Lipid-anchored cell-derived vesicles and uses thereof

Abstract

The invention discloses a lipid-anchored cell-derived vesicle and application thereof, the lipid-anchored cell-derived vesicle is prepared from monoacyl phosphatidylcholine and a cell membrane, and the monoacyl phosphatidylcholine is constructed by taking choline glycerophosphate as a hydrophilic head group and connecting an ester bond with a hydrophobic group. The invention designs a method for enhancing the stability of the cell-derived vesicle by anchoring the cell-derived vesicle with monoacyl phosphatidylcholine, which can prolong the blood circulation half-life period of the drug and improve the delivery effect of the tumor site, thereby enhancing the overall treatment effect of the drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a lipid-anchored cell-derived vesicle and its application. Background Technology

[0002] Driven by emerging and rapidly developing biomimetic technologies, the focus of nanomedicine research is gradually shifting from artificially synthesized nanocarriers to biomimetic nanocarriers with unique functionalities. Biomimetic nanotechnology utilizes endogenous substances such as cell membranes to construct nanocarriers, providing them with endogenous membrane lipids, proteins, and polysaccharides that mimic cell membrane surface features, thus enabling natural intercellular interactions and ultimately maximizing the multifunctionality of biomimetic nanomedicine systems. Based on this concept, cell membranes have been directly designed and developed into highly functional cell-derived vesicles (MVs). These MVs possess a phospholipid bilayer structure similar to liposomes, while retaining endogenous membrane lipids, proteins, and polysaccharides that mimic cell membrane surface features, providing natural intercellular interactions and maximizing the multifunctionality of biomimetic nanomedicine systems, showing broad application prospects. However, cell-derived vesicles lack renewal and repair capabilities, their intracellular supporting proteins are stripped and destroyed, and they have a greater surface curvature, resulting in poor physical stability. Over time, they are easily destroyed or undergo vesicle fusion, greatly limiting their long-term stable application.

[0003] Existing solutions to this problem include: using synthetic nanoparticles with stable physicochemical properties to provide physical support for fragile vesicles; however, carriers prepared using different methods and materials exhibit significant quality variations, making in vitro and in vivo evaluation of such drug delivery systems extremely difficult; using hydrophilic polymers to form a protective hydration layer on the surface of cell-derived vesicles to prevent vesicles from agglomerating during storage and being damaged by shear forces during blood circulation; however, this polymer surface modification strategy can significantly hinder the interaction between the cell membrane and target cells; fusing cholesterol or other small-molecule hydrophobic lipids with bimodal phospholipid effects to vesicles through co-incubation, freeze-thaw cycles, sonication, or extrusion is also a reasonable approach; however, the anchoring of hydrophobic regulatory lipids inevitably requires the use of organic solvents, which cannot avoid damaging the production of surface proteins on cell-derived vesicles. Summary of the Invention

[0004] To address the problem of insufficient stability of cell-derived vesicles, this invention proposes using monoacylphosphatidylcholine with a rigid ring structure to anchor cell-derived vesicles, regulate the cell membrane phospholipid bilayer, and increase vesicle stability.

[0005] One objective of this invention is to provide a lipid-anchored cell-derived vesicle made of monoacylphosphatidylcholine and cell membrane, wherein the mass ratio of monoacylphosphatidylcholine to cell membrane protein is 0.01:1-10:1.

[0006] The monoacylphosphatidylcholine is obtained by linking glycerophosphate choline and a hydrophobic molecule through an ester bond, wherein the hydrophobic molecule is a molecule with a rigid ring structure.

[0007] Furthermore, the hydrophobic molecule is selected from cholesterol, 10-(benzyloxy)-10-oxodecanoic acid, all-trans retinoic acid, abiraterone, belinositol, or vorinositol.

[0008] Furthermore, the cell membrane is a red blood cell membrane, platelet membrane, white blood cell membrane, tumor cell membrane, or mesenchymal stem cell membrane.

