A biomimetic photoimmunotherapy nanodrug delivery system for macrophage membranes and its preparation method

Abstract

This invention discloses a macrophage membrane biomimetic photoimmunotherapy nanoparticle drug delivery system and its preparation method. The drug delivery system is prepared by forming cationic liposome nanoparticles from dehydrochlorinated anthracycline antitumor antibiotics, photosensitizers, cationic phospholipids, neutral phospholipids, and cholesterol, and then preparing macrophage membrane biomimetic nanoparticles by co-extrusion with purified macrophage membranes. Finally, the nanoparticles are loaded onto soluble microneedles. By combining nanotechnology and transdermal drug delivery technology, chemotherapy drugs and photosensitizers are precisely delivered to the tumor site, which overcomes the defect of traditional nanoparticle drug delivery systems that are highly dependent on the EPR effect to specifically accumulate at the tumor site, thus inducing immunogenic cell death of tumor cells, activating the body's immune response, effectively inhibiting the growth of solid breast cancer tumors, and accelerating the tumor cell death process.

Description

Technical Field

[0001] This invention relates to a photoimmunotherapy nanodelivery system and its preparation method, and more particularly to a macrophage membrane biomimetic photoimmunotherapy nanodelivery system and its preparation method. Background Technology

[0002] Breast cancer has surpassed lung cancer to become the leading cause of cancer death worldwide, threatening the lives and health of women globally. Current treatments for breast cancer primarily include chemotherapy, radiotherapy, surgery, targeted therapy, and immunotherapy. However, single-modality therapies have certain drawbacks, such as cardiotoxicity, drug resistance, and high recurrence rates. For example, anthracycline hydrochloride antitumor drugs have strong cardiotoxicity, easily develop drug resistance, and cause systemic toxic side effects. Multimodal combination therapy is currently a research direction, and one method is the development of nanomedicine delivery systems. Currently, the main treatments for breast cancer are chemotherapy and surgical resection. Doxorubicin hydrochloride remains the most widely used first-line chemotherapy drug for breast cancer treatment; however, the severe toxic side effects it causes, leading to a decline in patients' quality of life, are a major drawback in its application.

[0003] However, traditional nanomedicine delivery systems are highly dependent on the EPR effect for specific accumulation at the tumor site. The EPR effect depends on a favorable vascular environment at the tumor site. In some types of tumors, such as prostate cancer and pancreatic cancer, low vascular permeability may render the EPR effect ineffective. Therefore, it is necessary to modify nanomedicine delivery systems using biomimetic techniques to enhance their targeting and increase the accumulation of nanoparticles at the tumor site. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a macrophage membrane biomimetic photoimmunotherapy nanodelivery system that enables transdermal drug delivery, precise drug delivery, high biocompatibility, and inhibition of tumor growth, and also provides a method for preparing the above-mentioned drug delivery system.

[0005] Technical solution: The macrophage membrane biomimetic photoimmunotherapy nanoparticle drug delivery system of the present invention is formed by forming biomimetic nanoparticles from a macrophage membrane modified with a macrophage membrane, consisting of dehydrochlorinated anthracycline antitumor antibiotics, photothermal responsive agents, cationic phospholipids and cholesterol, and a photomimetic nanoparticle loaded onto a soluble microneedle tip; wherein the anthracycline antitumor antibiotic is one of daunorubicin, doxorubicin, epirubicin, pirarubicin, mitoxantrone, and idarubicin.

[0006] Preferably, the mass ratio of cationic phospholipids to neutral phospholipids is 1:2 to 2:1; the mass ratio of phospholipids to cholesterol is 8:1 to 4:1; the mass ratio of dehydrochlorinated anthracycline antitumor antibiotics to photosensitizers is 1:15 to 1:25; the mass ratio of phospholipids to total drug content is 7.5:1 to 45:1; and the mass ratio of macrophage membranes to cationic liposomes is 1:2 to 2:1.

[0007] Preferably, the photothermal responsive agent is one of photosensitizer, diporphyrin ether, metal phthalocyanine, gold nanoparticles (AuNPs), graphene, carbon nanorods, copper sulfide (CuS), zinc sulfide (ZnS), indocyanine green (ICG), Prussian blue, and ultra-micro black phosphorus quantum dots (BPQD).

[0008] Preferably, the cationic phospholipid is one of the following: trimethyl-2,3-diolenooxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), dimethyl-2,3-diolenooxypropyl-2-(2-sperminecarbamoylamino)ethylammonium trifluoroacetate (DOSPA), trimethyl-dodecylammonium bromide (DTAB), and dimethyl-2-hydroxyethyl-2,3-diolenooxypropylammonium bromide (DORIE).

[0009] Preferably, the macrophage membrane is derived from one of the following: mouse mononuclear macrophage leukemia cells (RAW264.7), bone marrow-derived macrophages (BMDM), peritoneal macrophages (PM), peripheral blood-derived macrophages, or induced pluripotent stem cell-derived macrophages.

[0010] The method for manufacturing the above-mentioned drug delivery system includes the following steps:

[0011] (1) The hydrochlorinated anthracycline antitumor antibiotic was dissolved in an organic solvent by ultrasonication, and triethylamine was added to remove the hydrochloric acid through an acid-base neutralization reaction;

[0012] (2) Dehydrochlorinated anthracycline antitumor antibiotics, cationic phospholipids, neutral phospholipids and cholesterol are ultrasonically dissolved in an organic solvent, the organic solvent is removed by rotary evaporation to form a phospholipid bilayer, an aqueous solution containing a photothermal responsive agent is added, hydrated and ultrasonically dispersed, filtered and granulated to obtain cationic liposome nanoparticles.

[0013] (3) Take macrophage membranes, centrifuge and lyse them, sonicate them, and then obtain purified macrophage membranes by density gradient centrifugation;

[0014] (4) Take the purified macrophage membrane, mix it with the cationic liposome nanoparticle probe by ultrasound, and then co-extract it with a liposome extruder to obtain biomimetic nanoparticles.

[0015] (5) Take biomimetic nanoparticles and soluble microneedle matrix material, mix them evenly by ultrasonication, place them in microneedles, load the base material, dry and demold to obtain macrophage membrane biomimetic photoimmunotherapy nanodelivery system.

[0016] Preferably, in step (1), the mass-to-volume ratio of the hydrochlorinated anthracycline antitumor antibiotic to triethylamine is 1:10 to 1:500.

[0017] Preferably, in step (2), the dehydrochlorinated anthracycline antitumor antibiotic is doxorubicin, and the photothermal responsive agent is black phosphorus quantum dots.

[0018] Preferably, in step (3), the ultrasonic crusher has a crushing power of 30-300W and a density gradient centrifugation time of 30-120min.

