Biological membrane fusion nano-micelle, medicine-carrying biological membrane fusion nano-micelle as well as preparation method and application of medicine-carrying biological membrane fusion nano-micelle

Abstract

The invention provides a biological membrane fusion nano-micelle, a drug-loaded biological membrane fusion nano-micelle as well as a preparation method and application thereof, and belongs to the technical field of medicines. The biological membrane fusion nano-micelle comprises an elastin-like polypeptide nano-micelle, a cancer cell membrane coating the surface of the elastin-like polypeptide nano-micelle, L-arginine located between the elastin-like polypeptide nano-micelle and the cancer cell membrane, and a fat-soluble photosensitizer located between phospholipid bilayers of the cancer cell membrane. The biological membrane fusion nano-micelle can bypass a traditional endocytosis way, and the treatment effect can be improved; the hydrophilic and hydrophobic structure of the elastin-like polypeptide nano-micelle is utilized to facilitate encapsulation of fat-soluble chemotherapeutic drugs (such as rapamycin or adriamycin), and a fat-soluble photosensitizer (such as a photosensitizer IR780 or a photosensitizer ICG) can be embedded into a phospholipid bilayer of a cancer cell membrane by utilizing the hydrophobic effect of the elastin-like polypeptide nano-micelle. Cancer synergistic chemotherapy, photodynamic therapy and gas therapy can be realized.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a biomembrane fused nanomicelle, a drug-loaded biomembrane fused nanomicelle, its preparation method and application. Background Technology

[0002] Nanoparticles, exhibiting strong therapeutic loading capacity, prolonged blood circulation, improved penetration of biological barriers, and enhanced tumor targeting, have become a promising cancer treatment method. Polymer micelles, with their spherical structure of a hydrophobic core and a hydrophilic crown, have attracted significant attention due to their unique properties. Micellarization can spontaneously occur in aqueous environments through the self-assembly of amphiphilic molecules at concentrations above the critical micelle concentration (CMC). This simple manufacturing method facilitates the scalable production and clinical translation of polymer micelles. Furthermore, the hydrophobic core of polymer micelles can encapsulate hydrophobic cancer chemotherapy drugs (such as the hydrophobic breast cancer chemotherapy drugs paclitaxel, doxorubicin, and docetaxel), thereby overcoming key limitations associated with low solubility and low bioavailability. However, the potential cytotoxicity of synthetic polymers in micelle preparation remains a concern, limiting their clinical application.

[0003] Natural polymers are widely used in the manufacture of micelles for drug delivery applications due to their superior biocompatibility compared to synthetic polymers. In particular, elastin-like peptides (ELPs) composed of repeating Val-Pro-Gly-X-Gly pentapeptides (where X can be any amino acid except proline) have attracted significant attention due to their unique thermal reactivity. ELPs remain monomeric in aqueous solutions below their transition temperature (Tt), but hydrophobically aggregate when heated above Tt. Notably, the properties of ELPs, including Tt and hydrophilicity / hydrophobicity, can be precisely controlled on demand through precise manipulation of their genetic sequence. For example, when ELPs are genetically engineered into diblock peptides with a Tt below room temperature, they can spontaneously assemble into micelles in an aqueous environment at 37°C. This strategy allows hydrophobic cancer chemotherapy drugs to be encapsulated in the hydrophobic core of the micelles and functional molecules to be bound to a hydrophilic crown. The ELP micelle structure can be further stabilized by covalent cross-linking after introducing reactive residues (such as cysteine ​​or lysine) into the ELP. However, like most nanoparticles, ELP-based micelles are primarily internalized into cells via endocytosis, causing them to become trapped in lysosomal compartments of endosomes. As a result, less than 2% of the encapsulated nanoparticles can successfully escape these organelles, severely impairing their therapeutic efficacy. Summary of the Invention

[0004] The purpose of this invention is to provide a biomembrane fusion nanomicelle, a drug-loaded biomembrane fusion nanomicelle, its preparation method and application. The biomembrane fusion nanomicelle provided by this invention can bypass the traditional endocytosis pathway, which is beneficial to improving the therapeutic effect.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a biomembrane fusion nanomicelle, comprising elastin-like polypeptide nanomicelles, a cancer cell membrane coated on the surface of the elastin-like polypeptide nanomicelles, L-arginine located between the elastin-like polypeptide nanomicelles and the cancer cell membrane, and a lipid-soluble photosensitizer located between the phospholipid bilayer of the cancer cell membrane.

[0006] Preferably, the ratio of the cancer cell membrane, elastin-like polypeptide nanomicelles, lipid-soluble photosensitizer and L-arginine is 0.15~0.3mg:0.1~0.2mg:0.4~1.5nmol:5mg.

[0007] Preferably, the lipid-soluble photosensitizer includes photosensitizer IR780 or photosensitizer ICG.

[0008] Preferably, the cancer cell membrane includes the cell membrane of breast cancer cells.

[0009] This invention provides a drug-loaded biomembrane fused nanomicelle, comprising the biomembrane fused nanomicelle described above and a lipid-soluble chemotherapeutic drug encapsulated within elastin-like polypeptide nanomicelles in the biomembrane fused nanomicelle.

[0010] Preferably, the ratio of elastin-like polypeptide nanomicelles to lipid-soluble chemotherapeutic drugs in the drug-loaded biomembrane fused nanomicelles is 0.1~0.2mg:5~13nmol.

[0011] Preferably, the lipid-soluble chemotherapeutic agent includes rapamycin or doxorubicin.

[0012] This invention provides a method for preparing the drug-loaded biomembrane fused nanomicelles described above, comprising the following steps: An elastin-like polypeptide solution was mixed with a lipid-soluble chemotherapy drug solution and encapsulated. The resulting encapsulated material was then cross-linked to obtain a drug-loaded nanomicelle solution. The drug-loaded nanomicelle solution was mixed with an L-arginine solution to obtain a drug-loaded nanomicelle solution containing L-arginine. The cancer cell membrane solution was mixed with a lipid-soluble photosensitizer and then co-extruded for the first time to obtain a cancer cell membrane solution loaded with a lipid-soluble photosensitizer. The drug-loaded nanomicelles containing L-arginine were mixed with a cancer cell membrane solution loaded with a lipid-soluble photosensitizer, and then co-extruded a second time to obtain the drug-loaded biomembrane fused nanomicelles.

