inhibit the migration and invasion of human lung cancer cells by downregulating miR-21 and MMP expression. However, there are currently no studies investigating the inhibitory effects of this drug on the two therapeutic targets of MMPs and PD-L1 in nasopharyngeal carcinoma cells.

[0005] In recent years, the rapid development of photothermal therapy and immunotherapy has provided more new treatment options for tumors. However, there is currently no nanoparticle delivery system designed to combine the two therapies, specifically tailored to the unique tumor microenvironment characteristics of nasopharyngeal carcinoma. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the primary objective of this invention is to provide a biomimetic membrane-coated drug-loaded nanosystem, which is a system that co-delivers traditional Chinese medicine as an immunotherapy drug and a carrier that can act as a photothermal agent.

[0007] Another objective of this invention is to provide a method for constructing the above-mentioned biomimetic membrane-coated drug-loaded nanosystem.

[0008] Another object of the present invention is to provide an application of the above-mentioned biomimetic membrane-coated drug-loaded nanosystem for providing a new method for treating metastasis and recurrence of nasopharyngeal carcinoma.

[0009] The objective of this invention is achieved through the following technical solution.

[0010] The application of sinomenine in the preparation of drugs for treating nasopharyngeal carcinoma: This invention is the first to discover the inhibitory effect of sinomenine on PD-L1 expressed in nasopharyngeal carcinoma cells.

[0011] A biomimetic membrane-encapsulated drug-loaded nanosystem includes mesoporous polydopamine (MPDA), a drug, and a cell membrane; the drug is loaded within the mesoporous polydopamine to form drug-loaded nanoparticles; the cell membrane encapsulates the drug-loaded nanoparticles.

[0012] Preferably, the cell membrane is a tumor cell membrane.

[0013] Preferably, the drug is sinomenine (SIN); the cell membrane is TC1-EBNA1; and the mesoporous polydopamine has a particle size of 134±2.4 nm.

[0014] Preferably, the mass ratio of the mesoporous polydopamine to the drug is 1:1-2; the mass ratio of the cell membrane to the drug-loaded nanoparticles is 1:1-2.

[0015] More preferably, the mass ratio of the mesoporous polydopamine to the drug is 1:1; the mass ratio of the cell membrane to the drug-loaded nanoparticles is 1:1.

[0016] A method for constructing a biomimetic membrane-coated drug-loaded nanosystem as described in any of the above claims includes the following steps: (1) Disperse mesoporous polydopamine and the drug in ethanol and stir to obtain drug-loaded nanoparticles; (2) The cell membrane and drug-loaded nanoparticles were mixed evenly to obtain a biomimetic membrane-coated drug-loaded nanoparticle system.

[0017] Preferably, in step (1), the synthesis process of the mesoporous polydopamine is as follows: Figure 1 As shown.

[0018] Preferably, in step (1), the method for preparing the mesoporous polydopamine includes the following steps: F127 (polyether F127) and HCl·DA (dopamine hydrochloride) were added to ultrapure water and ethanol, and dissolved and mixed by ultrasonication. Trimethylbenzene was slowly added dropwise, and a white emulsion was formed by ultrasonication. Ammonia water was then added, and the mixture was stirred at 50-60℃ for 1-3 h. After centrifugation, washing, and drying, mesoporous polydopamine was obtained. Preferably, the volume ratio of the ultrapure water to ethanol is 1:1; Preferably, the ratio of F127, HCl·DA, trimethylbenzene, and ammonia is 0.1 g: 0.15 g: 200 ul: 375 ul.

[0019] Preferably, the stirring temperature is 50°C and the stirring time is 2 hours.

[0020] Preferably, in step (1), mesoporous polydopamine and the drug are dispersed in ethanol, stirred at 600-700 r / min for 24 h, the supernatant is collected, and the unloaded drug is removed by centrifugation with pure water to obtain drug-loaded nanoparticles.

[0021] In a further preferred embodiment, in step (1), mesoporous polydopamine and the drug are dispersed in ethanol, stirred at 700 r / min for 24 h, the supernatant is collected, and the unloaded drug is removed by centrifugation with pure water to obtain drug-loaded nanoparticles.

