Hollow dopamine nano-hemisphere material modified by hybrid cell membrane coating as well as preparation method and application of hollow dopamine nano-hemisphere material

Abstract

The invention discloses a hybrid cell membrane coating modified hollow dopamine nano-hemisphere material as well as a preparation method and application thereof. Pancreatic cancer cells and cell membranes of macrophages are heterozygally coated on a hollow dopamine nano-hemisphere, and the hollow dopamine nano-hemisphere modified by a heterozygous cell membrane coating, which not only can target pancreatic cancer but also can target a tumor microenvironment, and has good biocompatibility and photo-thermal therapy performance, is provided. The nano material prepared by the invention can efficiently identify multiple target spots, realize homing of tumors and microenvironments thereof and in-vivo biological intervention or removal of escaping of the tumors, and improve enrichment of the nano material at the tumors, so that the photo-thermal treatment effect is fully exerted, and the treatment effect of pancreatic cancer is enhanced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a hollow dopamine nanosphere material modified with a hybrid cell membrane coating, its preparation method, and its application. Background Technology

[0002] Exploring safe and effective new strategies for cancer treatment is a pressing clinical challenge. Currently, nanomedicine is developing rapidly, and the clinical use of nanomedicines such as liposomes and albumin-bound paclitaxel has brought new opportunities for precision cancer treatment research.

[0003] However, targeted and precise delivery of nanomedicines remains a major challenge in current research on precision oncology. Currently, there are two main methods for achieving targeted delivery of nanomaterials: passive targeting and active targeting. Passive targeting involves nanomaterials that require no modification, relying directly on their inherent physicochemical properties and the unique microenvironment of the diseased tissue to achieve retention and aggregation at the tumor site. This targeting effect is very low, with most of the nanomaterials being diluted or cleared through blood circulation. Active targeting relies on ligand-receptor interactions, using specific biomolecules such as antibodies, small peptides, and small molecule compounds to bind to specific receptors on the tumor, thus delivering the nanomedicine. However, ligand-modified nanomedicines, once in the bloodstream, rapidly adsorb proteins in the plasma, forming a protein crown on their surface that obscures the ligand, potentially causing them to lose their targeting ability. Further ligand modification can increase the overall size of the carrier or potential systemic toxicity. Furthermore, foreign substances are easily recognized and cleared by the reticuloendothelial system. Neither passive nor active targeting can effectively identify multiple tumor targets or escape biological interference within the body, hindering the full therapeutic benefits of nanomedicines.

[0004] Effective identification of multiple tumor targets, enhanced targeted and precise delivery of nanomedicines, and evasion of biological interference within the body hold promise for improving the efficacy of tumor treatment. Transcellular transport via endocytosis mediated by specific ligands (including antibodies, lipoproteins, peptides, or other endogenous molecules) and receptor recognition may improve the delivery efficiency of nanomedicines to solid tumors. However, clinical trials of nanomedicines with active targeted transport have not yielded significant results, primarily due to the highly heterogeneous microenvironment concealing therapeutic targets; and the interference of biological processes upon entry into the body by exogenous carriers, such as adsorption protein coronas obscuring ligands or capture and clearance by the reticuloendothelial system. These factors have repeatedly hindered molecular targeted therapy research.

[0005] Cell membranes, as natural biomaterials, offer advantages such as easy accessibility and relatively simple procedures for encapsulating nanoparticles. Separated biomembranes retain the distribution and biomass of membrane proteins to the greatest extent possible, maintaining various protein-mediated activities. Furthermore, biomembranes exhibit specific affinity for source cells of the same type as their original origin, i.e., functionalized homology homing or targeting effects. Therefore, biomembrane-coated nanomaterials can simultaneously retain the multifunctionality of both the source cells and the material, making them unique in the field of tumor therapy. Current research on bioactivity largely focuses on the single-molecule level, but biological functions in biological systems are often assembled from specific, ordered molecular structures. For malignant tumors such as pancreatic cancer, due to their dense and complex stromal tumor microenvironment, a composite cell membrane framework may be needed for multi-pathway targeting to achieve more favorable therapeutic outcomes.