[0009] In one embodiment of the present invention, the mass ratio of monoacylphosphatidylcholine (MCPC) to erythrocyte membrane proteins is 0.2:1-1.5:1.

[0010] In another embodiment of the present invention, the mass ratio of monoacylphosphatidylcholine (MAPC) to erythrocyte membrane proteins is 0.08:1-0.5:1.

[0011] In a third embodiment of the present invention, the mass ratio of monoacylphosphatidylcholine (MAPC) to platelet membrane proteins is 0.2:1-0.6:1.

[0012] Furthermore, the aforementioned lipid-anchored cell-derived vesicles also contain an active drug, which is a water-soluble drug.

[0013] Furthermore, the molar ratio of the monoacylphosphatidylcholine to the active drug is 0.5:1-10:1.

[0014] In one embodiment of the present invention, doxorubicin (DOX) was used as a model drug to investigate the inhibitory effect of erythrocyte-derived vesicles anchored to MCPCs on triple-negative breast cancer (TNBC).

[0015] In a second embodiment of the present invention, the photothermal therapeutic agent indocyanine green (ICG) was used as a model drug to investigate the inhibitory effect of erythrocyte-derived vesicles anchored to MCPCs on melanoma.

[0016] In a third embodiment of the present invention, doxorubicin (DOX) was used as a model drug to investigate the inhibitory effect of erythrocyte-derived vesicles anchored with MAPC on TNBC tumors rich in tumor stem cells (CSCs).

[0017] In a fourth embodiment of the present invention, doxorubicin (DOX) was used as a model drug to investigate the inhibitory effect of platelet-derived vesicles anchored to MAPC on TNBC tumors rich in tumor stem cells (CSC).

[0018] The second objective of this invention is to provide a method for preparing the above-mentioned lipid-anchored cell-derived vesicles, wherein monoacylphosphatidylcholine is dissolved in an aqueous solution, then added to a cell membrane, incubated, centrifuged, the precipitate is collected, and extruded through a liposome extruder to obtain the lipid-anchored cell-derived vesicles.

[0019] In one embodiment of the present invention, the aqueous solution is a phosphate buffer solution.

[0020] A third objective of this invention is to provide the application of the above-mentioned lipid-anchored cell-derived vesicles in the preparation of tumor therapeutic drugs.

[0021] The lipid-anchored cell-derived vesicles proposed in this invention exhibit high in vitro physical stability, prolonging the drug's blood circulation half-life and improving delivery to tumor sites, thereby enhancing the overall therapeutic effect. The lipid prodrug with the original drug's pharmacodynamics, used to stabilize cell-derived vesicles, can exert a dual effect of promoting vesicle stabilization and tumor stem cell differentiation.

[0022] The method provided by this invention does not rely on organic solvents and can achieve lipid anchoring under mild conditions in an aqueous buffer solution, thereby improving the therapeutic effect of cell-derived vesicle drug delivery. Attached Figure Description

[0023] Figure 1 In the text, a represents several MPC synthesis methods and structural formulas in Example 1; b represents the MAPC synthesis method and structural formula in Example 1.

[0024] Figure 2 In Example 1, a represents the change in particle size of different types of MPC-anchored EMV over time under different treatment conditions; b represents the change in particle size of cPMV over time under different treatment conditions; and c represents the change in particle size of cMMV over time under different treatment conditions.

[0025] Figure 3 The fluorescence polarization values ​​are caused by different types and concentrations of MPC anchoring EMV in Example 1.

[0026] Figure 4 In Example 1, a is the bilayer diagram of phospholipids mixed with different MPC and POPC as simulated by AMD; b is the change of order parameters over time after mixing different MPC and POPC as calculated by AMD in Example 1; c is the change of average lipid area over time after mixing different MPC and POPC as calculated by AMD in Example 1.