[0022] Preferably, in step (2), the cell membrane and drug-loaded nanoparticles are mixed evenly, and the 400 nm pore size polycarbonate membrane is extruded 4-6 times on an extruder, and then the 200 nm pore size membrane is extruded 2-4 times. Then, the uncomposite cell membrane is removed by centrifugation to obtain a biomimetic membrane-coated drug-loaded nanoparticle system.

[0023] In a further preferred embodiment, in step (2), the cell membrane and drug-loaded nanoparticles are mixed evenly, and the mixture is first extruded 6 times using a 400 nm pore size polycarbonate membrane on an extruder, and then extruded 6 times using a 200 nm pore size membrane. The uncomposite cell membrane is then removed by centrifugation to obtain a biomimetic membrane-coated drug-loaded nanoparticle system.

[0024] The application of any of the above-described biomimetic membrane-coated drug-loaded nanosystems in the preparation of drugs for treating nasopharyngeal carcinoma.

[0025] This invention designs a nanoparticle delivery system, MPDA@SIN&CM (abbreviated as M@SIN&CM), which uses MPDA as a carrier and a photothermal agent to load the small molecule drug sinomenine (SIN). By combining cell membrane camouflage, it can more efficiently target and accumulate at the tumor site, increase the bioavailability of small molecule drugs, and also provide a new solution for the treatment of metastatic nasopharyngeal carcinoma.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) The mesoporous polydopamine (MPDA) of the present invention can be used as a photothermal agent and as a carrier, and has good drug loading and biocompatibility.

[0027] (2) The system of the present invention can not only improve the disadvantage of MPDA easy aggregation, but also effectively target the lesion site and enhance the targeting.

[0028] (3) The system in this invention releases sinomenine under photothermal action. This drug can induce mitochondrial damage in tumor cells, induce tumor cell apoptosis, and activate immune damage-related molecular patterns through photothermal therapy with mesoporous polydopamine. It can also activate the immune system and synergistically inhibit PD-L1. The small molecule drug sinomenine can effectively treat the problem of easy lymph node metastasis in nasopharyngeal carcinoma by inhibiting its migration.

[0029] (4) The system in this invention can make the maximum use of sinomenine, increase the amount of drug that can effectively reach the lesion, and achieve better therapeutic effect. Attached Figure Description

[0030] Figure 1 This is a synthetic route diagram for the carrier polymer MPDA; Figure 2 This is a particle size distribution diagram of the drug carrier system; Figure 3 This is a potential diagram of the drug carrier system; Figure 4 Transmission electron microscopy image of the drug-loaded system obtained; Figure 5 The graph shows the photothermal conversion performance of the MPDA carrier. Figure 6 This is the UV spectrum of drug loading in MPDA; Figure 7 The effect of different concentrations of sinomenine on the migration of nasopharyngeal carcinoma cells is shown in the figure. Figure 8 Figure showing the effect of different drug concentrations on PD-L1 expression on the surface of nasopharyngeal carcinoma cells; Figure 9 The effect of different concentrations of sinomenine on the inhibitory effect of PD-L1 expressed on the surface of nasopharyngeal carcinoma cells is shown in the figure. Figure 10 This is a diagram of a cellular uptake experiment; Figure 11 This is a diagram of a live / dead cell detection experiment. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of protection claimed by the present invention.

[0032] Example 1 Preparation of mesoporous polydopamine (MPDA): 5 ml of ultrapure water and ethanol were placed in a round-bottom flask, and 0.1 g of F127 and 0.15 g of HCl·DA were added. The mixture was dissolved and homogenized by sonication. 200 μL of trimethylbenzene was slowly added dropwise, and the mixture was sonicated for 10 min to form a white emulsion. 375 μL of ammonia was added, and the mixture was stirred at 50 °C for 2 h. The product was collected by centrifugation at 11,000 r / min for 10 min, washed three times with ethanol and acetone, and then washed three times with deionized water. After drying at 50 °C, a gray-black powder was obtained and labeled as MPDA. Particle size analysis showed that the average particle size of MPDA was 134 ± 2.4 nm.

[0033] Drug loading: 5 mg MPDA and 5 mg sinomenine were dispersed and dissolved in ethanol. After 24 h at 700 r / min, the supernatant was collected and washed three times with pure water to remove unloaded sinomenine, yielding drug-loaded nanoparticles labeled MPDA@SIN (abbreviated M@SIN). The absorbance was measured using a UV spectrophotometer, and the drug loading rate was calculated from the drug standard curve.