[0013] This invention provides the application of the biomembrane fused nanomicelles described in the above technical solutions, the drug-loaded biomembrane fused nanomicelles described in the above technical solutions, or the drug-loaded biomembrane fused nanomicelles prepared by the preparation method described in the above technical solutions in the preparation of cancer therapeutic agents.

[0014] Preferably, the cancer includes breast cancer.

[0015] Beneficial Effects: The biomembrane fusion nanomicelles provided by this invention include elastin-like polypeptide nanomicelles, a cancer cell membrane coated on the surface of the elastin-like polypeptide nanomicelles, L-arginine located between the elastin-like polypeptide nanomicelles and the cancer cell membrane, and a lipophilic photosensitizer located between the phospholipid bilayer of the cancer cell membrane. The biomembrane fusion nanomicelles of this invention are mainly composed of a cancer cell membrane, elastin-like polypeptide nanomicelles, a lipophilic photosensitizer, and L-arginine. The hydrophilic-hydrophobic structure of the elastin-like polypeptide nanomicelles facilitates the encapsulation of lipophilic chemotherapeutic drugs (such as rapamycin or doxorubicin), and its hydrophobic effect allows lipophilic photosensitizers (such as photosensitizer IR780 or photosensitizer ICG) to be embedded in the phospholipid bilayer of the cancer cell membrane, resulting in an encapsulated structure. The biomembrane-fused nanomicelles provided by this invention, based on membrane fusion properties, utilize a membrane biomimetic method to enable elastin-like polypeptide nanomicelles coated with cancer cell membranes to bypass the traditional endocytosis pathway and achieve direct cytosolic delivery, thereby improving intracellular transport efficiency. Simultaneously, the biomembrane-fused nanomicelles can target cancer tissue through homologous membrane fusion, thereby improving therapeutic efficacy. The biomembrane-fused nanomicelles provided by this invention (such as CCM / IR780@Arg / ELP / Rapa in the examples) can achieve synergistic chemotherapy, photodynamic therapy, and gas therapy for cancers (such as breast cancer), with excellent therapeutic effects. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the spatial structure of the biomembrane-fused nanomicelles (CCM / IR780@Arg / ELP / Rapa) in an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the construction method of the pET-ELP recombinant plasmid. Figure 3 TEM images of ELP nanomicelles and CCM@Arg / ELP nanomicelles in Test Example 1; Figure 4The results of particle size and potential tests for ELP nanomicelles, CCM, and CCM@Arg / ELP nanomicelles in Test Example 1 are shown in the figure. Figure 5 The figure shows the stability test results of ELP nanomicelles and CCM@Arg / ELP nanomicelles in Test Example 1. Figure 6 The figure shows the glutathione responsive release test results of ELP / Rapa nanomicelles in Test Example 2; Figure 7 This is a diagram showing the isomorphic membrane fusion that occurred after 2 hours of incubation of nanomicelles (CCM-NBD@Arg / ELP-Cy5 or ELP-Cy5) with MCF-7 cells in Test Example 3. Figure 8 A graph showing cell viability in test example 4; Figure 9 This is a diagram of the biological targeting test in Test Example 5; Figure 10 The graph shows the efficacy test results of the antitumor biological treatment in Example 6. Figure 11 Representative images of tumors obtained from each group on day 14 in test case 6; Figure 12 Representative photographs of tumor-bearing mice treated with nanomicelles or free compounds on days 0, 7, and 14 in Test Example 6. Detailed Implementation

[0017] The present invention provides a biomembrane fusion nanomicelle, comprising elastin-like peptide (ELP) nanomicelles, a cancer cell membrane (CCM) coated on the surface of the elastin-like peptide nanomicelles, L-arginine (L-Arg) located between the elastin-like peptide nanomicelles and the cancer cell membrane, and a lipid-soluble photosensitizer located between the phospholipid bilayer of the cancer cell membrane.

[0018] In one embodiment of the present invention, the ratio of cancer cell membrane, elastin-like polypeptide nanomicelles, lipid-soluble photosensitizer and L-arginine in the biomembrane fusion nanomicelles can be 0.15~0.3mg:0.1~0.2mg:0.4~1.5nmol:5mg, or more specifically 0.15~0.2mg:0.1~0.15mg:0.4~1nmol:5mg, or more specifically 0.15mg:0.1mg:0.4nmol:5mg.

[0019] In one embodiment of the present invention, the elastin-like polypeptide nanomicelles can be formed from elastin-like polypeptides, specifically diblock elastin-like polypeptides. The diblock elastin-like polypeptide of the present invention can be obtained by overexpression of pET-ELP recombinant plasmid (purchased from Suzhou Genewise Biotechnology Co., Ltd.) in BL21(DE3) Escherichia coli cells followed by purification via reversible switching cycle (ITC). In one embodiment of the present invention, the sequence of the diblock elastin-like polypeptide is shown in SEQ ID NO.1, as detailed below: GVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVP GIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGSGV PGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSG VPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGVPGSGYGGKKGGKKGGCC; The abbreviation is G[VPGIG]48[VPGSG]48Y-GGKKGGKKGGCC.

[0020] The [VPGIG]48 is a hydrophobic segment, and the [VPGSG]48 is a hydrophilic segment. The GGKKGGKKGGCC sequence at the hydrophilic end of the hydrophilic segment is used for micelle crosslinking and functional modification. The lysine K contains a free amino group, and each diblock ELP molecule has four Ks at its C-terminus, meaning it has four amino groups for micelle crosslinking. The two outermost cysteine ​​Cs of the hydrophilic end contain free thiol groups, which are used to couple with molecules containing maleimide groups, thereby achieving functional modification of the micelle surface.