[0006] Hybrid membrane fusion technology enables hybrid membranes to inherit multiple cell membrane properties, endowing membrane-coated nanomaterials with multiple functions through the functions of multiple cell membranes. It can also effectively enrich multiple antigens, exhibit diverse physicochemical properties and cell-specific functions, reduce off-target toxicity, and enhance the homing or targeting ability of nanomedicines.

[0007] Photothermal therapy (PTT) in nanomedicine is a safe, radiation-free, and effective treatment method for locally ablating and killing tumors. The principle of PTT is similar to clinically used thermal ablation techniques; it raises the local temperature, directly destroying and killing tumor cells while minimizing damage to normal tissues. With the continuous development of nanomaterials technology, PTT is expected to become one of the important means of precision cancer treatment. Summary of the Invention

[0008] The first technical problem to be solved by the present invention is to provide a hollow dopamine nanosphere material modified with a hybrid cell membrane coating, which can target both pancreatic cancer and the tumor microenvironment, and has good biocompatibility and photothermal therapeutic properties.

[0009] The second technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned hollow dopamine nanosphere material modified with a hybrid cell membrane coating.

[0010] The third technical problem to be solved by the present invention is to provide the application of the above-mentioned hollow dopamine nanosphere material modified with a hybrid cell membrane coating.

[0011] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0012] A method for preparing a hollow dopamine nanosphere material modified with a hybrid cell membrane coating includes the following steps:

[0013] (1) Etch silica-dopamine nanoparticles and then coat them with dopamine to obtain dopamine nano-hemispherical particles.

[0014] (2) After the first centrifugation, pancreatic cancer cells and macrophages were collected separately. The pancreatic cancer cells and macrophages were swelled with water and then centrifuged a second time to collect the cell precipitate. The cell precipitate was sonicated to obtain the cell membranes of pancreatic cancer cells and macrophages, respectively.

[0015] (3) The cell membranes of the pancreatic cancer cells and the macrophages are mixed with the dopamine nanospheres, ultrasonically treated, and then extruded to obtain hollow dopamine nanospheres modified with a hybrid cell membrane coating.

[0016] In step (1), the silica-dopamine nanoparticles are prepared according to the following steps:

[0017] (i) Using hexadecyltrimethylammonium bromide and tetraethoxysilane as raw materials, the mixture was stirred, centrifuged, washed, placed in an ethanol solution containing concentrated hydrochloric acid, stirred, washed, centrifuged, and dispersed in an ethanol solution to obtain an ethanol dispersion of silica.

[0018] (ii) The ethanol dispersion of silica was mixed with polyethyleneimine, and then allowed to stand, washed, and centrifuged to obtain a mixed solution;

[0019] (iii) The mixed solution is reacted with 1,5-glutaraldehyde, washed, reacted with dopamine, washed, and then silica-dopamine nanoparticles are obtained.

[0020] In step (1), the etching is performed using an aqueous NaOH solution, wherein the concentration of the aqueous NaOH solution is 0.1M-0.2M (preferably 0.1M);

[0021] In step (1), the mass ratio of silica-dopamine nanoparticles to dopamine is 4:5-5:5 (preferably 4:5). After the silica-dopamine nanoparticles are etched by NaOH, the silica is removed to form a hollow structure. Because the hollow structure has a large cavity and thin walls, it will be concave into a hemispherical shape. Then, dopamine is wrapped on the outside to form dopamine nano-hemispherical particles with photothermal therapeutic effect.

[0022] In step (2), the first centrifugation is performed at a speed of 1000-1200 rpm (preferably 1200 rpm) for 3-10 min (preferably 3 min); the second centrifugation is performed at a temperature of 3-5℃ (preferably 4℃), a speed of 13000-16000 rpm (preferably 14000 rpm) for 15-30 min (preferably 20 min).

[0023] In steps (2) and (3), the ultrasound is performed for 3-5 minutes (preferably 3 minutes).

[0024] In step (3), the mass ratio of the cell membrane of the pancreatic cancer cells and the cell membrane of the macrophages to the dopamine nanospheres is 1:1:0.5-5 (preferably 1:1:1).

[0025] In step (3), the extrusion is performed using a polycarbonate film micro extruder, and the number of times is 20-25 times (preferably 20 times).

[0026] The hollow dopamine nanospheres modified with hybrid cell membrane coatings prepared by the above method are also within the scope of protection of this invention.