[0019] Preferably, in step (4), the liposome extruder performs 10 to 40 extrusions.

[0020] Preferably, in step (5), the soluble microneedle matrix material is one of hyaluronic acid (HA), chondroitin sulfate, chitosan (CS), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), carboxymethyl cellulose (CMC), and polylactic acid-glycolic acid copolymer (PLGA); further, the soluble microneedle matrix material is a PVP K90 solution with a mass-volume percentage of 10% to 40%; the soluble microneedle base material is a mixed solution of polyvinyl alcohol 1788 and PVP K30, with a mass ratio of polyvinyl alcohol 1788 to PVP K30 of 5 to 15: 10 to 30.

[0021] Preferably, step (1) specifically involves: dissolving doxorubicin hydrochloride in an organic solvent by ultrasonication, adding triethylamine, stirring and reacting overnight at room temperature in the dark to obtain a dark red solution, and removing the organic solvent by rotary evaporation to obtain doxorubicin (DOX).

[0022] Preferably, step (2) specifically involves: dissolving cationic phospholipids, neutral phospholipids, cholesterol, and doxorubicin in an organic solvent using ultrasound, placing the solution in a flask, and removing the organic solvent by rotary evaporation in a 40°C water bath for 15 minutes to form a uniform phospholipid bilayer. The solution is then dried overnight in a vacuum desiccator in the dark. An aqueous solution of BPQD is added, and the solution is hydrated in a 40°C water bath for 30 minutes to allow the phospholipid bilayer to detach from the flask wall. The resulting solution is then ultrasonically dispersed in an ultrasonic disruptor for 15 minutes. The resulting solution is then passed through 0.45μm and 0.22μm aqueous microporous membranes for granulation to obtain cationic liposome nanoparticles BD@L loaded with DOX and BPQD.

[0023] Preferably, step (3) specifically involves: collecting and counting cultured mouse mononuclear macrophage leukemia cells (RAW264.7), centrifuging them at 4°C and 5000 rpm for 3 min in a refrigerated centrifuge to obtain cell pellets, resuspending them in Tris-magnesium sulfate buffer (TM buffer), and adjusting the cell density to 1.0–2.5 × 10⁻⁶ cells / year. 7 Cells / ml. The cells were sonicated in an ultrasonic cell homogenizer on ice. 1M sucrose solution was added to the homogenized cells to form a 0.25M sucrose suspension. After centrifugation at 2000g for 10 min at 4°C, the supernatant was collected and centrifuged again at 3000g for 30 min. The supernatant was discarded. The precipitate was resuspended in TM buffer containing 0.25M sucrose at 4°C and purified by centrifugation at 3000g for 30 min. The resulting precipitate was the purified macrophage membrane.

[0024] Preferably, step (4) specifically involves: mixing BD@L with the purified macrophage membrane in a ratio of 2:1 to 1:2 using ultrasound, and then extruding the mixture 10 to 40 times using a liposome extruder to finally obtain macrophage membrane-encapsulated cationic drug-loaded liposomes BD@LM biomimetic nanoparticles.

[0025] Preferably, step (5) is as follows: BD@LM biomimetic nanoparticles and PVP K90 aqueous solution are mixed evenly at a mass ratio of 1:20 to 3:10. 500 μl is added to the PDMS microneedle mold and dried overnight in a vacuum dryer. Excess matrix material on the surface of the PDMS microneedle mold is removed. 600 μl of PVA and PVP K30 aqueous solution mixed in a certain proportion is added and dried overnight in an electric thermostatic drying oven at 40°C. After demolding, drug-loaded soluble microneedles BD@LM MN are obtained.

[0026] Invention Principle: This invention utilizes macrophage membrane-modified cationic liposomes to load dehydrochlorinated anthracycline antitumor antibiotics and water-soluble photothermal responsive agents to form a drug-loaded biomimetic nanocomposite. This drug-loaded biomimetic nanocomposite is then loaded into the tip of a soluble microneedle, forming a macrophage membrane-modified photoresponsive antitumor nanodelivery system loaded with anthracycline antitumor drugs. This system precisely targets tumor sites via transdermal delivery, enhancing intratumoral drug penetration and reducing the systemic toxicity of anthracycline drugs.

[0027] Doxorubicin hydrochloride, an anthracycline antitumor antibiotic, can bind to DNA and RNA, inhibiting their synthesis. However, it exhibits strong cardiotoxicity, readily develops drug resistance, and impairs bone marrow hematopoietic function, leading to a decrease in white blood cells and platelets. When prepared as liposomes, its high water solubility results in low encapsulation efficiency. Therefore, an acid-base neutralization reaction is used to dehydrochlorinate it, improving the encapsulation efficiency. Furthermore, dehydrochlorinated doxorubicin is positively charged, enabling it to form cationic liposomes with the negatively charged photoresponsive agent BPQD encapsulated in the water cavity of the liposomes. This increases the structural stability of the liposomes while simultaneously improving the encapsulation efficiency and drug loading of the water-soluble photoresponsive agent BPQD, thereby enhancing the photothermal conversion effect of the nanoparticles.

[0028] The study utilizes macrophage membranes as biomimetic membranes. Macrophages are a type of innate immune cell and the most abundant cell type in the tumor microenvironment, accounting for approximately 50% of solid tumors. They can be recruited to the tumor site by cytokines released by tumor cells and the postoperative inflammatory environment. Furthermore, integrins α4 and β1 on the macrophage membrane can specifically bind to vascular adhesion molecule 1 on breast cancer cells, inhibiting their metastasis. Due to their structural similarity to phospholipids, macrophage membranes can fuse with each other, precisely delivering loaded chemotherapeutic drugs and photoresponsive agents to the tumor site, reducing the immunogenicity of nanoparticles, and preventing them from being recognized and cleared by the body's immune system. Compared to other photothermal responsive agents, black phosphorus (BP) exhibits good biodegradability, with degradation products being non-toxic water and phosphate ions. BP, as an emerging nanomaterial, exists in various forms, among which ultra-micro quantum dots possess stronger photothermal conversion efficiency and a larger specific surface area. However, they are highly susceptible to degradation upon exposure to air or aqueous environments, leading to a decrease in photothermal conversion efficiency.

[0029] A biomimetic nanoparticle drug delivery system for transdermal drug administration in breast cancer was prepared by loading dehydrochlorinated doxorubicin and water-soluble BPQD onto cationic liposomes modified with macrophage membranes, forming nanoparticles BD@LM, and then loading these nanoparticles onto the tips of soluble microneedles. This nanodelivery system simultaneously addresses the problems of low encapsulation efficiency and significant systemic toxicity of doxorubicin hydrochloride liposomes, easy degradation of BPQD, and easy recognition and clearance of cationic liposomes by the immune system, resulting in short circulation time in vivo. It achieves targeted delivery of DOX and expands the application scope of BPQD, improving its photothermal efficacy. Overall, this nanodelivery system, combined with multimodal treatment approaches such as chemotherapy, phototherapy, and immunotherapy, effectively inhibits tumor growth and prevents tumor recurrence and metastasis.