[0034] Encapsulation: TC1-EBNA1 tumor cell membranes were vortexed with the drug-loaded carrier MPDA@SIN at a 1:1 mass ratio. The mixture was first extruded 4-6 times using a 400 nm pore size polycarbonate membrane on a liposome extruder, followed by 2-4 extrusions using a 200 nm pore size membrane. The mixture was centrifuged at 8000 rpm at 4°C to remove uncombined cell membranes, yielding a biomimetic membrane-encapsulated drug-loaded nanosystem, labeled MPDA@SIN&CM (abbreviated as M@SIN&CM), and stored at 4°C for later use. The nanosystem was dispersed in PBS, and 6 μL was used to prepare an electron microscopy sample. The external morphology of the encapsulated nanosystem was photographed using TEM.

[0035] Figure 2 The particle size test showed that the concentration of all three groups was 2 ug / ml. It can be seen that the particle size of the system increased after drug coating and membrane coating, indicating that the biomimetic membrane (TC1-EBNA1 membrane) was successfully coated on the surface of the nanoparticles.

[0036] Figure 3 It is a zeta potential test. The potential is measured according to the particle size and concentration. After encapsulation, the negative potential can be seen to rise. This is because the positive charge of the nasopharyngeal carcinoma cell membrane (TC1-EBNA1 membrane) neutralizes the negative charge on the carrier surface.

[0037] Figure 4 The image is a transmission electron microscope (TEM) image, which clearly shows the mesoporous structure on the carrier. After encapsulation, a different colored band appears around the membrane, further proving that the membrane protein was successfully encapsulated on the nanoparticles.

[0038] Figure 5 The image shows a photothermal experiment to investigate the different photothermal conversion efficiencies of carriers with different concentrations under 808nm near-infrared light irradiation at different powers within a certain time period. It can be seen that with the increase of concentration and power, the higher the temperature, the higher the photothermal conversion efficiency, indicating that the carrier has good photothermal performance.

[0039] Figure 6 The results are from a UV-Vis spectrophotometer test at a 1:1 mass ratio. The characteristic peak of SIN (261 nm) can be observed on M@SIN. Based on the drug solution standard curve, the drug loading rate is calculated to be 38.95%, indicating that this carrier has excellent drug loading performance.

[0040] Figure 7 and Figure 8The image shows a cell scratch assay to investigate the effect of different concentrations of sinomenine on the migration of nasopharyngeal carcinoma cells (TC1-EBNA1). The experiment was conducted as follows: TC1-EBNA1 cells were seeded in complete culture medium and, after reaching confluence, scratches were artificially created. Different drug concentrations were applied, and the scratches were photographed under a microscope. The scratch area was statistically analyzed using ImageJ, and the final cell migration rate was calculated. Figure 7 It can be seen that the PBS group had the fastest cell migration rate and the area decreased. As the concentration of SIN drug increased, the rate of decrease in the scratch area slowed down, that is, cell migration slowed down.

[0041] Figure 9 This image shows an immunofluorescence assay. Laser confocal microscopy was used to investigate the inhibitory effect of different concentrations of sinomenine on PD-L1 expression on the surface of nasopharyngeal carcinoma cells (TC1-EBNA1). After treatment with different drug concentrations, the cells were fixed in 4% paraformaldehyde for 30 min, washed three times with PBS, blocked for 15 min with blocking buffer, incubated overnight at 4 °C with PD-L1 primary antibody, washed three times with PBS, incubated with Alexa Flour 488 secondary antibody for 1 h, washed three times with PBS, stained with DAPI for 5 min, washed three times, and then processed. Green represents PD-L1 membrane protein; blue represents the cell nucleus. The intensity of the green fluorescence indicates that sinomenine indeed has a strong inhibitory effect on PD-L1 expression on the surface of nasopharyngeal carcinoma cells.

[0042] Figure 10 The image shows the TC1-EBNA1 cell uptake experiment. After incubating cells in different groups for 8 hours, staining was performed using Nile Red with red fluorescence instead of the coated drug. Blue represents DAPI, and cell nuclei are marked. +L indicates irradiation with an 808 nm infrared laser for 5 minutes. It can be seen that after the carrier is coated, the red fluorescence uptake by the cells is enhanced. After light irradiation, cell permeability increases, resulting in more red fluorescence uptake, indicating that this system has strong targeting capabilities.