[0027] The application of the above-mentioned hybrid cell membrane coating-modified hollow dopamine nanospheres in the preparation of drugs for treating pancreatic cancer is also within the scope of protection of this invention.

[0028] The aforementioned drug for treating pancreatic cancer has good targeting properties for pancreatic tumors.

[0029] Beneficial effects:

[0030] (1) Compared with the prior art, the hollow dopamine nanospheres prepared by the present invention have good water solubility, low toxicity and biocompatibility in vivo. The carrier also has a highly efficient photothermal therapy (PTT) effect, which can cause the temperature of the tumor site to rise, thereby enhancing the therapeutic effect of pancreatic cancer.

[0031] (2) Two types of cell membranes were extracted in this study: one is the tumor cell membrane and the other is the cell biomembrane highly expressed in the tumor microenvironment. This achieved multi-pathway targeting that targets both the tumor and the tumor microenvironment, improved the enrichment of nanomaterials at the tumor site, and optimized the targeting performance and the impact on the material itself.

[0032] (3) The hybrid cell membrane extraction and coating method of this patent can improve the targeting of nanomaterials in mice.

[0033] (4) The biofilm coating nanomaterials prepared in this study have good biocompatibility and pharmacokinetic properties. They can escape the recognition and clearance of the reticuloendothelial system, prolong its blood circulation time, and significantly reduce edge effects and potential toxicity. Attached Figure Description

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0035] Figure 1Material characterization of the dopamine-coated dopamine nanospheres (DNHs) and the uncoated dopamine nanospheres (DNHs(1)) prepared in this invention. Among them, (a) is the transmission electron microscopy of DNHs and DNHs(1); (b) is the scanning electron microscopy of DNHs and DNHs(1); (c) is the photothermal properties of DNHs and DNHs(1); and (d) is the cytotoxicity experiment of DNHs and DNHs(1).

[0036] Figure 2 Characterization of the DNHs, hybrid membrane-coated dopamine nanospheres (DNHs@PM), and single-membrane-coated dopamine nanospheres (DNHs@P, DNHs@M) prepared in this invention. (a) is a transmission electron microscope image of DNHs and DNHs@PM; (b) is the hydration particle size analysis of DNHs and DNHs@PM; (c) is the Zeta potential analysis of DNHs and DNHs@PM; and (d) is the SDS-PAGE electrophoresis analysis of DNHs@P, DNHs@M, and DNHs@PM.

[0037] Figure 3 The photothermal properties of DNHs@PM are characterized. Among them, (a) shows the temperature rise curves of DNHs@PM under different laser powers; (b) shows the corresponding thermograms of DNHs@PM under different laser powers; (c) shows the temperature rise curves of DNHs@PM under different material concentrations; and (d) shows the corresponding thermograms of DNHs@PM under different material concentrations.

[0038] Figure 4 Photothermal stability experiment of DNHs@PM.

[0039] Figure 5 The photothermal therapeutic effects of DNHs and DNHs@PM on pancreatic cancer cells in vitro.

[0040] Figure 6 This study demonstrates the therapeutic effect of nanomedicine on pancreatic cancer in vivo. (a) shows optical imaging at different time points after nanomedicine injection in an animal model of subcutaneous pancreatic cancer; (b) shows the relative fluorescence intensity of the tumor site at different time points; (c) shows the temperature rise curve and thermogram of the tumor site after photothermal therapy following nanomedicine injection in the animal model of pancreatic cancer; and (d) shows the tumor growth curve of the animal model of pancreatic cancer after photothermal therapy with nanomedicine.

[0041] Figure 7 Cytotoxicity experiments of two types of dopamine nanospheres prepared by etching with hydrofluoric acid or sodium hydroxide.

[0042] Figure 8 Transmission electron microscopy image of hybrid membrane-coated dopamine nanospheres DNHs@PM(2) prepared by ultrasonication.

[0043] Figure 9 Optical imaging images of pancreatic cancer animal models after injection of DNHs@PM(2) and DNHs@PM. Detailed Implementation

[0044] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0045] Example 1: Preparation and characterization of dopamine nanospheres with hybrid film coating.