[0030] As a superficial tumor, breast cancer benefits from transdermal microneedles, which can directly penetrate the skin's stratum corneum to deliver drugs directly to the lesion or into the bloodstream via the dermis, significantly enhancing drug penetration and absorption. As a localized drug delivery method, microneedles increase the concentration of nanoparticles at the tumor site, enhancing their penetration into the tumor and reducing systemic toxicity caused by DOX. This nanodelivery system, with BD@LM located in the tip of a soluble microneedle, directly releases the drug to the tumor site or allows it to enter the bloodstream through the rich blood vessels of the dermis. It then penetrates deep into the tumor via the specific binding of integrin receptors on macrophage membranes to breast cancer cells. Under irradiation with an 808nm near-infrared laser, the chemotherapy drug doxorubicin and the photothermal responsive agent BPQD are released from the biomimetic nanocomposite. Doxorubicin enters the nucleus of breast cancer cells, inhibiting DNA and RNA synthesis and suppressing breast cancer cell proliferation. BPQD converts light energy into heat energy and instantaneously generates a large amount of reactive oxygen species, inducing oxidative damage to tumor cells. The two work synergistically to induce tumor immunogenic cell death (ICD) and release damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), high-mobility histone 1 (HMGB-1), and adenosine triphosphate (ATP). DAMPs promote the recruitment and maturation of dendritic cells, further enhancing T cell infiltration at the tumor site, improving the immunosuppressive microenvironment at the tumor site, and initiating the body's immune response. Due to the unique drug delivery method of the microneedles, BD@LM is directly delivered to tumor cells, producing excellent tumor growth effects and avoiding the systemic toxicity of anthracycline antitumor drugs.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) Combining active and passive targeting increases the retention and penetration of antitumor drugs at the tumor site; local transdermal administration avoids the cardiotoxicity and drug resistance of anthracycline drugs; protects BPQD from oxidative decomposition, and utilizes its outstanding photothermal conversion effect to induce tumor cell immunogenic cell death, activate the body's immune response, and improve the antitumor effect; (2) Loading biomimetic nanocomposites onto soluble microneedles solves the limitation of low efficiency of nano-drug delivery systems in targeting tumors through the EPR effect, enhances the penetration and retention of biomimetic nanoparticles at the tumor site, and combines chemotherapy, phototherapy, and immunotherapy to synergistically inhibit tumor growth in multiple modes; (3) The preparation method of the present invention increases the encapsulation rate of doxorubicin by 2-3 times. Attached Figure Description

[0032] Figure 1 Figure A shows the characterization results of BD@L nanoparticles in Example 1, where Figure A is the particle size distribution and transmission electron microscopy (TEM) image of BD@L nanoparticles, and Figure B is the TEM image of BPQD.

[0033] Figure 2 This is a transmission electron microscope image of macrophage membrane vesicles from Example 2;

[0034] Figure 3 Figure A shows the characterization results of BD@LM biomimetic nanoparticles in Example 2. Figure A shows the particle size distribution and transmission electron microscopy image of BD@LM biomimetic nanoparticles, and Figure B shows the Zeta potential of BD@L nanoparticles and BD@LM biomimetic nanoparticles.

[0035] Figure 4 This is an SDS-PAGE gel electrophoresis image of the BD@LM biomimetic nanoparticles in Example 2;

[0036] Figure 5 The flow cytometry colocalization map of the BD@LM biomimetic nanoparticles in Example 2 is shown.

[0037] Figure 6 The figures shown are the results of the photothermal performance evaluation of BD@LM biomimetic nanoparticles in Example 3, where Figure A is a photothermal imaging image and Figure B is a time-temperature change graph.

[0038] Figure 7 Figure 4 shows the results of the in vitro cytotoxicity study of BD@LM biomimetic nanoparticles. Figure A shows the cellular uptake of BD@LM biomimetic nanoparticles, and Figure B shows the cytotoxicity results of BD@LM biomimetic nanoparticles on 4T1 cells.

[0039] Figure 8 Figure 5 shows the results of BD@LM biomimetic nanoparticles inducing 4T1 apoptosis, where Figure A is the flow cytometry result and Figure B is the quantitative result.

[0040] Figure 9 The images show the in vitro photodynamic effects of BD@LM biomimetic nanoparticles in Example 6, where Figure A is an inverted fluorescence microscope image of ROS generated in 4T1 cells, and Figure B is a flow cytometry quantification of ROS.

[0041] Figure 10 Figure A shows the flow cytometry characterization of the in vitro maturation of DC cells induced by BD@LM biomimetic nanoparticles in Example 7, where Figure A is the flow cytometry result and Figure B is the flow cytometry quantification.

[0042] Figure 11 The images shown are characterization diagrams of BD@LM MN in Example 8, where Figure A is a stereomicroscope observation image, Figure B is a scanning electron microscope observation image, and Figure C is a laser confocal microscope scan image.

[0043] Figure 12 Figure A shows the mechanical characterization of BD@LM MN in Example 8, where Figure A is the result of the skin penetration test and Figure B is the result of the electric pressure testing machine test.

[0044] Figure 13The distribution of BD@LM in tumor-bearing mice in Example 9 is shown in Figure A, which is the in vivo imaging result from 0 to 48 hours, and Figure B is the quantitative fluorescence intensity map of the tumor site from 0 to 48 hours.

[0045] Figure 14 The images show the in vivo pharmacodynamics of tumor-bearing mice treated with different groups of drugs in Example 10. Figure A shows the growth curve of mouse tumor volume, and Figure B shows the quantitative graph of mouse tumor weight.