[0027] Figure 5 In Example 1, a is a phospholipid bilayer diagram of the mixture of MAPC and POPC simulated by AMD; b is the change of order parameters of the mixture of MAPC and POPC calculated by AMD over time in Example 1; c is the change of average lipid area of ​​the mixture of MAPC and POPC calculated by AMD over time in Example 1.

[0028] Figure 6 In Example 1, a represents the EPR scan spectra of 16-DSA-labeled EMVs; b represents the order parameters calculated based on the 16-DSA-labeled EPR scan spectra in Example 1; c represents the isotropic hyperfine coupling constant calculated based on the 16-DSA-labeled EPR scan spectra in Example 1; d represents the EPR scan spectra of 5-DSA-labeled EMVs in Example 1; and e represents the average lipid area calculated based on the 5-DSA-labeled EPR scan spectra in Example 1.

[0029] Figure 7 The particle size distribution and transmission electron microscopy results of cEMV in Example 1 are shown in the figure. Scale bar: 50 nm.

[0030] Figure 8 In the figures, a represents the pharmacokinetic curves of Cy5-EMV and Cy5-cEMV in Example 2; b represents the fluorescence intensity values ​​of different tissues collected from 4T1 tumor-bearing mice after administration of Cy5-EMV and Cy5-cEMV in Example 2; c represents the tumor growth curves of 4T1 tumor-bearing mice after different treatments in Example 3; d represents tumor photographs 14 days after different treatments in Example 3; e represents the tumor weight 14 days after different treatments in Example 3; f represents the tumor inhibition rate 14 days after different treatments in Example 3; g represents the HE staining examination of tumors 14 days after different treatments in Example 3 (scale bar: 100 μm); and h represents the weight changes of mice 14 days after different treatments in Example 3.

[0031] Figure 9 The images show fluorescence imaging of different tissues collected from 4T1 tumor-bearing mice after administration of Cy5-EMV and Cy5-cEMV in Example 2.

[0032] Figure 10 In Example 4, a represents the absorbance value change of ICG / cEMV in the range of 700-900nm; b represents the temperature change of ICG / cEMV irradiated at different powers in Example 4; c represents the temperature change of continuous irradiation of ICG or ICG / cEMV in Example 4; and d represents the temperature change of continuous irradiation of ICG / cEMV with different ICG concentrations in Example 4.

[0033] Figure 11In Example 4, a is a thermal imaging image of melanoma-bearing mice after ICG / cEMV administration; b is the temperature change of melanoma-bearing mice after ICG / cEMV administration.

[0034] Figure 12 In Example 4, a represents the individual tumor growth curves of B16F10 tumor-bearing mice after different treatments; b represents the average tumor growth curves of each group of B16F10 tumor-bearing mice after different treatments in Example 4; and c represents the HE staining examination of the tumor and the skin near the tumor 14 days after different treatments in Example 4, with a scale bar of 500 μm.

[0035] Figure 13 The survival rate of cells treated with DOX / aEMV containing different MAPC:DOX molar ratios in Example 5 is shown.

[0036] Figure 14 In the text, 'a' represents the ALDH levels of cells after different treatments in Example 5. + and percentage of cell population; b represents Sca-1 cells after different treatments in Example 5. + Percentage of cell population; c represents Nanog of cells after different treatments in Example 5. + Percentage of the cell population.

[0037] Figure 15 In the table, a represents the in vitro limited dilution analysis of cells after different treatments in Example 5; b represents the change in the number of tumor spheres (diameter > 50 μm) after different treatments in Example 5.

[0038] Figure 16 In Example 5, a represents the tumor growth curves of CSC-rich 4T1 tumor-bearing mice after different treatments; b represents the tumor images of CSC-rich 4T1 tumor-bearing mice 14 days after different treatments; c represents the tumor weight 14 days after different treatments; d represents the tumor inhibition rate 14 days after different treatments; and e represents the HE staining examination of the tumors 14 days after different treatments in Example 5. Scale bar: 50 μm.

[0039] Figure 17 Nanog immunohistochemical staining of tumors was performed 14 days after different treatments in Example 5. Scale bar: 100 μm.