[0046] (1) Preparation of dopamine nanospheres

[0047] In a 35°C water bath, add 75 mL of water, 30 mL of anhydrous ethanol, and 0.5 mL of ammonia water, and stir at 500 rpm. Add 0.16 g of hexadecyltrimethylammonium bromide to the mixture, followed by 0.5 mL of tetraethoxysilane, and stir for 3 hours. Collect the precipitate by high-speed centrifugation at 10,000 rpm for 10-15 minutes. Wash the precipitate three times with anhydrous ethanol and disperse it in 10 mL of anhydrous ethanol. In a 60°C water bath, add 400 μL of concentrated hydrochloric acid to 200 mL of anhydrous ethanol, and disperse the 10 mL solution containing the precipitate into the mixture. Stir at 500 rpm for 3 hours. Wash the product twice with anhydrous ethanol, then twice with water, and centrifuge at 10,000 rpm for 10-15 minutes. Repeat the entire process 2-3 times to obtain 30-40 mg of silica nanoparticles dispersed in anhydrous ethanol.

[0048] Mix 10 mL of a 2 mg / mL silica-ethanol dispersion with 10 mL of a 2 mg / mL polyethyleneimine solution. Let stand at room temperature for 20-30 minutes. Wash the product 2-3 times with water. Centrifuge at 10,000 rpm for 10-15 minutes to obtain 15 mL of the mixture. Add 3 mL of a 2 mg / mL 1,5-glutaraldehyde solution to this mixture and react. Shake at room temperature for 12-24 hours at 200 rpm. Wash the product 2-3 times with water and disperse in 15 mL of deionized water. Add 10 mL of a 5 mg / mL dopamine solution and continue shaking at room temperature for 12-24 hours at 200 rpm. Wash the product 2-3 times with water and disperse in 10 mL of deionized water to obtain silica-dopamine nanoparticles.

[0049] 40 mg of prepared silica-dopamine nanoparticles were added to 20 mL of 100 mM NaOH aqueous solution, etched on a shaker for 5 minutes, washed with water once, centrifuged at 10,000 rpm for 10-15 minutes, dispersed in 15 mL of Tris alkaline solution, and then 50 mg of dopamine was added and shaken on a shaker for 12-24 h at a speed of 200 times / min to obtain a black product. The product was washed with water 3 times and finally dispersed in 10 mL of water to obtain dopamine nanohemispheres (DNHs) with photothermal therapeutic effect. The nanomaterials without 50 mg of dopamine coating were washed, collected, and used as experimental control group, labeled as DNHs (1).

[0050] (2) Cell Culture

[0051] Pancreatic cancer cells PANC02 were cultured in 1640 medium containing 1% penicillin / streptomycin and 10% fetal bovine serum at a passage ratio of 1:2 and incubated at 37°C in a 5% CO2 incubator. Macrophages RAW 264.7 were cultured in DMEM medium containing 1% penicillin / streptomycin and 10% fetal bovine serum at a passage ratio of 1:2 and incubated at 37°C in a 5% CO2 incubator. Cells were digested by pipetting or scraping with a scraper.

[0052] (3) Extraction of cell biomembranes

[0053] The count is 10 6 Pancreatic cancer cells (P) and macrophages (M) in their active growth phase were washed twice with PBS. Cells were then digested with trypsin or scraped off with a scraper. The cell pellet was collected by centrifugation at 1200 rpm for 3 minutes. 10 mL of water was added to the pellet, and the cells were gently dispersed and placed in a 4°C freezer for 3-4 hours. After the cells absorbed water and ruptured, they were further centrifuged at 4°C at 14000 rpm for 20 minutes. 3 mL of water was added to the cell pellet, and the pellet was gently agitated and sonicated for 3 minutes to obtain the cell membranes of pancreatic cancer cells and macrophages (P or M).

[0054] (4) Hybrid cell membrane coated dopamine nanospheres

[0055] Pancreatic cancer cell membranes (P) and macrophage membranes (M) were mixed with dopamine nanospheres at specific mass ratios (μg:μg = 300:150, 300:300, 300:600, or 300:1200), or pancreatic cancer cell membranes (P) and macrophage membranes (M) were mixed together with dopamine nanospheres at specific mass ratios (μg:μg:μg = 300:300:150, 300:300:300, 300:300:600, or 300:300:1200). The mixtures were then sonicated for 3 minutes and extruded sequentially through polycarbonate membrane micro-extruders with pore sizes of 400 nm and 200 nm, 20 times, to obtain single-film coated dopamine nanospheres (DNHs@P or DNHs@M) or hybrid-film coated dopamine nanospheres (DNHs@PM).