[0046] Figure 15 The image shows the HE staining results of the PBS group and the BD@LM MN(+) group after treatment in Example 11. Detailed Implementation

[0047] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0048] Example 1

[0049] Preparation and characterization of BD@L nanoparticles

[0050] (1) Preparation of BD@L nanoparticles

[0051] 10.0 mg of doxorubicin hydrochloride was ultrasonically dissolved in 10 ml of methanol. The resulting solution was placed in a brown round-bottom flask, and 10–500 μl of triethylamine solution was added. The mixture was stirred overnight on a magnetic stirrer, and the solution changed from orange-red to dark red. The round-bottom flask was then dried under reduced pressure in a 40.0°C water bath to remove the methanol solution. The resulting dark red solid was doxorubicin. The dark red solid was then subjected to a full-wavelength UV scan using a UV spectrophotometer. Based on the scan results, the optimal ratio of doxorubicin hydrochloride to triethylamine was determined, and the ratio with no triethylamine residue was selected as the final formulation. Cationic lipid DOTAP, neutral lipid DOPE, and cholesterol in a mass ratio of 1:1:0.5 were accurately weighed and dissolved thoroughly in methanol. DOX was added, and the mixture was then transferred to a 50 ml brown round-bottom flask. The organic solvent was evaporated under reduced pressure in a 40.0°C water bath until a uniform lipid film formed on the flask wall. The mixture was then dried overnight in a vacuum desiccator. Add BPQD aqueous solution to a brown eggplant-shaped flask and hydrate at 40.0℃ until a suspension is formed and the membrane is completely detached from the wall. Sonicate with a probe for 15 min in an ice bath, and then filter sequentially through 0.45 μm and 0.22 μm aqueous filters to obtain an aqueous solution of drug-loaded cationic liposome nanoparticles (BD@L).

[0052] (2) Characterization of BD@L nanoparticles

[0053] The BD@L nanoparticles prepared using the optimal formulation process were characterized. The particle size and polydispersity index of the BD@L nanoparticle aqueous solution were determined using dynamic light scattering (DLS), and the results are shown in the attached figure. Figure 1As shown in Figure A, the average particle size of the nanoparticles is 90.77 ± 2.65 nm, and the PDI is 0.253 ± 0.022. Transmission electron microscopy (TEM) was used to image the BPQD and BD@L nanoparticles, and the results are shown in the attached figure. Figure 1 As shown in B, the BPQD particle size is 5–10 nm, which falls within the quantum dot size range; see attached... Figure 1 As shown in Figure A, the BD@L nanoparticles have a near-spherical structure with BPQD in the internal water cavity. The nanoparticle size is slightly smaller than the particle size of the aqueous solution determined by dynamic light scattering, which is consistent with the actual size, indicating that the BD@L nanoparticles were successfully prepared. After dehydrochlorination, lipid-soluble doxorubicin can be obtained, which can be encapsulated between phospholipid bilayers. In Example 1, the encapsulation efficiency of doxorubicin was measured to be 80.83% ± 2.20%.

[0054] Example 2

[0055] Preparation and characterization of BD@LM biomimetic nanoparticles

[0056] (1) Extraction and purification of macrophage membranes

[0057] RAW264.7 cells cultured in DMEM high-glucose medium were collected and accurately counted. The cells were centrifuged at 4°C and 5000 rpm for 3 min in a refrigerated centrifuge to obtain a cell pellet. The pellet was resuspended in TM Buffer and the cell density was adjusted to 2.5 × 10⁻⁶ cells / mL. 7 Cells / ml; place the cell suspension in an ultrasonic cell disruptor and sonicate to disrupt the cells. Add 1M sucrose solution to the sonicated cell homogenate to adjust the sucrose concentration to 0.25M. Centrifuge at 4℃ and 2000g for 10 min in a refrigerated centrifuge, discard the precipitate, and retain the supernatant. Take the supernatant and centrifuge at 4℃ and 3000g for 30 min in a refrigerated centrifuge to obtain the precipitate, which is the macrophage membrane. Resuspend the obtained precipitate in TM Buffer solution containing 0.25M sucrose at 4℃ and repeat the above step to obtain the purified macrophage membrane.

[0058] (2) Characterization of macrophage membrane

[0059] The extracted macrophage membranes were resuspended in PBS, and then extruded several times using a liposome extruder through a 200nm polycarbonate membrane to obtain macrophage membrane vesicles, as shown in the attached image. Figure 2 As shown in the TEM, macrophage membranes can form vesicles with a particle size of less than 200 nm after being extruded by a liposome extruder.

[0060] (3) Preparation of BD@LM

[0061] The BD@L nanoparticle aqueous solution and macrophage membrane were mixed at a mass ratio of 1:1. The macrophage membrane was uniformly dispersed in the BD@L nanoparticle aqueous solution by ultrasonic cell disruption for 5 minutes. The BD@LM biomimetic nanoparticle aqueous solution was obtained by extruding a 200nm polycarbonate membrane back and forth several times using a liposome extruder.

[0062] (4) Characterization of BD@LM biomimetic nanoparticles

[0063] The particle size and dispersive index of BD@LM biomimetic nanoparticles were determined using dynamic light scattering (DLS), and the results are shown in the attached figure. Figure 3 As shown in Figure A, the particle size of BD@LM was 146.17±7.62 nm, and the PDI was 0.24±0.018. TEM results showed a white bright ring on the outer layer of the liposomes, indicating that the macrophage membrane was successfully encapsulated on the liposomes. The potentials of BD@L and BD@LM were measured using a Malvern particle size analyzer, and the results are shown in the attached figure. Figure 3 As shown in Figure B, the zeta potential of the cationic liposomes was 36.8 ± 11.0 mV. After encapsulation by the macrophage membrane, the potential reversed to -23.16 ± 3.48 mV. This negative potential increased the biocompatibility of the biomimetic nanoparticles. The full-protein spectrum of BD@LM was characterized by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE), and the results are shown in the attached figure. Figure 4 As shown, the purified macrophage cell membrane and the prepared BD@LM biomimetic nanoparticles have a full protein spectrum. Figure 1 The functional protein bands were located in the same positions as those in macrophages, demonstrating the retention of functional proteins on the BD@LM biomimetic nanoparticles.

[0064] Blank cationic liposomes (C6-Lip) were labeled with the lipid-soluble dye coumarin 6 (C6) and mixed with prescribed amounts of DiI-labeled macrophage membranes. The liposome-macrophage membrane mixture was extruded several times using a liposome extruder to form (C6-Lip@DiI-M). The fluorescence intensity of C6 and DiI was detected by flow cytometry. The biomimetic nanoparticles formed by different liposome-to-macrophage membrane mass ratios were investigated, and the results are shown in the appendix. Figure 5 As shown, the mass ratio of C6-Lip to DiI-labeled macrophage membranes was set to 2:1, 1:1, and 1:2, respectively. When the mass ratio of liposomes to macrophage membranes was 2:1, the percentage of C6 and DiI double positivity was the highest, at 92.6%, indicating that the yield of biomimetic nanoparticles was high, with only a small portion of macrophage membrane remaining, and the vast majority of macrophage membranes able to bind tightly to BD@L.