[0040] Figure 18In the figures, a represents bioluminescence images of mice after different treatments in Example 6; b represents the changes in individual bioluminescence intensity of mice after different treatments in Example 6; c represents the changes in average bioluminescence intensity of mice after different treatments in Example 6; d represents lung images 14 days after different treatments in Example 6; e represents the number of lung tumor lesions 14 days after different treatments in Example 6; and f represents HE staining examination of the lungs 14 days after different treatments in Example 6. Scale bar: 100 μm.

[0041] Figure 19 Nanog immunohistochemical staining of tumors was performed 14 days after different treatments in Example 6. Scale bar: 100 μm.

[0042] Figure 20 In Example 7, a represents the change in RBC count in mice after different treatments compared to saline treatment; b represents the change in PLT count in mice after different treatments compared to saline treatment; c represents the change in WBC count in mice after different treatments compared to saline treatment; d represents the change in serum ALT level in mice after different treatments compared to saline treatment; e represents the change in serum AST level in mice after different treatments compared to saline treatment; f represents the change in serum BUN level in mice after different treatments compared to saline treatment; and g represents the change in serum CRE level in mice after different treatments compared to saline treatment. Detailed Implementation

[0043] This invention designs a method for anchoring cell-derived vesicles (MVs) with monoacylphosphatidylcholine (MPC) to increase MV stability, thereby achieving lipid anchoring under mild conditions.

[0044] Specifically, the monoacylphosphatidylcholine (MPC) is constructed by linking a hydrophilic head group to a hydrophobic group via an ester bond using glycerophosphocholine as the hydrophilic head group. Since glycerophosphocholine is the most abundant hydrophilic head group of lipids in the cell membrane, the amphiphilic lipids constructed using it to anchor the cell membrane exhibit optimal biocompatibility. Although glycerophosphocholine has two hydroxyl groups that can be modified, this invention only modifies a single chain. The hydrophobic lipids, by linking their 1-OH groups via ester bonds to construct the monoacyl structure MPC, exhibit good water solubility and efficient membrane insertion. The hydrophobic group can be a long chain or a rigid ring structure such as cholesterol and 10-(benzyloxy)-10-oxodecanoic acid. When a lipid with a rigid ring structure is selected, the MPC formed by linking it with glycerophosphocholine exerts an ordering and aggregation effect on the phospholipid bilayer of the cell membrane, thereby improving the overall stability of the cell membrane structure. MPC can be prepared as an aqueous solution and simply incubated with the cell membrane to achieve anchoring. The anchored MV exhibits higher in vitro and in vivo stability. In addition to general hydrophobic lipids, MPC lipid prodrugs can also be synthesized from clinically used drugs such as all-trans retinoic acid, abiraterone, belinositol, vorinositol, or other drug molecules containing rigid ring structures. The rigid rings of these molecules conform to the effect structure rules of stable MPCs, and they have the dual functions of therapeutic agents and MV stabilizers.