[0043] Figure 11 The image shows the live / dead cell detection assay. Cells treated with different groups (TC1-EBNA1) were incubated at 37 ℃ in the dark for 15 min using a propidium iodide-calcein live / dead cell detection kit. After gently washing away the dye with PBS, images were captured using a live-cell workstation. PI is red, indicating dead cells; Calcine-AM is green, indicating live cells; +L indicates cells irradiated with 808 nm infrared laser for 5 min; MS represents M@SIN; MSC represents M@SIN&CM; MC+L represents M&CM+L; MS+L represents M@SIN +L; and MSC+L represents M@SIN&CM+L. The image shows that the M@SIN&CM+L group induced the most apoptosis and had the best effect.

Claims

1. Use of sinomenine in the preparation of a drug for treating nasopharyngeal carcinoma.

2. A biomimetic membrane-coated drug-loaded nanosystem, characterized in that, The mesoporous polydopamine, a drug, and a cell membrane; the drug is loaded in the mesopores of the mesoporous polydopamine to form drug-loaded nanoparticles; and the cell membrane wraps the drug-loaded nanoparticles.

3. The biomimetic membrane-coated drug-loaded nanosystem according to claim 2, characterized in that, The cell membrane is a tumor cell membrane.

4. The biomimic membrane-coated drug-loaded nanosystem according to claim 2 or 3, characterized in that, The drug is sinomenine; the cell membrane is TC1-EBNA1; and the particle size of the mesoporous polydopamine is 134 ± 2.4 nm.

5. The method for constructing a biomimetic membrane-coated drug-loaded nanosystem according to claim 2, characterized in that, The mass ratio of the mesoporous polydopamine to the drug is 1:1-2; and the mass ratio of the cell membrane to the drug-loaded nanoparticles is 1:1-2.

6. The method for constructing a biomimetic membrane-coated drug-loaded nanosystem according to any one of claims 2-5, characterized in that, The method comprises the following steps: (1) dispersing the mesoporous polydopamine and the drug in ethanol, stirring to obtain drug-loaded nanoparticles; (2) mixing the cell membrane and the drug-loaded nanoparticles uniformly to obtain a biomimetic membrane-coated drug-loaded nanoparticle system.

7. The method for constructing a biomimetic membrane-coated drug-loaded nanosystem according to claim 6, characterized in that, In step (1), the method for preparing the mesoporous polydopamine comprises the following steps: F127 and HCl·DA are added to ultrapure water and ethanol, ultrasonically dissolved and mixed, tri-methylbenzene is slowly added dropwise, a white emulsion is formed under ultrasonication, ammonia water is added, stirring is performed at 50-60°C for 1-3 h, centrifugation, washing, and drying are performed to obtain the mesoporous polydopamine; The volume ratio of the ultrapure water to the ethanol is 1:1; The amount ratio of the F127, HCl·DA, tri-methylbenzene, and ammonia water is 0.1 g:0.15 g:200 ul:375 ul.

8. The method for constructing a biomimetic membrane-coated drug-loaded nanosystem according to claim 6, characterized in that, In step (1), the mesoporous polydopamine and the drug are dispersed in ethanol, stirring is performed at 600-700 r / min for 24-36 h, the supernatant is collected, and the drug not loaded is removed by centrifugation and washing with pure water to obtain the drug-loaded nanoparticles.

9. The method for constructing a biomimetic membrane-coated drug-loaded nanosystem according to claim 6, characterized in that, In step (2), the cell membrane and the drug-loaded nanoparticles are mixed uniformly, first extruded 4-6 times on an extruder using a polycarbonate membrane with a pore size of 400 nm, then extruded 2-4 times using a membrane with a pore size of 200 nm, and finally centrifuged to remove the uncomplexed cell membrane to obtain the biomimetic membrane-coated drug-loaded nanoparticle system.

10. Use of the biomimetic membrane-coated drug-loaded nanoparticle system of any one of claims 2-5 in the preparation of a drug for treating nasopharyngeal carcinoma.

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