[0021] In one embodiment of the present invention, the cancer cell membrane may include the cell membrane of breast cancer cells, specifically the cell membrane of MCF-7 cells. The present invention does not specifically limit the source of the cancer cell membrane; any cancer cell membrane from a source well known to those skilled in the art can be used, and it can be extracted using methods well known to those skilled in the art. In this embodiment of the present invention, a commercial membrane protein extraction kit can be used to isolate the cancer cell membrane from MCF-7 cells, specifically by extracting approximately 4 × 10⁻⁶ cells. 8 One MCF-7 cell was suspended in 2 mL of membrane protein extraction buffer containing 1 mM benzosulfonyl fluoride (PMSF), cooled on ice for 15 min, and then lysed by three freeze-thaw cycles (liquid nitrogen freezing, thawing at 37 °C, repeated 3 times) to obtain lysates. The lysates were centrifuged at 700 g and 4 °C for 10 min to remove cell nuclei and cell debris. The supernatant was collected and further centrifuged at 14000 g and 4 °C for 30 min to obtain CCM. The CCM was mixed with RIPA lysis buffer, and the integrity of the membrane proteins was verified by 12% SDS-PAGE gel electrophoresis. The prepared CCM was then stored at -80 °C for later use.

[0022] In one embodiment of the present invention, the lipid-soluble photosensitizer may include photosensitizer IR780 or photosensitizer ICG.

[0023] The biomembrane-fused nanomicelles provided by this invention, based on membrane fusion properties, utilize a membrane biomimetic method to enable elastin-like polypeptide nanomicelles coated on cancer cell membranes to bypass the traditional endocytosis pathway and achieve direct cytosolic delivery, thereby improving intracellular transport efficiency. Simultaneously, the biomembrane-fused nanomicelles can target cancer tissue through homologous membrane fusion, thereby improving therapeutic efficacy. The biomembrane-fused nanomicelles of this invention can serve as drug delivery carriers. The elastin-like polypeptide nanomicelles can be used to encapsulate lipid-soluble chemotherapeutic drugs, enhancing cancer treatment efficacy based on chemotherapy. The L-arginine, located between the elastin-like polypeptide nanomicelles and the cancer cell membrane, acts as a solubilizer and serves as a NO precursor, enhancing cancer treatment efficacy based on NO gas therapy. The lipid-soluble photosensitizer can enhance cancer treatment efficacy based on photodynamic therapy.

[0024] This invention provides a drug-loaded biomembrane fused nanomicelle, comprising the biomembrane fused nanomicelle described above and a lipid-soluble chemotherapeutic drug encapsulated within elastin-like polypeptide nanomicelles in the biomembrane fused nanomicelle.

[0025] In one embodiment of the present invention, the ratio of elastin-like polypeptide nanomicelles to lipid-soluble chemotherapeutic drugs in the drug-loaded biomembrane fused nanomicelles can be 0.1~0.2mg:5~13nmol, further can be 0.1~0.15mg:6~9nmol, and specifically can be 0.1mg:6.5nmol.

[0026] As one embodiment of the present invention, the lipid-soluble chemotherapy drug may include rapamycin (Rapa) or doxorubicin (DOX).

[0027] In one embodiment of the present invention, the drug-loaded biomembrane fused nanomicelles are spherical with a particle size of 130-180 nm, or more specifically 170-180 nm.

[0028] This invention provides a method for preparing the drug-loaded biomembrane fused nanomicelles described above, comprising the following steps: An elastin-like polypeptide solution was mixed with a lipid-soluble chemotherapy drug solution and encapsulated. The resulting encapsulated material was then cross-linked to obtain a drug-loaded nanomicelle solution. The drug-loaded nanomicelle solution was mixed with an L-arginine solution to obtain a drug-loaded nanomicelle solution containing L-arginine. The cancer cell membrane solution was mixed with a lipid-soluble photosensitizer and then co-extruded for the first time to obtain a cancer cell membrane solution loaded with a lipid-soluble photosensitizer. The drug-loaded nanomicelles containing L-arginine were mixed with a cancer cell membrane solution loaded with a lipid-soluble photosensitizer, and then co-extruded a second time to obtain the drug-loaded biomembrane fused nanomicelles.

[0029] In this invention, unless otherwise specified, all raw materials used are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0030] This invention involves mixing an elastin-like peptide solution with a lipid-soluble chemotherapeutic drug solution, encapsulating the mixture, and then cross-linking the resulting encapsulated material to obtain a drug-loaded nanomicelle solution. In one embodiment of this invention, the concentration of the elastin-like peptide solution can be 0.2-0.4 mg / mL, and the solvent can be PBS; the concentration of the lipid-soluble chemotherapeutic drug solution can be 48-50 μM, and the solvent can be acetonitrile; the volume ratio of the elastin-like peptide solution to the lipid-soluble chemotherapeutic drug solution can be 3-3.5:6.5-7, specifically 3:7. In another embodiment of this invention, the encapsulation process may include: mixing the elastin-like peptide solution and the lipid-soluble chemotherapeutic drug solution, then rotary evaporating and vacuum drying at 38-40°C for 10-12 h; mixing the resulting residue with PBS, and then rotary evaporating at 35-37°C for 15-20 min to ensure that the components are fully dispersed in the PBS and to achieve encapsulation. In one embodiment of the present invention, the encapsulation treatment preferably further includes: centrifuging the encapsulated material to remove free lipid-soluble chemotherapy drugs, and collecting the precipitate as the encapsulated material; the centrifugation temperature can be 37°C, the rotation speed can be 10000 rpm, and the time can be 10 min. In another embodiment of the present invention, the crosslinking treatment is carried out in the presence of a 1,2-ethylenedimethyl dimethyl thiosulfonate (MTS-2-MTS) solution; specifically, the encapsulated material can be mixed with the MTS-2-MTS solution for the crosslinking treatment; the concentration of the MTS-2-MTS solution can be 28~30 mg / mL, and the solvent can be N,N-dimethylformamide (DMF); the amount of MTS-2-MTS solution used is sufficient to ensure sufficient crosslinking of the terminal thiol groups of the elastin-like peptide; the crosslinking treatment temperature can be 37°C, and the time can be 25~30 min.