[0045] First, this invention synthesized a series of MPCs. Among these synthesized MPCs, monocholesterol phosphatidylcholine (MCPC), monobenzyloxydecyl phosphatidylcholine (MBDPC), and the lipid prodrug monoall-trans phosphatidylcholine (MAPC) with rigid acyl ring structures were screened to show significant MV stabilizing effects. Then, the fluorescence polarization values ​​of erythrocyte-derived vesicles (EMVs) anchored by different MPCs were measured using a 1,6-diphenyl-1,3,5-hextriene (DPH) probe. Atomic-molecular dynamics simulations (AMD) were used to calculate the parameter changes caused by MPC anchoring, and electron paramagnetic resonance (EPR) probes were used to measure the changes in EMV physicochemical parameters caused by different MPC anchoring. Then, after preparing MCPC-anchored EMV (cEMV), Cy5-cEMV was obtained by fluorescently labeling cEMV with Cy5, and similarly, Cy5-EMV was obtained by fluorescently labeling EMV with Cy5. The pharmacokinetics and tumor targeting in mice were investigated using Cy5-EMV and Cy5-cEMV. Using a hypotonic method, the chemotherapeutic drug doxorubicin (DOX) was encapsulated in EMV and cEMV, respectively, to obtain DOX / EMV and DOX / cEMV, to investigate the effect of DOX / cEMV on inhibiting the growth of triple-negative breast cancer (TNBC) tumors. Using a hypotonic method, the photothermal therapeutic agent indocyanine green (ICG) was encapsulated in EMV and cEMV, respectively, to obtain ICG / EMV and ICG / cEMV, to investigate the photothermal effect of ICG / cEMV and its efficacy in the treatment of melanoma. MAPC-anchored EMV (aEMV) was prepared, and DOX was encapsulated using a hypotonic method to prepare DOX / aEMV. The in vitro and in vivo effects of DOX / aEMV on inhibiting the growth of tumor stem cell-rich TNBC tumors were investigated. Platelet-derived vesicles (PMVs) were prepared using platelet membranes, and aPMVs were obtained by MAPC anchoring. DOX was then encapsulated in aPMVs using a hypotonic method to prepare DOX / aPMVs. Similarly, DOX was encapsulated in PMVs using a hypotonic method to prepare DOX / PMVs. The in vivo effects of DOX / aPMV on inhibiting CSC-rich TNBC lung metastases were investigated. Finally, the in vivo safety of DOX / cEMV, ICG / cEMV, DOX / cEMV, and DOX / aPMV was evaluated.

[0046] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0048] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0049] Example 1

[0050] Screening of MPCs and study on the mechanism of MV stabilization

[0051] I. Synthesis and Characterization of MPC

[0052] like Figure 1 As shown in Figure a, 0.582 g of stearic acid was mixed with excess thionyl chloride and heated under reflux for 6 h. After the reflux was completed, the excess thionyl chloride was removed by rotary evaporation to obtain stearoyl chloride. 0.5 g of glycerophosphate choline and 0.51 g of dibutyltin oxide were added to isopropanol and heated under reflux. After reflux was completed, 0.1 mL of triethylamine was added and stirred thoroughly. Stearoyl chloride was then dissolved in dichloromethane and added dropwise to the reaction solution, and the reaction continued for 16 h. The mixture was purified using a silica gel column to obtain monostearoylphosphatidylcholine (MSPC). Similarly, by replacing stearic acid with palmitic acid (0.525 g), cinnamic acid (0.467 g), lauric acid (0.41 g), oleic acid (0.604 g), transoleic acid (0.604 g), linoleic acid (0.601 g), and 10-(benzyloxy)-10-oxodecanoic acid (0.626 g), MPPC, MTPC, MDPC, MOPC, MLPC, MEPC, and MBDPC were prepared using the same method. MCPC was synthesized by replacing stearoyl chloride with cholesterol chloroformate (0.959 g).

[0053] like Figure 1 As shown in Figure b, 0.45 g of all-trans retinoic acid and 0.585 g of N,N-carbazyldiimidazole (CDI) were added to dichloromethane and heated to reflux. 0.31 g of glycerophosphate choline and 0.456 g of 1,8-diazobispirocyclo[5.4.0]undecyl-7-ene (DBU) were dissolved in dimethyl sulfoxide and then heated to reflux. Subsequently, the two solutions were mixed and heated to reflux for 18 h. Preliminary purification was performed using silica gel column chromatography, followed by preparative liquid chromatography purification to obtain MAPC.

[0054] All the MPCs prepared above were confirmed to be correct by high-resolution mass spectrometry, proton nuclear magnetic resonance (NMR) spectroscopy, and carbon NMR spectroscopy.