[0065] Example 3

[0066] Photothermal Properties Study of BD@LM Biomimetic Nanoparticles

[0067] Take 1 ml of PBS, BPQD, BD@L, and BD@LM into 1.5 ml EP tubes respectively, and use a laser emitter (808 nm, 1.5 W / cm²). 2 The EP tube was irradiated for 10 minutes. A thermal imager was used to capture images of the EP tube's thermal radiation during the irradiation process, and the temperature was recorded. A time-temperature curve was plotted. The results are attached. Figure 6 As shown in Figure A, after being encapsulated by macrophage membranes, BD@L and BD@LM exhibit higher photothermal conversion efficiency compared to naked BPQD, reaching a temperature of 52.3℃ after 10 minutes of irradiation, demonstrating their ability to kill tumor cells. The temperature change curves of the nanoparticles over time are attached. Figure 6 As shown in Figure B, the results indicate that the encapsulation of macrophage membranes does not affect the photothermal conversion efficiency of BPQDs. Furthermore, due to the encapsulation of the cell membranes, BPQDs are isolated from oxygen in the air, reducing their oxidation and thus achieving a higher heating effect.

[0068] Example 4

[0069] In vitro cytotoxicity study of BD@LM biomimetic nanoparticles

[0070] (1) Study on the uptake effect of BD@LM biomimetic nanoparticles by tumor cells 4T1

[0071] Add 1 ml of 10 to each well of a 12-well plate 4 Four T1 cells were cultured overnight in a complete culture medium in an incubator. After complete cell adhesion, the original culture medium was aspirated, and the cells were washed with PBS. 1 ml of incomplete culture medium solution containing DOX, BD@L, and BD@LM was added to each well, with a blank incomplete culture medium solution added as a control. After incubation for 2–8 hours, the solution in the wells was aspirated, and the cells were washed three times with PBS. 500 μl of 4% paraformaldehyde was added for fixation for 20 min, followed by three washes with PBS. 500 μl of DAPI staining solution was added to each well for staining for 10 min, followed by three washes with PBS. Uptake was observed using an inverted fluorescence microscope. Results are attached. Figure 7 As shown in Figure A, the uptake of BD@LM biomimetic nanoparticles was significantly greater than that of BD@L nanoparticles, indicating that encapsulation by macrophage membranes can enhance the uptake by 4T1 tumor cells.

[0072] (2) Cytotoxicity study of BD@LM biomimetic nanoparticles against 4T1 tumor cells

[0073] Add 100 μl of 5 × 10⁻⁶ ppm solution to each well of a 96-well plate. 3 After culturing in 4T1 complete medium for 24 hours, the original medium was removed, and 100 μl of incomplete medium solution containing DOX and BD@LM was added. A PBS group was set as the blank group, and the untreated group as the control group. After incubation for 6 hours, the wells containing BD@LM were incubated at 808 nm (1.5 W / cm²).2 Cells were irradiated with near-infrared laser for 10 min (near-infrared laser irradiation is indicated by (+)), and incubated for 24 h. The culture medium was then aspirated, and the cells were washed twice with PBS. 20 μl of incomplete culture medium solution with a concentration of 5 mg / ml MTT was added to each well, and the cells were incubated at 37℃ in a 5% CO2 cell culture incubator for 4 h. The original culture medium was then aspirated, and 150 μl of dimethyl sulfoxide (DMSO) was added. After shaking for 5 min with a microplate reader, the absorbance (OD value) was measured at 570 nm. Cell viability was calculated according to the formula below. Results are attached. Figure 7 As shown in B, the IC of free DOX 50 The IC50 concentration of BD@LM(+) was 2.4 μg / ml. 50 The concentration was 0.68 μg / ml, which was significantly lower than that of free DOX. BD@LM(+) significantly enhanced the killing effect of 4T1 tumor cells, indicating that the chemotherapy effect of DOX and the phototherapy effect of BPQD can be significantly enhanced after delivery by biomimetic nanoparticles, producing a synergistic effect.

[0074] Cell viability = (A Sample -A PBS ) / (A Control -A PBS )×100%

[0075] A Sample —Absorbance of the drug-treated cell group

[0076] A PBS —Absorbance of PBS solution

[0077] A Control —Absorbance of untreated cell groups

[0078] Example 5

[0079] Investigation of the effect of BD@LM biomimetic nanoparticles on inducing apoptosis in tumor cells 4T1

[0080] 4T1 cells at 1×10 5 Inoculate one sample per well into a 6-well plate and incubate overnight to allow adhesion. Remove the original culture medium and add a diluted drug-containing solution (incomplete culture medium). The drug administration groups are Control, DOX, BPQD, BD@L, and BD@LM, with three replicates per group. Six hours after drug administration, groups requiring illumination are treated with 808 nm light at 1.5 W / cm². 2Cells were irradiated with laser for 10 min, incubated for another 6 h, the drug-containing culture medium was discarded, cells were washed with PBS, digested with trypsin, and centrifuged at 1000 rpm for 3 min to obtain cell pellet. The pellet was resuspended in 100 μl of binding buffer, and 2.5 μl of Annexin V-FITC and 2.5 μl of PI staining solution were added to each well. After gentle vortexing, the cells were incubated at room temperature in the dark for 20 min. Then, 400 μl of binding buffer was added and mixed thoroughly. The cells were then analyzed by flow cytometry. Results are attached. Figure 8 As shown in Figure A, both free DOX and free BPQD(+) can induce apoptosis. BD@L has a stronger apoptosis-inducing effect than free DOX or BPQD. After being encapsulated by the macrophage membrane, the apoptosis-inducing ability of BD@LM is further enhanced. Compared with other groups, the degree of apoptosis induced in 4T1 cells by the BD@LM(+) group is significantly increased, as shown in the attached figure. Figure 8 As shown in Figure B, the percentage of early-age cells was 11.2%, while the percentage of late-age cells increased to 29.5%, indicating that BD@LM combined with phototherapy has excellent anti-tumor effects.

[0081] Example 6

[0082] Research on the in vitro photodynamic effect of BD@LM biomimetic nanoparticles

[0083] The effect of BD@LM on the generation of reactive oxygen species (ROS) under near-infrared laser irradiation was investigated. Four T1 cells were cultured in 24-well plates for 24 h. After 12 h of incubation with BPQD, BD@L, and BD@LM, cells in each well were washed three times with incomplete culture medium. Cells were then stained with H2DCFDA (10 μM) and cultured in a 37°C, 5% CO2 incubator for 30 min. The culture medium was aspirated from the wells, and cells were then irradiated with 1.5 W / cm² of medium. 2 Cells were irradiated with an 808 nm near-infrared laser for 10 min, washed three times with incomplete culture medium, and the fluorescence of DCF was imaged using an inverted fluorescence microscope. The fluorescence of the generated DCF was then detected by flow cytometry. Results are attached. Figure 9 As shown in Figure A, BPQD can generate ROS under near-infrared light excitation. The BD@LM(+) group showed the strongest ROS generation ability, and the cell morphology of the BD@LM(+) group changed, indicating that the large amount of ROS generated by the BD@LM biomimetic nanoparticles after laser irradiation can kill tumor cells. Flow cytometry results are attached. Figure 9 As shown in Figure B, the DCF fluorescence of the BD@LM(+) group is stronger than that of other groups, further verifying that the BD@LM biomimetic nanoparticles have a good photodynamic effect.