[0056] (5) Material characterization

[0057] After material preparation, 1 mL of 100 μg / mL concentrations of DNHs, DNHs(1), and DNHs@PM (prepared by combining pancreatic cancer cell membrane (P) and macrophage membrane (M) with dopamine nanospheres at a mass ratio of μg:μg:μg = 300:300:300) were placed in cuvettes for UV curve detection using a UV spectrophotometer. Additionally, both materials were placed in dedicated cuvettes and analyzed for zeta potential and hydrated particle size using a zeta potential analyzer. Cell membrane protein content was measured using a BCA protein assay kit. Membrane proteins were characterized by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), stained with Coomassie brilliant blue, washed with water, and then imaged for observation.

[0058] Transmission electron microscopy (TEM) of dopamine-coated dopamine nanospheres (DNHs) and uncoated dopamine nanospheres (DNHs) (1). Figure 1 a) and scanning electron microscope image ( Figure 1 (b) From a morphological perspective, there are no significant differences between the two materials in terms of appearance, structure, dispersibility, and size. Figure 1 c) The photothermal properties of the two materials were compared, with H2O as the control group. The results showed that the temperature of the dopamine-coated dopamine nanospheres (DNHs) rose to 44.7℃ after 300 seconds of irradiation with an 808nm laser, while the uncoated dopamine nanospheres (DNHs) and the control group H2O did not show significant temperature increases, proving that the dopamine-modified DNHs have photothermal therapeutic effects. Furthermore, the two materials were co-incubated with pancreatic cancer cells at different concentration gradients for 24 hours, and cell viability was measured using the CCK8 assay. Figure 1d) The results showed that as the concentration increased, the cell survival rate co-incubated with DNHs(1) decreased to 49.9%, while the cell survival rate co-incubated with DNHs remained at 85%. This demonstrates that the dopamine nanospheres coated with dopamine have low toxicity and good cell biocompatibility, and can be used for subsequent hybrid membrane coating of dopamine nanospheres and in vivo and in vitro photothermal therapy experiments.

[0059] Transmission electron microscopy (TEM) analysis of DNHs@PM revealed a clear morphology, good dispersion, a hollow, hemispherical structure, and a high specific surface area. The biofilm layer coated on the DNHs@PM was very prominent and uniformly coated on the surface of the dopamine nanospheres, with a particle size of approximately 200 nm. Figure 2 a). Hydrated particle size was measured for DNHs and DNHs@PM materials. The hydrated particle size of DNHs was approximately 340.6 nm, while the hydrated particle size of DNHs@PM coated with a cell membrane was approximately 411.4 nm. The membrane coating increased the particle size of the nanomaterials by approximately 70 nm. Figure 2 b). Zeta potential analysis of the two materials showed that the potential of DNHs was approximately 6.73 mV, while the potential of DNHs@PM coated with the cell membrane was approximately 18.11 mV. Modification of the biomembrane also altered the potential. Figure 2 c). Because the cell membrane is rich in various protein components, we immediately performed SDS-PAGE protein electrophoresis analysis on the single-membrane-coated dopamine nanospheres (DNHs@P, DNHs@M) and DNHs@PM. By comparing the electrophoresis results, we found that DNHs@PM was rich in various single-membrane characteristic protein bands. Figure 2 d). The above results indicate that the hybrid film has been successfully coated onto the surface of dopamine nanospheres, and its performance is stable.

[0060] (6) Photothermal properties of the material

[0061] Compared with traditional hollow dopamine nanomaterials, the dopamine nanospheres synthesized in this patent are encapsulated with a layer of dopamine after being etched into a hollow structure. Since dopamine is a normal chemical molecule present in the human body, this design not only improves its biological stability and biocompatibility, but also enhances its photothermal therapy (PTT) performance, making it not only a carrier, but also a key therapeutic component that plays an important role in thermally ablating and killing tumors.

[0062] The DNHs@PM used in this experiment was prepared by combining pancreatic cancer cell membranes (P) and macrophage membranes (M) with dopamine nanospheres in a mass ratio of μg:μg:μg = 300:300:300.