[0055] II. Screening of MPCs with MV stabilization function

[0056] Preparation of erythrocyte membrane: Blood was collected from the canthi of BALB / c mice and placed in test tubes pre-treated with EDTA anticoagulation. The obtained whole blood was centrifuged at 600g for 5 min to remove plasma and the intermediate leukocyte layer. The erythrocyte pellet at the bottom was collected and washed twice with cold PBS. The erythrocyte pellet was then resuspended in hypotonic 0.25×PBS at a volume ratio of 1:9 and incubated on ice for 30 min to allow for complete lysis. The lysed erythrocyte solution was centrifuged at 10000g for 10 min, the supernatant containing hemoglobin was discarded, and the solution was washed three times with PBS. The pink precipitate obtained at the bottom of the centrifuge tube was the erythrocyte membrane.

[0057] Platelet membrane preparation: Whole blood from BALB / c mice was collected and placed in EDTA-anticoagulated tubes. After centrifugation at 100g for 20 min, the supernatant containing platelet-rich plasma was collected. 10 mL of the platelet-rich plasma was further centrifuged at 800g for 20 min to allow platelets to settle, and the precipitate was collected. The collected platelet precipitate was dispersed in 10 mL of PBS containing 1 mM EDTA, 2 μM prostaglandin E1, and one protease inhibitor tablet, and subjected to three freeze-thaw cycles between -80℃ and 25℃. The frozen-thawed platelet solution was centrifuged at 4000g for 3 min, the supernatant was discarded, and the precipitate was washed three times with PBS to obtain the platelet membrane precipitate.

[0058] Mononuclear cell membrane preparation: BALB / c mice were euthanized by cervical dislocation, and the tibia and femur were obtained under aseptic conditions. The bones were cut open at both ends with surgical scissors, and the bone marrow cavity was flushed with sterile PBS. The flushed solution was filtered through gauze to remove bone fragments, yielding bone marrow cells. The bone marrow cells were placed in erythrocyte lysis buffer and incubated at 4°C for 15 min, followed by centrifugation at 800g for 5 min. The cell pellet was collected and washed twice with sterile PBS. The purified and washed cells were seeded into 6-well ultra-low adhesion culture plates in RPMI 1640 medium containing 20 ng / mL macrophage colony-stimulating factor and 10% fetal bovine serum, and cultured for 5 days in a cell culture incubator with 1% oxygen. Subsequently, the obtained mononuclear cells (2.5 × 10⁻⁶) were... 7 Mononuclear cells were dispersed in 5 mL of a hypotonic lysis aqueous solution composed of Tris-HCl (30 mM), glucose (75 nM), mannose (225 mM), EDTA (0.5 mM), and half a tablet of protease inhibitor and incubated for 2 h. The cells were then lightly sonicated using an ultrasonic cell disruptor. The treated solution was centrifuged at 10,000 g for 25 min, and the supernatant containing cell membrane fragments was collected. This supernatant was further centrifuged at 120,000 g for 40 min. After high-speed centrifugation, a white precipitate was obtained at the bottom of the centrifuge tube, which is the mononuclear cell membrane.

[0059] 1 mg of cell membrane was dispersed in 2 mL of PBS, and then gently sonicated at low power using an ultrasonic cell disruptor. The unanchored MV was obtained by repeatedly extruding the MV using a liposome extruder.

[0060] Using an ultrasonic cell disruptor, 1.5 mg of different MPCs were dissolved in 1 mL of PBS, and then 1 mg of cell membrane was dispersed in the solution. The mixture was incubated at 37°C for 30 min. The incubated solution was centrifuged at 10000 g, the supernatant was discarded, and the bottom precipitate was collected and washed three times with PBS. The obtained precipitate was then dispersed again in 2 mL of PBS, and gently sonicated using a low-power ultrasonic cell disruptor. The precipitate was then extruded multiple times using a liposome extruder to obtain MPC-anchored MV.