[0084] Example 7

[0085] Study on the in vitro promotion of dendritic cell maturation by BD@LM biomimetic nanoparticles

[0086] Dendritic cells (DC 2.4) were cultured in 12-well plates under 5% CO2 and 37°C cell culture conditions, with a cell count of 2.5 × 10⁻⁶ cells per well. 5 After discarding the original culture medium, the cells were washed twice with PBS, and 1 ml of complete culture medium solution containing DOX, BD@L, and BD@LM was added respectively. The group with added PBS was set as the control group. After incubation for 6 hours, the cells were incubated at 808 nm (1.5 W / cm²). 2 Near-infrared laser irradiation for 10 min, followed by incubation for 24 h, was then discarded. Cells were washed twice with PBS, and FITC-anti-CD80 antibodies and PE-anti-CD86 antibodies were added. Cells were incubated at 4°C in the dark for 30 min, washed with an appropriate amount of PBS, and resuspended in 500 μl PBS. CD80 and CD86 expression were detected by flow cytometry. Results are attached. Figure 10 As shown in Figure A, near-infrared laser irradiation can induce immunogenic cell death (ICD) in tumor cells. The released related molecular patterns (DAMPs) such as adenosine triphosphate (ATP), calreticulin (CRT), and high-mobility histone 1 (HMGB-1) can activate the immune response and promote the maturation of dendritic cells (DCs). The synergistic effect of chemotherapy and phototherapy significantly promoted the maturation of DC 2.4. The percentage of mature DC 2.4 in the BD@LM(+) group was 21.6%, significantly higher than other groups. Flow cytometry results are attached. Figure 10 As shown in Figure B, the BD@LM biomimetic nanoparticles demonstrate synergistic effects with chemotherapy, phototherapy, and immunotherapy, exhibiting excellent anti-tumor activity.

[0087] Example 8

[0088] Construction and evaluation of the BD@LM transdermal drug delivery system

[0089] (1) Construction of soluble microneedles loaded with BD@LM biomimetic nanoparticles (BD@LM MN)

[0090] Needle tip: Accurately weigh 1.5g of PVP K90, dissolve it in 10ml of deionized water, and remove air bubbles from the solution by sonication. Add BD@LM biomimetic nanoparticles, mix thoroughly, and store in the dark for later use.

[0091] Substrate: Accurately weigh 1.5g PVA, swell in 10ml deionized water overnight, dissolve in 80℃ hot water, add PVPK30, dissolve completely, and then sonicate to remove air bubbles from the solution. Store for later use.

[0092] Add 500 μl of the needle tip solution to a PMDS microneedle mold, place it in a glass vacuum desiccator, and use a circulating water multi-purpose vacuum pump to set the vacuum level to 0.1 MPa. Dry for 1–4 hours, and scrape off excess needle tip material from the substrate with a scraper. Add substrate material to the mold, place it in a 40°C oven to dry overnight, and demold to obtain drug-loaded microneedles.

[0093] (2) Characterization of BD@LM MN morphology

[0094] Blank microneedles (Blank MN) and BD@LM MN were observed using a stereomicroscope, as shown in the attached figure. Figure 11 As shown in Figure A, the drug-loaded microneedles appear pale red, with a tip height of approximately 800 μm and a pyramidal side length of approximately 380 μm. The tip morphology remains intact and shows no change after drug loading. Observation was performed using a scanning electron microscope (SEM), and the results are shown in the attached figure. Figure 11 As shown in Figure B, the obtained microneedles have a complete three-dimensional morphology, a smooth surface, and are free of cracks or breaks. The results, observed using a laser confocal microscope, are shown in the attached figure. Figure 11 As shown in C, the red fluorescence represents DOX, proving that BD@LM can be uniformly distributed in the needle tip, indicating that BD@LM MN was successfully constructed.

[0095] (3) Study on the penetrability effect of BD@LM MN

[0096] SD rats were euthanized by cervical dislocation. Skin from the back was removed, and subcutaneous fat and adhesions were carefully removed. After removing surface hair with a pet shaver, the BD@LM MN needle was quickly inserted into the rat skin and pressed for 10 minutes. Immediately after removing the needle, it was stained with 0.4% trypan blue solution for 3–5 minutes. Excess trypan blue solution was wiped away with a cotton swab dipped in physiological saline or PBS, and the needle tip insertion was observed. Results are attached. Figure 12 As shown in Figure A, the microneedles can be successfully inserted into the skin of rats. The microneedle array is 15×15, and the needle holes are clearly visible after trypan blue staining, indicating that BD@LM MN can break through the skin barrier and deliver the drug percutaneously to the site of action to exert its effect.

[0097] (4) Research on the mechanical properties of BD@LM MN

[0098] The mechanical strength of Blank MN and BD@LM MN was determined using an electric compression testing machine. The maximum force was set to 40 N, and the lifting speed was 5 mm / s. The microneedle array was placed on the electric compression testing machine with the tips facing upwards. The mechanical strength of BD@LM MN was measured, and the results are attached. Figure 12As shown in Figure B, the results indicate that the needle tip did not break during the test. When the deformation reached 400 μm, the pressure on each needle tip of the BlankMN and BD@LM MN patches was 0.79±0.03 N and 0.67±0.01 N, respectively, both of which are much higher than the minimum pressure of 0.058 N required for the needle tip to penetrate human skin. This demonstrates that the microneedles have sufficient mechanical strength to be used in in vivo studies.