[0031] After obtaining the drug-loaded nanomicelle solution, the present invention mixes the drug-loaded nanomicelle solution with an L-arginine solution to obtain a drug-loaded nanomicelle solution containing L-arginine. In one embodiment of the present invention, the concentration of the L-arginine solution can be 10-12 g / 100 mL (i.e., 100-120 mg / mL), and the amount of L-arginine solution used is sufficient to ensure that the final concentration of L-arginine in the obtained drug-loaded nanomicelle solution containing L-arginine meets the requirements. In another embodiment of the present invention, the concentration of elastin-like polypeptide in the drug-loaded nanomicelle solution containing L-arginine can be 0.2-0.4 mg / mL, the concentration of L-arginine can be 1-1.2 g / 100 mL (i.e., 10-12 mg / mL), and the concentration of the lipid-soluble chemotherapeutic drug can be 10-26 μM, more specifically 13-20 μM.

[0032] This invention involves mixing a cancer cell membrane solution with a lipid-soluble photosensitizer and performing a first co-extrusion to obtain a cancer cell membrane solution loaded with the lipid-soluble photosensitizer. In one embodiment, the concentration of the cancer cell membrane solution can be 0.3~0.6 mg / mL, and the solvent can be PBS; the concentration of the lipid-soluble photosensitizer in the mixture obtained after mixing the cancer cell membrane solution with the lipid-soluble photosensitizer can be 45~50 μM. In another embodiment, the porous membranes used in the first co-extrusion are preferably sequentially a 1µm polycarbonate porous membrane and a 400nm polycarbonate porous membrane; preferably, 10 cycles are performed for each type of porous membrane during co-extrusion. In yet another embodiment, the first co-extrusion is preferably followed by ultrafiltration centrifugation to remove free lipid-soluble photosensitizer; the ultrafiltration centrifugation can be performed twice, with each ultrafiltration centrifugation speed being 4500~5000 rpm and the time being 25~30 min. In one embodiment of the present invention, the concentration of the cancer cell membrane in the cancer cell membrane solution loaded with the lipid-soluble photosensitizer can be 0.3~0.6 mg / mL, and the concentration of the lipid-soluble photosensitizer can be 0.8~3 μM.

[0033] After obtaining the drug-loaded nanomicelle solution containing L-arginine and the cancer cell membrane solution loaded with a lipid-soluble photosensitizer, the present invention mixes the drug-loaded nanomicelle solution containing L-arginine and the cancer cell membrane solution loaded with a lipid-soluble photosensitizer, and performs a second co-extrusion to obtain the drug-loaded biomembrane fused nanomicelles. As one embodiment of the present invention, the volume ratio of the drug-loaded nanomicelle solution containing L-arginine to the cancer cell membrane solution loaded with a lipid-soluble photosensitizer can be 1:1; the porous membrane used in the second co-extrusion can be a 400nm polycarbonate porous membrane, and preferably 10 cycles are performed when using a 400nm polycarbonate porous membrane for co-extrusion.

[0034] The preparation method of the biomembrane fusion nanomicelles described in this invention can be operated with reference to the above method, the difference being that the lipid-soluble chemotherapy drugs are omitted, and will not be described in detail here.

[0035] This invention provides the application of the biomembrane fused nanomicelles described in the above technical solutions, the drug-loaded biomembrane fused nanomicelles described in the above technical solutions, or the drug-loaded biomembrane fused nanomicelles prepared by the preparation method described in the above technical solutions in the preparation of cancer therapeutic agents.

[0036] In one embodiment of the present invention, the cancer may include breast cancer.

[0037] In biological systems, membrane fusion, the fusion of intercellular membrane components, is considered to play a crucial role in intercellular communication and substance transport. During membrane fusion, the donor cell is bound to its membrane and comes into contact with the target cell, subsequently forming a continuous fused lipid bilayer. Thus, membrane components are mixed, and the internal contents of the donor cell are directly transported into the cytoplasm of the target cell. Inspired by this natural phenomenon, fused cell membranes can be used to encapsulate nanoparticles for drug delivery. This biomimetic approach allows encapsulated nanoparticles to bypass the traditional endocytosis pathway, achieving direct cytosolic transport and improving intracellular transport efficiency. Simultaneously, lipid diffusion and mixing during membrane fusion enable the engineering of target cell membranes. Furthermore, the genetic biological characteristics of target cells, including immune escape, long-term circulation, and tissue targeting, can enhance the pharmacokinetic properties of encapsulated nanoparticles. These characteristics make nanoparticles coated with fused cell membranes promising drug delivery carriers.

[0038] Inspired by natural membrane fusion processes, this invention designs a biomembrane-fusion nanomicelle, a membrane-promoting nanomicelle, capable of enhancing the therapeutic effects of photodynamic, chemodynamic, and nitric oxide (NO) gas therapy for cancer. Specifically, in this embodiment, the biomembrane-fusion nanomicelle is prepared by encapsulating ELP nanomicelles loaded with photosensitizer IR780, L-arginine (L-Arg), and rapamycin (Rapa) into the cancer cell membrane (CCM) of MCF-7 cells, where MCF-7 is a human cancer cell line with intrinsic isomorphic fusion capability. In this invention, the biomembrane-fusion nanomicelle can target cancer tissue through homologous membrane fusion, allowing the photosensitizer IR780 to anchor in the CCM while simultaneously releasing the L-Arg and Rapa-loaded ELP nanomicelles directly into the cytoplasm. Under near-infrared light irradiation, the CCM-anchored photosensitizer IR780 triggers photodynamic therapy (PDT), which simultaneously disrupts the CCM and catalyzes the conversion of L-Arg into tumor-suppressive NO gas, thereby improving therapeutic efficacy. Meanwhile, excessive intracellular glutathione (GSH) can cleave disulfide bonds in ELP nanomicelles loaded with Rapa, triggering controlled Rapa release to enhance the chemotherapy effect.

[0039] Figure 1 This is a schematic diagram of the spatial structure of the biomembrane-fused nanomicelles (CCM / IR780@Arg / ELP / Rapa) in an embodiment of the present invention, where the left side is a three-dimensional structural diagram and the right side is a cross-sectional structural diagram; Figure 1It is known that the CCM / IR780@Arg / ELP / Rapa includes elastin-like polypeptide (ELP) nanomicelles, a cancer cell membrane (CCM) coated on the surface of the ELP nanomicelles, L-arginine (L-Arg) located between the ELP nanomicelles and the cancer cell membrane, photosensitizer IR780 located between the phospholipid bilayers of the cancer cell membrane, and lipophilic chemotherapeutic drug rapamycin (Rapa) encapsulated inside the ELP nanomicelles in the biomembrane fusion nanomicelles.