[0061] The hydrodynamic particle size changes of erythrocyte-derived vesicles (EMVs) anchored by different MPCs were investigated under three different treatment conditions. Specifically, T1: MVs were diluted 40-fold with PBS, thoroughly mixed, and the particle size of the diluted samples was measured; T2: MVs were sterilized and incubated at 4°C for 21 days, and the particle size was measured afterward; T3: MVs were frozen at -20°C for 4 hours and thawed at 20°C for 2 hours, repeated 3 times, and the particle size was measured after each freeze-thaw cycle. The untreated group was designated as the nT group, and the percentage change in particle size for each group compared to the nT group was calculated. Figure 2 As shown in Figure a, the MPC anchoring with a rigid ring segment at the hydrophobic end helps improve the stability of EMV, and MBDPC and MCPC were initially screened out.

[0062] Platelet-derived vesicles (PMVs), MCPC-anchored PMVs (cPMVs), monocyte-derived vesicles (MMVs), and MCPC-anchored MMVs (cMMVs) were prepared separately, and their hydrodynamic particle size changes under three different treatment conditions were investigated using the same method. Specifically, T1: MVs were diluted 40-fold with PBS, thoroughly mixed, and the particle size of the diluted samples was measured; T2: MVs were sterilized and incubated at 4°C for 21 days, and the particle size of the samples was measured; T3: MVs were frozen at -20°C for 4 hours and thawed at 20°C for 2 hours, repeated 3 times, and the particle size was measured after each freeze-thaw cycle. The percentage change in particle size of MVs in each group compared to the untreated group was calculated, such as... Figure 2 As shown in Figures b and c, MCPC-mediated MV stability is not model-dependent, but rather exhibits significant universality.

[0063] III. The Mechanism of MV Stabilization

[0064] 1. Investigation of MV cell membrane fluidity

[0065] Using an ultrasonic cell disruptor, different types and concentrations of MPC were dissolved in 1 mL of PBS, and 1 mg of erythrocyte membrane was dispersed in the solution. The mixture was incubated at 37°C for 30 min. The incubated solution was centrifuged at 10000 g, the supernatant was discarded, and the bottom precipitate was collected and washed three times with PBS. The precipitate was then dispersed again in PBS and gently sonicated using a low-power ultrasonic cell disruptor. The precipitate was then extruded multiple times using a liposome extractor to obtain MPC-anchored EMV. EMVs anchored with different concentrations and types of MPC were incubated with 2 μM DPH at 37°C for 0.5 h, washed twice with PBS, and then the fluorescence polarization values ​​were measured using a multi-mode microplate reader at an excitation wavelength of 362 nm and an emission wavelength of 432 nm.

[0066] like Figure 3 As shown, different concentrations and types of monolinear MPC anchoring to the cell membrane did not reduce cell membrane fluidity. However, as the anchoring amount of MBDPC and MCPC increased within a certain range, cell membrane fluidity gradually decreased. There is an optimal amount of MCPC anchoring to reduce fluidity (MCPC incubation concentration of 1.5 mg / mL).