[0099] Example 9

[0100] Distribution of BD@LM MN transdermal drug delivery system in tumor-bearing mice in vivo and in vitro tissues and organs

[0101] Balb / c mice (female, 6–8 weeks old) were obtained from the Comparative Medicine Center of Yangzhou University. 4T1 cells were cultured until confluence reached 70%–80%, digested with trypsin, centrifuged at 1000 rpm for 3 min to obtain cell pellets, resuspended in PBS, counted, and seeded with 1 × 102 cells onto the right lower fourth fat pad of each mouse. 6 4T1 cells were used to induce tumor growth by suturing the wound. After 14 days, mice with the 4T1 orthotopic model were randomly divided into three groups. The groups received 40 μl of DiR-labeled BD@L and BD@LM via tail vein injection, and the group received 1 μg of DiR-labeled BD@LM MN patch. Mice were anesthetized by intraperitoneal injection of 2% chloral hydrate at 0, 2, 4, 8, 12, 24, and 48 hours. In vivo fluorescence distribution was recorded using a small animal in vivo imaging system. Mice were sacrificed 48 hours later, and the heart, liver, spleen, lung, kidney, and tumor were removed. Fluorescence intensity of the isolated organs was recorded using a small animal in vivo imaging system. Results are attached. Figure 13 As shown in Figure A, compared with BD@L without macrophage membrane, the BD@LM group showed stronger fluorescence at the tumor site. The BD@LM MN group also showed higher fluorescence intensity at the tumor site compared to the intravenous injection group. Quantitative data showed that the tumor fluorescence value was significantly higher in the BD@LM group than in the intravenous injection group. (See attached figure) Figure 13 (B) Furthermore, compared to the intravenous injection group, no fluorescence was observed in the liver region of the BD@LM MN group, indicating that BD@LM was distributed only at the tumor site and did not enter other tissues and organs, thus avoiding the cardiotoxicity and bone marrow suppression caused by systemic DOX administration. Therefore, this demonstrates that the application of BD@LM MN can enhance the drug's targeting to the tumor site, reduce the damage of chemotherapy drugs to major organs, and improve the safety of the drug delivery system.

[0102] Example 10

[0103] Study on the inhibitory effect of BD@LM MN transdermal drug delivery system on in situ breast cancer tumors

[0104] Balb / c mice (female, 6–8 weeks old) were obtained from the Comparative Medicine Center of Yangzhou University. 4T1 cells were cultured until confluence reached 70%–80%, digested with trypsin, centrifuged at 1000 rpm for 3 min to obtain cell pellets, resuspended in PBS, counted, and seeded with 1 × 102 cells onto the right lower fourth fat pad of each mouse. 6 Four T1 cells were collected. All mice were divided into five groups: PBS group, BD@L group, BD@L(+) group, BD@LM(+) group, and BD@LM MN group, with five mice in each group. Tumors were cultured until they reached a volume of 100 mm². 3 Day 0 was recorded as the time of administration. The drug was administered eight times on Days 0, 2, 4, 6, 8, 10, 12, and 14. The first four groups received intravenous injections. BD@LM MN was a microneedle patch; during administration, the patch was inserted into the tumor site and pressed for 20 minutes to dissolve the needle tip. Six hours after administration, an 808nm laser with a power of 1.5W / cm² was used. 2 The tumors were irradiated with near-infrared laser for 10 minutes. During the treatment, changes in tumor volume were recorded in each group of mice, and the results are shown in the attached figure. Figure 14 As shown in Figure A, in the early stage of treatment, the tumor volume of mice in each group did not change significantly. After the fourth day, the tumor volume of mice in the PBS group and BD@L group increased rapidly, exceeding 1000 mm² by the end of treatment. 3 The mice in the PBS group and BD@L group had the largest tumor volume, exceeding 1000 mm at the end of treatment. 3 Tumor growth was significantly inhibited in both the BD@L(+) and BD@LM(+) groups, indicating the active targeting effect of the macrophage membrane and the synergistic effect of phototherapy-chemotherapy. Phototherapy-induced ICD activation of the body's immune response enhances the immune effect in mice, combating tumor growth through autoimmune action and significantly inhibiting tumor volume increase. The difference between the BD@LM MN(+) group and the PBS and BD@L groups was even more significant, with tumor volume gradually decreasing in the later stages of drug administration, demonstrating the best therapeutic effect. This proves that the percutaneous drug delivery system can precisely deliver BD@LM biomimetic nanoparticles to the tumor site, increasing drug accumulation at the tumor site and promoting drug penetration into the tumor tissue, showcasing the significant advantages of microneedle patches in the treatment of breast cancer. After treatment, mice in each group were sacrificed, tumors were removed and weighed, and the tumor weight results were consistent with the tumor volume results, as shown in the attached figure. Figure 14 As shown in B, the BD@LM MN(+) group had the smallest tumor weight, which was significantly different from the PBS group.

[0105] Example 11

[0106] In vivo safety study of the BD@LM MN transdermal drug delivery system

[0107] In Example 10, after the mice were treated, their hearts, livers, spleens, lungs, and kidneys were removed, fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The staining was photographed using a scanner, and the results are shown in the attached figure. Figure 15 As shown, the cells in the major organs of mice in each group had normal morphology, normal cell nucleus size and intact morphology, and no obvious nuclear shrinkage or intercellular cracks were observed. No obvious damage was observed in the major organs of the mice, indicating that BD@LM MN has good biocompatibility and that BD@LM biomimetic nanoparticles do not produce cardiotoxicity or other organ toxicity.

[0108] Comparative Example 1

[0109] Compared to Example 1, doxorubicin hydrochloride was used for the preparation of liposomes:

[0110] Accurately weigh DOTAP, DOPE, cholesterol, and doxorubicin hydrochloride, dissolve them thoroughly in methanol solution, and then transfer the solution to a 50 ml brown oval flask. Evaporate the organic solvent under reduced pressure in a 40.0 °C water bath until a uniform lipid film forms on the flask wall. Dry overnight in a vacuum desiccator. Add BPQD aqueous solution to the brown oval flask and hydrate at 40.0 °C until a suspension forms and the film completely detaches from the flask wall. Sonicate for 15 min under ice bath conditions, and filter sequentially through 0.45 μm and 0.22 μm aqueous filters to obtain a drug-loaded cationic liposome nanoparticle BD@L aqueous solution.

[0111] Liposomes prepared by thin-film dispersion are suitable for encapsulating lipid-soluble drugs. For water-soluble drugs like doxorubicin hydrochloride, the encapsulation rate is only 30%–50% because the drug is encapsulated in the aqueous cavity of the liposome. However, the encapsulation rate of the dehydrochlorinated drug in Example 1 was 80.83% ± 2.20%, which significantly improved the encapsulation rate and avoided drug waste.

[0112] Comparative Example 2

[0113] Compared to Example 1, the mass ratio of DOTAP to DOPE and the mass ratio of phospholipids to cholesterol were changed in the preparation of BD@L nanoparticles:

[0114] BD@L nanoparticles were prepared according to the method described in Example 1 using DOTAP and DOPE in mass ratios of 2:1, 1:1, and 1:2, and phospholipids and cholesterol in mass ratios of 8:1, 6:1, 3:1, and 2:1, respectively. The hydrated particle size, PDI, Zeta potential, DOX encapsulation efficiency, and drug loading of the obtained BD@L nanoparticles were measured. The optimal formulation for BD@L nanoparticle preparation was selected based on small particle size and PDI, Zeta potential less than 40 mV, and high DOX loading and encapsulation efficiency.