[0040] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0041] Preparation Example 1 ELP extraction: Under the induction of isopropyl-β-D-thiogalactoside (IPTG), diblock ELP (pET-ELP recombinant plasmid purchased from Suzhou Genewise Biotechnology Co., Ltd.) was overexpressed in BL21(DE3) Escherichia coli cells, and then the obtained diblock ELP was purified by reversible switching cycle (ITC). All peptides were detected by SDS-PAGE, and the prepared diblock ELP was stored in 15 vol% glycerol PBS solution at -20℃ for later use.

[0042] The specific sequence of the binary block ELP described in this invention is as follows: G[VPGIG]48[VPGSG]48Y-GGKKGGKKGGCC (SEQ ID NO. 1).

[0043] The [VPGIG]48 is a hydrophobic segment, and the [VPGSG]48 is a hydrophilic segment. The GGKKGGKKGGCC sequence at the hydrophilic end of the hydrophilic segment is used for micelle crosslinking and functional modification. The lysine K contains a free amino group, and each diblock ELP molecule has four Ks at its C-terminus, meaning it has four amino groups for micelle crosslinking. The two outermost cysteine ​​Cs of the hydrophilic end contain free thiol groups, which are used to couple with molecules containing maleimide groups, thereby achieving functional modification of the micelle surface.

[0044] Figure 2This diagram illustrates the construction method of the pET-ELP recombinant plasmid. Specifically, the DNA sequence of the biblock ELP is inserted between the Nde1 and Xhol restriction sites of the pET-31b plasmid to construct the pET-ELP recombinant plasmid. Then, the corresponding biblock ELP is expressed in E. coli cells according to the aforementioned method.

[0045] Preparation Example 2 Extraction of cancer cell membrane (CCM): CCM was isolated from MCF-7 cells using a commercial membrane protein extraction kit, specifically by extracting approximately 4 × 10⁻⁶ cells. 8 One MCF-7 cell was suspended in 2 mL of membrane protein extraction buffer containing 1 mM benzosulfonyl fluoride (PMSF), cooled on ice for 15 min, and then lysed by three freeze-thaw cycles (liquid nitrogen freezing, thawing at 37 °C, repeated 3 times) to obtain lysates. The lysates were centrifuged at 700 g and 4 °C for 10 min to remove cell nuclei and cell debris. The supernatant was collected and further centrifuged at 14000 g and 4 °C for 30 min to obtain CCM. The CCM was mixed with RIPA lysis buffer, and the integrity of the membrane proteins was verified by 12% SDS-PAGE gel electrophoresis. The prepared CCM was then stored at -80 °C for later use.

[0046] Example 1 (1) Preparation of L-Arg-containing ELP / Rapa nanomicelle solution: A PBS solution of 0.2 mg / mL diblock ELP (prepared in Preparation Example 1) was mixed with a 50 μM rapamycin (Rapa) acetonitrile solution at a volume ratio of 3:7. The mixture was rotary evaporated at 40 °C and vacuum dried for 12 h to obtain an ELP / Rapa membrane. The ELP / Rapa membrane was mixed with PBS and rotary evaporated at 37 °C for 20 min to gradually disperse ELP and Rapa in PBS and achieve ELP encapsulation of Rapa. The membrane was then centrifuged at 37 °C and 10,000 rpm for 10 min to remove free Rapa. The precipitate was collected and 1 μL was added to the PBS solution. A solution of 1,2-ethylenedimethyl dimethyl thiosulfonate (MTS-2-MTS) at a concentration of 30 mg / mL in N,N-dimethylformamide (DMF) was mixed and incubated at 37°C for 30 min to achieve crosslinking of the terminal thiol groups of ELP, resulting in an ELP / Rapa nanomicelle solution. The ELP / Rapa nanomicelle solution was then mixed with a 10% (w / v) L-Arg solution to bring the final L-Arg concentration to 1% (w / v), resulting in an ELP / Rapa nanomicelle solution containing L-Arg (ELP concentration: 0.2 mg / mL, L-Arg concentration: 1% (w / v), Rapa concentration: 13 μM). (2) Preparation of CCM / IR780 solution: CCM (prepared in Preparation Example 2) was dissolved in PBS to a final concentration of 0.3 mg / mL to obtain a CCM solution; photosensitizer IR780 was added to the CCM solution to a final concentration of IR780 of 50 μM, and the solution was extruded through a 1 µm polycarbonate porous membrane for 10 cycles, then extruded through a 400 nm polycarbonate porous membrane for 10 cycles, and then ultrafiltered and centrifuged twice at 5000 rpm to remove free IR780. Each ultrafiltration and centrifugation time was 30 min to obtain a CCM solution loaded with IR780 (denoted as CCM / IR780 solution, with a CCM concentration of 0.3 mg / mL and a photosensitizer IR780 concentration of 0.8 μM). (3) Preparation of drug-loaded biomembrane fused nanomicelles (i.e., CCM-coated ELP-based nanomicelles): 500 μL of the L-Arg-containing ELP / Rapa nanomicelle solution was mixed with 500 μL of the CCM / IR780 solution. The resulting mixture was extruded through a 400 nm polycarbonate porous membrane for 10 cycles to obtain a CCM-coated ELP-based nanomicelle (denoted as CCM / IR780@Arg / ELP / Rapa nanomicelles) solution, wherein the CCM concentration was 0.15 mg / mL, the ELP concentration was 0.1 mg / mL, the IR780 concentration was 0.4 μM, the Rapa concentration was 6.5 μM, and the L-Arg concentration was 0.5% (w / v).

[0047] Test Example 1 The ELP nanomicelles used in this test example were prepared as follows: 1 mL of PBS solution containing 0.2 mg / mL diblock ELP (prepared in Preparation Example 1) and 1 μL of N,N-dimethylformamide (DMF) solution containing 30 mg / mL 1,2-ethylenedimethyldimethylthiosulfonate (MTS-2-MTS) were mixed and incubated at 37°C for 30 min to obtain the ELP nanomicelle solution.