[0063] Adjust different power levels (0, 0.2, 0.5, 1, 1.5, 2 W / cm).2 The temperature change of DNHs@PM at the same concentration (100 μg / mL) was measured after irradiation with an 808 nm laser for 300 seconds. Different concentrations (12.5, 25, 50, 100, 200, or 500 μg / mL) were also measured at the same power (1 W / cm²). 2 The temperature change of DNHs@PM after excitation at 808 nm for 300 seconds was investigated, and the relationship between the temperature change and concentration of the nanomedicine was observed. DNHs@PM was irradiated with an 808 nm laser for 300 seconds and then cooled for 600 seconds. The photothermal stability of DNHs@PM was analyzed after repeating this process for 6 cycles.

[0064] Temperature control was performed on 100 μg / mL DNHs@PM under lasers of different powers. As the laser power increased, the temperature mediated by DNHs@PM gradually increased from room temperature. When the laser power was 1 W / cm², the temperature mediated by DNHs@PM increased. 2 After 300 seconds of irradiation, the temperature of DNHs@PM can rise to 44.7℃; when the laser power is 2W / cm². 2 After 300 seconds of irradiation, the temperature of DNHs@PM can rise to 57.1℃. Figure 3 a, b). Then, the laser power was adjusted to 1 W / cm². 2 Different material concentrations were set to observe temperature changes. The results showed that as the concentration increased, the mediated temperature also increased; when the material concentration was 500 μg / mL, the temperature rose to 50.1℃ after 300 seconds of irradiation. Figure 3 (c, d) The above results confirm that DNHs@PM can induce temperature changes under laser irradiation, which can be further used for thermal ablation to kill tumors. After six cycles of laser irradiation with heating and cooling, DNHs@PM can still rapidly heat up to the cell lethal temperature, indicating that DNHs@PM has good and stable photothermal properties, can be reused multiple times, and can efficiently kill tumor cells. Figure 4 ).

[0065] Example 2: Application of hollow dopamine nanospheres modified with a hybrid cell membrane coating.

[0066] (1) Effects of DNHs and DNHs@PM on in vitro photothermal therapy of pancreatic cancer cells

[0067] The DNHs@PM used in this experiment was prepared by combining pancreatic cancer cell membranes (P) and macrophage membranes (M) with dopamine nanospheres in a mass ratio of μg:μg:μg = 300:300:300.

[0068] Pancreatic cancer cells PANC02 were cultured and dispersed in 96-well plates. Once the confluence reached 80%, different concentration gradients of DNHs and DNHs@PM (6.25, 12.5, 25, 50, and 100 μg / mL) were added. After incubation for 24 hours, photothermal therapy (808nm laser+, 300 seconds, 1W / cm²) was performed. 2 The PBS photothermal therapy group (laser+) served as the control group, and the survival rate of pancreatic cancer cells was detected by the CCK8 method.

[0069] The results showed that cell viability decreased after incubation with DNHs and DNHs@PM followed by photothermal therapy (laser+). As the concentration increased from 6.25 μg / mL to 100 μg / mL, the cell viability after DNHs (laser+) treatment decreased to 54.0%, and the cell viability after DNHs@PM (laser+) treatment decreased to 13.3%, demonstrating that DNHs@PM had a better killing effect on pancreatic cancer cells. Analysis attributed this result to its high targeting and cellular uptake capabilities. Figure 5 ).

[0070] (2) Construction of animal models of pancreatic cancer

[0071] Pancreatic cancer cells PANC02 were cultured and collected. Female black mice aged 6-8 weeks, weighing approximately 18g, were subcutaneously injected with approximately 5 × 10⁵ cells on the right side of each mouse. 6 A subcutaneous pancreatic cancer animal model was established using PANC02 cells, with tumor formation time of approximately one month.

[0072] (3) In vivo distribution of nanomedicines

[0073] The DNHs@P used in this experiment was prepared from pancreatic cancer cell membranes (P) and dopamine nanospheres at a mass ratio of μg:μg=300:300; DNHs@M was prepared from macrophage membranes (M) and dopamine nanospheres at a mass ratio of μg:μg=300:300; and DNHs@PM was prepared from a combination of pancreatic cancer cell membranes (P) and macrophage membranes (M) and dopamine nanospheres at a mass ratio of μg:μg:μg=300:300:300.