[0115] The mass ratio of DOTAP to DOPE affects the charge of BD@L nanoparticles. When the zeta potential of the nanoparticles is greater than 40 mV, they can bind to plasma proteins after entering the body, preventing them from exerting their drug effect at the target site and also causing toxic reactions in the body. By screening the mass ratio of DOTAP to DOPE, cationic drug-loaded liposome nanoparticles with a potential of 36.8 ± 11.0 mV were obtained. This avoids the toxic side effects caused by positive charge and allows for tight binding to the negatively charged macrophage membrane. Simultaneously, the flowability of the liposome nanoparticles needs to be investigated, as it affects the efficiency of nanoparticle entry into target tissues. Good flowability provides conditions for nanoparticle infiltration at the target site; however, excessive flowability can cause drug leakage and affect the stability of the nanoparticles. Cholesterol plays an important role in adjusting the flowability of nanoparticles. Therefore, by adjusting the mass ratio of phospholipids and cholesterol, a formulation with high drug loading, good flowability, and good stability was selected as the optimal formulation for preparing BD@LM biomimetic nanoparticles, which can improve drug efficacy while reducing raw material waste.

[0116] Comparative Example 3

[0117] Compared to Example 2, the mass ratio of BD@L nanoparticle aqueous solution to macrophage membrane was changed in the preparation of BD@LM:

[0118] The BD@L nanoparticle aqueous solution and macrophage membrane were mixed at a mass ratio of 5:1. The macrophage membrane was uniformly dispersed in the BD@L nanoparticle aqueous solution by ultrasonic cell disruption for 5 minutes. The BD@LM biomimetic nanoparticle aqueous solution was obtained by extruding a 200nm polycarbonate membrane back and forth several times using a liposome extruder.

[0119] Comparative Example 4

[0120] Compared to Example 2, the mass ratio of BD@L nanoparticle aqueous solution to macrophage membrane was changed in the preparation of BD@LM:

[0121] The BD@L nanoparticle aqueous solution was mixed with macrophage membrane at a mass ratio of 1:4. The macrophage membrane was uniformly dispersed in the BD@L nanoparticle aqueous solution by ultrasonic cell disruption for 5 minutes. The BD@LM biomimetic nanoparticle aqueous solution was obtained by extruding a 200nm polycarbonate membrane back and forth several times using a liposome extruder.

[0122] The mass ratio of liposomes to macrophage membranes determines the efficiency of biomimetic nanoparticle formation. As shown in Comparative Example 2, excessive liposomes prevent macrophage membranes from completely encapsulating the liposomes, resulting in low targeting of the biomimetic nanoparticle drug delivery system. Insufficient membrane proteins that can specifically bind to tumor cells prevent the system from exerting a good active targeting effect after entering the body. As shown in Comparative Example 4, excessive macrophage membranes result in biomimetic nanoparticles with excessively large particle sizes, making drug release difficult and hindering the EPR effect. At the same time, macrophage membranes that do not bind to liposomes also lead to a waste of resources.

Claims

1. A macrophage membrane biomimetic photoimmunotherapy nanodelivery system, characterized in that, The drug delivery system comprises cationic liposome nanoparticles composed of dehydrochlorinated anthracycline antitumor antibiotics, photothermal responsive agents, cationic phospholipids, neutral phospholipids, and cholesterol, and biomimetic nanoparticles formed by modifying the outer surface of the cationic liposome nanoparticles with macrophage membranes. The biomimetic nanoparticles are then loaded onto soluble microneedles. The anthracycline antitumor antibiotic is one of daunorubicin, doxorubicin, epirubicin, pirarubicin, mitoxantrone, and idarubicin. The photothermal responsive agent is black phosphorus quantum dots.

2. The drug delivery system according to claim 1, characterized in that, The mass ratio of cationic phospholipids to neutral phospholipids is 1:2 to 2:1; the mass ratio of phospholipids to cholesterol is 8:1 to 4:1; the mass ratio of dehydrochlorinated anthracycline antitumor antibiotics to photosensitizers is 1:15 to 1:25; the mass ratio of phospholipids to total drug content is 7.5:1 to 45:1; and the mass ratio of macrophage membranes to cationic liposomes is 1:2 to 2:

1.

3. The drug delivery system according to claim 1, characterized in that, The cationic phospholipid is one of the following: trimethyl-2,3-diolenooxypropylammonium chloride, trimethyl-2,3-dioleoyloxypropylammonium bromide, dimethyl-2,3-diolenooxypropyl-2-(2-sperminecarbamoyl)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, and dimethyl-2-hydroxyethyl-2,3-diolenooxypropylammonium bromide.

4. The method for manufacturing the drug delivery system according to claim 1, characterized in that, Includes the following steps: (1) The hydrochlorinated anthracycline antitumor antibiotic was dissolved in an organic solvent by ultrasonication, and triethylamine was added to remove the hydrochloric acid through an acid-base neutralization reaction; (2) Dehydrochlorinated anthracycline antitumor antibiotics, cationic phospholipids, neutral phospholipids and cholesterol are ultrasonically dissolved in an organic solvent, the organic solvent is removed by rotary evaporation to form a phospholipid bilayer, an aqueous solution containing a photothermal responsive agent is added, hydrated and ultrasonically dispersed, filtered and granulated to obtain cationic liposome nanoparticles. (3) Take macrophage membranes, centrifuge and lyse them, sonicate and disrupt them, and obtain purified macrophage membranes by density gradient centrifugation; (4) Take the purified macrophage membrane, mix it with the cationic liposome nanoparticle probe by ultrasound, and co-extract it with a liposome extruder to obtain biomimetic nanoparticles; (5) Take biomimetic nanoparticles and soluble microneedle matrix material, mix them evenly by ultrasonication, place them in microneedles, load the base material, dry and demold to obtain macrophage membrane biomimetic photoimmunotherapy nanodelivery system.

5. The manufacturing method according to claim 4, characterized in that, In step (1), the mass-to-volume ratio of the hydrochloric acid anthracycline antitumor antibiotic to triethylamine is 1:10 to 1:

500.

6. The manufacturing method according to claim 4, characterized in that, In step (2), the dehydrochlorinated anthracycline antitumor antibiotic is doxorubicin.

7. The manufacturing method according to claim 4, characterized in that, In step (3), the ultrasonic disruption is performed with a power of 30~300W and a density gradient centrifugation time of 30~120 min.

8. The manufacturing method according to claim 4, characterized in that, In step (4), the co-extrusion is performed 10 to 40 times.

9. The manufacturing method according to claim 4, characterized in that, In step (5), the soluble microneedle matrix material is one of hyaluronic acid, chondroitin sulfate, chitosan, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose or polylactic acid-hydroxyacetic acid copolymer, and the soluble microneedle base material is a mixed solution of polyvinyl alcohol 1788 and PVP K30.

Images