[0048] The preparation method of CCM@Arg / ELP nanomicelles used in this test example is as follows: the preparation method of CCM / IR780@Arg / ELP / Rapa nanomicelles in Example 1 is followed, except that IR780 and Rapa are omitted.

[0049] Figure 3 TEM images of ELP nanomicelles and CCM@Arg / ELP nanomicelles in Test Example 1, with a scale bar of 100 nm; Figure 3 It can be seen that both ELP nanomicelles and CCM@Arg / ELP nanomicelles exhibit a spherical morphology.

[0050] Figure 4The figures show the particle size and potential test results for ELP nanomicelles, CCM, and CCM@Arg / ELP nanomicelles in Test Example 1. The left side shows the particle size test results, and the right side shows the potential test results. Figure 4 It can be seen that the diameter of CCM@Arg / ELP nanomicelles is 176 nm, similar to that of the CCM control group, but larger than that of ELP nanomicelles (50 nm), indicating that CCM successfully encapsulates ELP nanomicelles; at the same time, Figure 4 It can be seen that the potential of CCM@Arg / ELP nanomicelles is -15mV.

[0051] Figure 5 The figure shows the stability test results of ELP nanomicelles and CCM@Arg / ELP nanomicelles in Test Example 1. Specifically, the newly prepared nanomicelle samples (i.e., the dispersion medium is PBS) were incubated at 4℃ for 0, 6 h, 12 h, 24 h and 48 h. The hydrodynamic diameter of ELP nanomicelles and CCM@Arg / ELP nanomicelles was measured by dynamic light scattering (DLS). The stability was characterized by the change in the sample diameter. The results show that, similar to ELP nanomicelles, CCM@Arg / ELP nanomicelles can still maintain their particle size well after incubation in PBS at 4℃ for 48 h, indicating that they have good stability.

[0052] Test Example 2 The ELP / Rapa nanomicelles used in this test example are specifically the L-Arg-containing ELP / Rapa nanomicelles prepared in step (1) of Example 1.

[0053] Figure 6The figure shows the glutathione-responsive release test results of ELP / Rapa nanomicelles in Test Example 2. Specifically, ELP / Rapa nanomicelles were encapsulated in 10 kDa dialysis bags (dialysis fluid was 1 mM PBS, and the volume ratio of dialysis fluid to external fluid was 1:50; the dialysis fluid in the glutathione (GSH) response group contained 10 mM GSH, and 10% fresh GSH-containing dialysis fluid was replaced every 2 hours). At regular intervals, a small amount of the dialysis bag fluid was mixed with acetonitrile at a volume ratio of 1:1. The Rapa content in the resulting mixture was determined by HPLC, and the change in Rapa content in the dialysis bag over time was calculated based on the Rapa standard curve, thereby calculating the release of Rapa over time. The results showed that in the presence of GSH, ELP / Rapa nanomicelles achieved a significantly faster release rate within 12 hours. Notably, at 2 h and 4 h, the release rates of the GSH-containing ELP / Rapa nanomicelles were 36% and 53%, respectively, which were twice the release rates of the GSH-free ELP / Rapa nanomicelles at the same time interval. These results indicate that the ELP nanomicelles in this invention can effectively encapsulate hydrophobic chemotherapeutic drugs (i.e., the lipid-soluble chemotherapeutic drug Rapa) in the core based on hydrophobic interactions, and rapidly release the payload through the responsive cleavage of disulfide bonds in the coronal layer in response to glutathione.

[0054] Test Example 3 To demonstrate that CCM-coated ELP nanomicelles can fuse with cancer cells and then deliver the payload into the cytoplasm, in this test case, the CCM shell and ELP core were labeled with NBD (green) and Cy5 (red), respectively. The labeled nanomicelles (CCM-NBD@Arg / ELP-Cy5 and ELP-Cy5) were incubated with MCF-7 cells for 2 h, and then imaged using CLSM and analyzed using ImageJ software.

[0055] The preparation method of CCM-NBD@Arg / ELP-Cy5 nanomicelles used in this test example is as follows: CCM was labeled with DSPE-NBD (the resulting labeled product is abbreviated as CCM-NBD). Specifically, 10 mg of DSPE-NBD was dissolved in chloroform and a membrane was prepared by thin-film hydration. Then, 1 mL of CCM in PBS solution (CCM concentration of 0.3 mg / mL) was added, and hydration was carried out at 37°C for 30 min. After hydration, the membrane was extruded through a 1 µm polycarbonate porous membrane for 10 cycles, followed by extrusion through a 400 nm polycarbonate porous membrane for 10 cycles to obtain CCM-NBD. ELP was modified with Cy5 and then self-assembled to form ELP-Cy5 nanomicelles. Specifically, Cy5 modification was performed during the ELP extraction process, that is, after the first ITC cycle, ELP and NHS-Cy5 solution (0.4 mg / mL) were mixed at pH=8. The reaction was carried out at 25℃ and 100 rpm for 1 hour, followed by a second ITC cycle for extraction and purification to obtain Cy5-modified ELP monomers. Then, using the Cy5-modified ELP monomers, ELP-Cy5 nanomicelles were finally prepared according to the preparation method of ELP nanomicelles. Subsequently, CCM-NBD@Arg / ELP-Cy5 nanomicelles were prepared by combining the ELP-Cy5 nanomicelles with CCM-NBD, according to the preparation method of CCM@Arg / ELP nanomicelles.

[0056] Figure 7 This image shows the isomorphic membrane fusion that occurred after 2 hours of incubation of nanomicelles (CCM-NBD@Arg / ELP-Cy5 or ELP-Cy5) with MCF-7 cells in Test Example 3. The left side is a fluorescence distribution image, and the right side is a grayscale quantization image of the red and green channels in the region indicated by the white dashed line in the fluorescence distribution image, generated using ImageJ software. Figure 7 It was observed that the green fluorescence signal corresponding to the CCM-NBD ​​coating layer diffused along the cell membrane in the CCM-NBD@Arg / ELP-Cy5 nanomicelle group, indicating membrane fusion. For the red fluorescence of the ELP-Cy5 core, some of it co-localized well with the green fluorescence of the CCM-NBD ​​coating layer, while the remainder was found to diffuse into the cytoplasm. This suggests that the CCM-NBD@Arg / ELP-Cy5 nanomicelles deliver the ELP-Cy5 core into MCF-7 cells via membrane fusion. In contrast, in the ELP-Cy5 nanomicelle group (nanomittles without the CCM coating layer), almost all the red fluorescence signal was located intracellularly, indicating an endocytic uptake pathway.