[0074] First, 20 subcutaneous pancreatic cancer animal models were established and divided into four groups of 5 animals each. At the same time, four materials, DNHs, DNHs@P, DNHs@M and DNHs@PM, were mixed with an equal mass of Cy7 dye for 24 hours. Then, 100 μL of the corresponding nanomedicine (i.e., DNHs, DNHs@P, DNHs@M and DNHs@PM treated with Cy7 dye) was injected into the tail vein of each of the four groups.

[0075] The distribution of nanomedicines in the heart, liver, spleen, lungs, kidneys, and tumors at different time points (1, 2, 4, 6, 8, 12, or 24 hours) after injection was observed using a small animal optical imaging system. Imaging signals from the tumor sites of each mouse were captured using appropriate imaging software, and fluorescence signal intensity was calculated to analyze the optimal time for nanomedicines to reach the tumor, thus guiding the timing of precise targeted therapy.

[0076] The results showed that the fluorescence signal at the tumor site gradually increased over time, demonstrating the high enrichment of the nanomaterials. The DNHs injection group, relying on passive targeted transport, showed a 9.6-fold increase in tumor signal intensity at 24 hours compared to 1 hour; DNHs@P, homologously homing to the tumor site, showed a 55.8-fold increase in tumor signal intensity at 24 hours compared to 1 hour; DNHs@M, homologously homing to the tumor microenvironment, showed a 63.1-fold increase in tumor signal intensity at 24 hours compared to 1 hour; and DNHs@PM, which simultaneously targeted both the tumor and its microenvironment, showed a 197.2-fold increase in tumor signal intensity at 24 hours compared to 1 hour. 24 hours after injection, the tumor signal intensity in the DNHs@PM injection group was 7.6 times, 3.0 times, and 2.7 times higher than that in the DNHs injection group, DNHs@P injection group, and DNHs@M injection group, respectively, with DNHs@PM showing the highest targeting efficiency. Figure 6 (a, b)

[0077] (4) In vivo therapy with nanomedicines

[0078] Fifteen subcutaneous pancreatic cancer tumor animal models were established and randomly divided into three groups of five mice each. Each group received a tail vein injection of 100 μL PBS, 100 μL 4 mg / mL Cy7-treated DNHs, and DNHs@PM, respectively. After the nanomedicines reached the tumor under optical imaging guidance, the mice were anesthetized using a small animal anesthesia machine and placed on a platform. The tumor site was then irradiated with an 808 nm near-infrared laser at a power of 1 W / cm². 2The mice were irradiated for 5 minutes, and the temperature changes at the tumor site were recorded using an infrared thermal imager. An irradiation time-tumor temperature change curve was plotted. After treatment, the mice's weight changes and health status were observed, and the tumor volume was measured every 3 days for a total of 18 days. The tumor volume was calculated using the following formula: Tumor volume = a × b 2 ×0.5 (a = maximum longitudinal diameter of the tumor, b = maximum transverse diameter of the tumor) to compare the treatment results of mice in each group.

[0079] The results showed that, 24 hours after injection of PBS, DNHs, and DNHs@PM, laser irradiation increased the tumor temperature by 4.8℃, 16.6℃, and 24.6℃, respectively. This increase was attributed to the targeted and photothermal effects of the materials, which induced a rise in local tumor temperature. Figure 6 c). We further monitored the three treatment groups for 18 days. Compared with the other two treatment groups, the DNHs@PM treatment group showed significant inhibition of tumor growth and the best treatment effect. Figure 6 d). The above results further validate the advantages of hybrid membrane-coated dopamine nanospheres in terms of targeting and therapy.

[0080] Comparative Example 1

[0081] The preparation method of the dopamine nanospheres in step (1) of this comparative example is the same as that in Example 1, except that hydrofluoric acid is used for etching. The cytotoxicity of the two types of dopamine nanospheres prepared by the two etching methods, namely hydrofluoric acid etching (DNHs-HF) and sodium hydroxide etching (DNHs-NaOH), is compared.

[0082] Pancreatic cancer cells were incubated at the same concentration for 24 hours, and cell viability was verified using the CCK8 assay. Results are as follows: Figure 7 As shown, dopamine nanospheres synthesized by etching with sodium hydroxide (NaOH) solution (DNHs-NaOH) did not show obvious toxicity. However, the cell survival rate of dopamine nanospheres (DNHs-HF) etched with hydrofluoric acid (HF) decreased from 99.5% to 48.6% as the material incubation concentration increased, indicating potential cytotoxicity.