[0057] Test Example 4 The targeted cytotoxicity of CCM-coated ELP nanomicelles on breast cancer cells was evaluated using MCF-7, HeLa, and 4T1 cells. Specifically, cells were incubated with nanomicelles or the free compound under near-infrared light for 2 h, then the nanomicelles and the free compound were removed, and the cells were cultured in a medium free of nanomicelles and the free compound for 46 h before viability was assessed.

[0058] The ELP / Rapa nanomicelles used in this test example are specifically the L-Arg-containing ELP / Rapa nanomicelles prepared in step (1) of Example 1.

[0059] The preparation method of CCM@Arg / ELP / Rapa nanomicelles used in this test example is as follows: the method for preparing CCM / IR780@Arg / ELP / Rapa nanomicelles in Example 1 is followed, except that IR780 is omitted.

[0060] Figure 8 This is a cell viability graph from Test Example 4. The left side shows the time-dependent viability of MCF-7 cells after treatment with nanomicelles or free drug, while the right side shows the viability of HeLa, 4T1, and MCF-7 cells after contact with nanomicelles or free drug. Figure 8 The results showed that treatment with CCM / IR780@Arg / ELP / Rapa nanomicelles resulted in cell viability rates of 38% for HeLa cells and 24% for 4T1 cells, while the cell viability rate of MCF-7 cells was only 7-9%, indicating that homologous membrane fusion has strong targeting specificity. CCM@Arg / ELP / Rapa nanomicelle treatment also significantly reduced the induced cell viability (50%) in MCF-7 cells compared to HeLa (75%) and 4T1 cells (76%), confirming CCM encapsulation-dependent targeting. In stark contrast, treatment with ELP / Rapa nanomicelles, IR780, or Rapa showed similar cytotoxicity in all three cell lines. These results confirm the selective cytotoxicity of CCM-encapsulated ELP nanomicelles on breast cancer cells.

[0061] Test Example 5 Based on the strong in vitro anticancer efficacy of CCM-coated ELP nanomicelles, this test case evaluates its tumor accumulation ability, i.e., biological targeting. Specifically, mice with unilateral tumors were intravenously injected with ICG-coupled nanomicelles (CCM@Arg / ELP-ICG and ELP-ICG) or free ICG, and then in vivo fluorescence imaging was performed under near-infrared irradiation for 24 hours.

[0062] The preparation method of ELP-ICG nanomicelles used in this test example is as follows: ICG is modified at the end of ELP and then self-assembled to form ELP-ICG nanomicelles. Specifically, ICG modification is performed during the ELP extraction process. After the first ITC cycle, ELP and NHS-ICG solution (0.4 mg / mL) are reacted at pH=8, temperature of 25℃ and stirring speed of 100 rpm for 1 h. Then, a second ITC cycle is performed to extract and purify the ELP monomer modified with ICG. Then, the ELP monomer modified with ICG is used and the preparation method of ELP nanomicelles is followed to finally prepare ELP-ICG nanomicelles.

[0063] The preparation method of CCM@Arg / ELP-ICG nanomicelles used in this test example is as follows: using the ELP-ICG nanomicelles, the preparation method of CCM@Arg / ELP nanomicelles is followed to finally prepare CCM@Arg / ELP-ICG nanomicelles.

[0064] Figure 9 The following are images from the biological targeting test in Test Example 5: (a) is a schematic diagram of the in vivo biodistribution assessment scheme; (b) is an in vivo fluorescence image; (c) is a quantitative intensity analysis of tumor-bearing mice at different time points after administration of CCM@Arg / ELP-ICG nanomicelles, ELP-ICG nanomicelles, or free ICG; (d) is an in vitro fluorescence image; and (e) is a quantitative intensity analysis of tumor tissue and major organs of tumor-bearing mice within 24 hours after administration of CCM@Arg / ELP-ICG nanomicelles, ELP-ICG nanomicelles, or free ICG (where H: heart; Li: liver; S: spleen; Lu: lung; K: kidney; T: tumor).

[0065] Depend on Figure 9 As shown in (b) and (c), 0.5 h post-injection, all groups (CCM@Arg / ELP-ICG nanomicelles, ELP-ICC nanomicelles, and ICG) exhibited fluorescent signals at the tumor site, indicating tumor delivery via systemic circulation. In the CCM@Arg / ELP-ICG nanomicelles group, the fluorescence intensity at the tumor site gradually increased, reaching a maximum at 6 h post-injection, indicating active tumor targeting mediated by CCM. Notably, even after 24 h, the tumor preferentially retained CCM@Arg / ELP-ICG nanomicelles. Although ELP-ICG nanomicelles showed similar accumulation at the tumor site due to passive EPR targeting, their tumor fluorescence intensity was significantly lower than that of CCM@Arg / ELP-ICG nanomicelles. Conversely, free ICG showed a rapid decay of fluorescence signal over time, indicating poor tumor retention.

[0066] like Figure 9As shown in (a), to further investigate the in vivo biodistribution of the nanomicelles, major organs and tumor tissues were collected 24 hours after injection for ex vivo imaging. Figure 9 As shown in (d), the free ICG group exhibited negligible fluorescence signals in all organs and tumor tissues, attributed to rapid clearance. CCM@Arg / ELP-ICG nanomicelles and ELP-ICC nanomicelles showed similar organ retention in the liver, lungs, kidneys, spleen, and heart, indicating a comparable clearance pathway via the reticuloendothelial system (RES). Notably, CCM@Arg / ELP-ICG nanomicelles produced stronger fluorescence in tumor tissues compared to ELP-ICG nanomicelles. Figure 9 The quantitative analysis results shown in (e) confirm that CCM@Arg / ELP-ICG nanomicelles are more effective. These results indicate that CCM-coated ELP nanomicelles exhibit effective tumor-selective accumulation and long-term retention, which are fundamental pharmacokinetic characteristics for anticancer efficacy.