[0083] Comparative Example 2

[0084] The count is 10 6Pancreatic cancer cells and macrophages in their vigorous growth phase were washed twice with PBS, digested with trypsin or scraped with a scraper, and the cell pellet was collected by centrifugation at 1200 rpm for 3 minutes. 500 μL of protein extraction buffer was added to the pellet, and the mixture was incubated on ice for 2-3 hours. After thorough mixing, the cells were fully lysed, and the cell membrane pellet was collected by centrifugation at 14000 rpm for 20 minutes. 3 mL of water was added to both cell membrane pellets, and the mixture was combined with dopamine nanospheres at a mass ratio of 1:1:1. After sonication for 30 minutes, hybrid membrane-coated dopamine nanospheres (DNHs@PM) were obtained.

[0085] The results are as follows Figure 8 As shown, transmission electron microscopy revealed that although this method can extract hybrid membranes and successfully encapsulate dopamine nanospheres with a particle size of approximately 200 nm, the encapsulation of the hybrid membranes is uneven, with overlapping portions of multiple cell membranes and incomplete encapsulation. This encapsulation method may affect the further in vivo and in vitro applications of nanomedicines.

[0086] Comparative Example 3

[0087] DNHs@PM and DNHs@PM(2), treated with Cy7 dye, were injected intravenously into a mouse model of subcutaneous pancreatic cancer. The tumor signal was tracked 24 hours after intravenous injection using an optical imaging system. Results are as follows: Figure 9 As shown, the tumor signal intensity of the hybrid membrane-coated dopamine nanospheres (DNHs@PM) injection group was higher than that of the DNHs@PM(2) injection group by about 4.1 times after 24 hours, which further confirms that the extraction and coating method of this patent can improve the targeting of nanomaterials in mice.

[0088] This invention provides a method for preparing and applying a hybrid cell membrane coating-modified hollow dopamine nanosphere material. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a hollow dopamine nanosphere material modified with a hybrid cell membrane coating, characterized in that, Includes the following steps: (1) Etch silica-dopamine nanoparticles and then coat them with dopamine to obtain dopamine nano-hemispherical particles. (2) The pancreatic cancer cells and macrophages were ruptured by adding water and centrifuged. The cell pellets were collected and sonicated to obtain the cell membranes of the pancreatic cancer cells and macrophages, respectively. (3) The cell membranes of the pancreatic cancer cells and the macrophages are mixed with the dopamine nanospheres, ultrasonically treated, and then extruded to obtain hollow dopamine nanospheres modified with a hybrid cell membrane coating.

2. The preparation method according to claim 1, characterized in that, In step (1), the silica-dopamine nanoparticles are prepared according to the following steps: (i) The ethanol dispersion of silica was mixed with polyethyleneimine, and then allowed to stand, washed, and centrifuged to obtain a mixed solution; (ii) The mixed solution is reacted with 1,5-glutaraldehyde, washed, reacted with dopamine, washed, and then silica-dopamine nanoparticles are obtained.

3. The preparation method according to claim 1, characterized in that, In step (1), the etching is performed using an aqueous NaOH solution with a concentration of 0.1M-0.2M.

4. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of the silica-dopamine nanoparticles to dopamine is 4:5-5:

5.

5. The preparation method according to claim 1, characterized in that, In step (2), the centrifugation is carried out at a temperature of 3-5℃, a speed of 13000-16000rpm, and a time of 15-30min.

6. The preparation method according to claim 1, characterized in that, In steps (2) and (3), the ultrasound is performed for 3-5 minutes.

7. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of the cell membrane of the pancreatic cancer cells and the cell membrane of the macrophages to the dopamine nanospheres is 1:1:0.5-5.

8. The preparation method according to claim 1, characterized in that, In step (3), the extrusion is performed using a polycarbonate film micro extruder, and the number of times is 20-25.

9. Hollow dopamine nanospheres modified with a hybrid cell membrane coating, prepared by the preparation method according to any one of claims 1-8.

10. The application of the hollow dopamine nanosphere material modified with the hybrid cell membrane coating as described in claim 9 in the preparation of drugs for treating pancreatic cancer.

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