[0067] Test Example 6 This test case evaluated the antitumor efficacy of CCM-coated ELP nanomicelles (i.e., CCM / IR780@Arg / ELP / Rapa nanomicelles) versus those without CCM-coated ELP nanomicelles (i.e., ELP / Rapa nanomicelles). Specifically, unilateral tumor-bearing mice were intravenously injected with 200 μL of CCM / IR780@Arg / ELP / Rapa nanomicelles, CCM@Arg / ELP / Rapa nanomicelles, ELP / Rapa nanomicelles, IR780, Rapa, or PBS. On days 0, 2, 4, and 6, all mice were exposed to near-infrared radiation for 8 minutes 6 hours after injection. Body weight and tumor volume were monitored every other day.

[0068] Figure 10 The following are the results of the antitumor efficacy test in Test Example 6: (a) is a schematic diagram of the in vivo antitumor performance evaluation scheme; (b) is a graph showing the weight monitoring results of tumor-bearing mice treated with nanomicelles or free compounds during the treatment period; (c) is a graph showing the tumor volume monitoring results of tumor-bearing mice under different treatment methods; and (d) is a graph showing the weight test results of tumors removed from mice under different treatment methods on day 14.

[0069] Figure 11 These are representative images of tumors obtained from each group on day 14 in test case 6. Figure 12 Representative photographs of tumor-bearing mice treated with nanomicelles or free compounds on days 0, 7, and 14 in Test Example 6.

[0070] like Figure 10As shown in (b), the weight loss in mice treated with nanomicelles was negligible, comparable to the PBS control group, indicating excellent biocompatibility. After 14 days of treatment, significant tumor necrosis was observed in the CCM / IR780@Arg / ELP / Rapa nanomicelle group, as shown in (b). Figure 12 As shown. Notably, tumors in the CCM / IR780@Arg / ELP / Rapa nanomicelle group showed slight growth before day 8, followed by gradual shrinkage starting from day 9. In contrast, treatment with CCM@Arg / ELP / Rapa nanomicelles, ELP / Raba nanomicelles, IR780, Rapa, or PBS showed limited tumor-suppressive activity, such as... Figure 10 As shown in (c) and (d), these results confirm the potent in vivo anticancer activity of CCM / IR780@Arg / ELP / Rapa nanomicelles.

[0071] To further evaluate the anticancer effect, all mice were sacrificed on day 14, and tumor tissue and major organs were isolated and collected. Figure 11 As shown in the representative photographs of isolated tumor tissues, the CCM / IR780@Arg / ELP / Rapa nanomicelles group had the smallest tumor volume compared to other treatment groups. The average tumor weight in the CCM / IR780@Arg / ELP / Rapa nanomicelles group was 0.2 g, a reduction of 87% compared to the PBS control group (1.5 g). In comparison, the tumor weight inhibition rates of the CCM@Arg / ELP / Rapa nanomicelles, ELP / Rapa nanomicelles, IR780, and Rapa groups were 0.6 g, 1.0 g, 0.5 g, and 1.1 g, respectively, with corresponding tumor weight inhibition rates of 56%, 34%, 67%, and 23%. These results clearly demonstrate the superior anticancer performance of CCM / IR780@Arg / ELP / Rapa nanomicelles.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A biomembrane fusion nanomicelle, comprising elastin-like polypeptide nanomicelles, a cancer cell membrane coated on the surface of the elastin-like polypeptide nanomicelles, L-arginine located between the elastin-like polypeptide nanomicelles and the cancer cell membrane, and a lipid-soluble photosensitizer located between the phospholipid bilayers of the cancer cell membrane.

2. The biomembrane-fused nanomicelles according to claim 1, characterized in that, The ratio of the amount of cancer cell membrane, elastin-like polypeptide nanomicelles, lipid-soluble photosensitizer and L-arginine is 0.15~0.3mg:0.1~0.2mg:0.4~1.5nmol:5mg.

3. The biomembrane fused nanomicelles according to claim 1 or 2, characterized in that, The lipid-soluble photosensitizer includes photosensitizer IR780 or photosensitizer ICG.

4. The biomembrane fused nanomicelles according to claim 1 or 2, characterized in that, The cancer cell membrane includes the cell membrane of breast cancer cells.

5. A drug-loaded biomembrane fused nanomicelle, comprising the biomembrane fused nanomicelle according to any one of claims 1 to 4 and a lipid-soluble chemotherapeutic drug encapsulated within elastin-like polypeptide nanomicelles in the biomembrane fused nanomicelle.

6. The drug-loaded biomembrane fused nanomicelles according to claim 5, characterized in that, The ratio of elastin-like polypeptide nanomicelles to lipid-soluble chemotherapeutic drugs in the drug-loaded biomembrane fused nanomicelles is 0.1~0.2mg:5~13nmol.

7. The drug-loaded biomembrane fused nanomicelles according to claim 5 or 6, characterized in that, The lipid-soluble chemotherapy drugs include rapamycin or doxorubicin.

8. A method for preparing drug-loaded biomembrane fused nanomicelles according to any one of claims 5 to 7, comprising the following steps: An elastin-like polypeptide solution was mixed with a lipid-soluble chemotherapy drug solution and encapsulated. The resulting encapsulated material was then cross-linked to obtain a drug-loaded nanomicelle solution. The drug-loaded nanomicelle solution was mixed with an L-arginine solution to obtain a drug-loaded nanomicelle solution containing L-arginine. The cancer cell membrane solution was mixed with a lipid-soluble photosensitizer and then co-extruded for the first time to obtain a cancer cell membrane solution loaded with a lipid-soluble photosensitizer. The drug-loaded nanomicelles containing L-arginine were mixed with a cancer cell membrane solution loaded with a lipid-soluble photosensitizer, and then co-extruded a second time to obtain the drug-loaded biomembrane fused nanomicelles.

9. The application of the biomembrane fused nanomicelles according to any one of claims 1 to 4, the drug-loaded biomembrane fused nanomicelles according to any one of claims 5 to 7, or the drug-loaded biomembrane fused nanomicelles prepared by the preparation method according to claim 8 in the preparation of cancer therapeutic agents.

10. The application according to claim 9, characterized in that, The cancers mentioned include breast cancer.

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