[0067] 2. AMD computing

[0068] AMD was used to simulate the parameters of MSPC, MDPC, MBDPC, MCPC, and MAPC after MV insertion, with cholesterol (Chol) as a control. Specifically, the GROMACS (version 2020.6) molecular dynamics package was used. 1-Palmitoyl-2-oleoyllecithin (POPC) and various other MPCs were described using the CHARMM36 force field, with water molecules simulated using a 3-point transferable intermolecular potential (TIP3P). The POPC bilayer was constructed using the CHARMM-GUI engine and reequilibrated for 100 ns after reaching energy minimization over several thousand steps. After reequilibration, MAPC was distributed within the POPC bilayer using a solvation-sparse strategy to construct a mixed lipid bilayer, which was then reequilibrated for 100 ns. The ordering parameters of POPC and the average lipid area of ​​the mixed lipid bilayer of POPC and MPC were directly calculated from the simulated model. The mean force potential (PMF) was calculated in real-time by pulling POPC lipid molecules out of the mixed bilayer using an umbrella sampling mechanism. The calculation of lipid binding free energy is simplified to the PMF value of POPC completely pulled into water minus the PMF value at the lipid bilayer initiation point. MSPC, MDPC, MBDPC, and MCPC simulations are as follows: Figure 4 In the middle a, MAPC such Figure 5 In section a. Further parameter calculations are as follows: Figure 4 China bc and Figure 5Compared to pure POPC, the anchoring of MBDPC, MCPC, and MAPC, similar to Chol, significantly increased the hydrophobic end order parameter and reduced the surface area occupied by lipid molecules in the mixed membrane. This resulted in a more regular molecular arrangement and tighter molecular stacking in the cell membrane, effects referred to as the ordering effect and aggregation effect, respectively, which overall increased the stability of the MV. Based on PMF calculations, the binding free energy of lipids after anchoring to the cell membrane by different MPCs was found. The anchoring of MBDPC, MCPC, and MAPC increased the binding free energy of the pure POPC membrane from 20.5 kcal / mol to 22.32 kcal / mol, 23.67 kcal / mol, and 22.8 kcal / mol, respectively. This clearly indicates that they have a solidification effect on the cell membrane, improving the physical stability of the MV and making it more resistant to environmental damage. In contrast, MDPC decreased slightly to 20.25 kcal / mol, but MSPC decreased to 17.79 kcal / mol.

[0069] 3. EPR Analysis and Calculation

[0070] EMVs anchored to different MPCs were incubated with 1 μM of 5-doxyl-stearic acid (5-DSA) or 16-doxyl-stearin (16-DSA) at 20 °C for 0.5 h. After incubation, the samples were washed twice with PBS and then placed into glass capillaries. Spectra were obtained using an electron paramagnetic resonance spectrometer at 25 °C and 9.8 GHz. The 5-DSA and 16-DSA spectra were obtained as follows: Figure 6 a and Figure 6 In the middle d. Molecular order parameters and isotropic hyperfine coupling constants were calculated using the obtained 16-DSA spectra, as shown below. Figure 6 The average molecular area is calculated using the obtained 5-DSA spectra, such as... Figure 6 After anchoring with MBDPC, MCPC, and MAPC, the ordered parameters of EMV significantly increased, the isotropic hyperfine coupling constant significantly decreased, and the average lipid area within the cell membrane significantly decreased. This indicates that the regularity of the arrangement of hydrophobic lipid chains within the cell membrane increased, the overall hydrophobicity of the cell membrane increased, the membrane surface became more compact, and the permeability of hydrophilic substances decreased.

[0071] IV. cEMV Characterization

[0072] The hydrodynamic particle size of MCPC-anchored EMV (cEMV) was determined using a potentiometric particle size analyzer. cEMV was appropriately diluted and added dropwise to a copper grid. After the solution evaporated, phosphotungstic acid staining solution was added for counterstaining. After evaporating the solvent again, the morphology of cEMV was characterized using transmission electron microscopy. The results are as follows: Figure 7The prepared monoacylphosphatidylcholine-anchored cell-derived vesicles have complete vesicle morphology, a hydrodynamic particle size of about 120 nm, and a uniform particle size distribution.

[0073] Example 2

[0074] In vivo pharmacokinetic and tumor-targeting evaluation of Cy5-cEMV with different MCPC anchors

[0075] I. In vivo pharmacokinetic evaluation of Cy5-cEMV

[0076] Red blood cell membranes containing 1 mg of membrane proteins were dispersed in 50 μM Sulfo-Cy5-NHS solution and incubated at 37 °C for 30 min. After incubation, the solution was centrifuged at 12000 g for 10 min, the supernatant was discarded, the precipitate was collected, and the membrane was washed three times with PBS. Fluorescently labeled red blood cell membranes were then dispersed in PBS buffer containing 1 mL of MCPC at concentrations of 0 mg / mL, 0.5 mg / mL, 1.5 mg / mL, and 3 mg / mL, respectively, and incubated at 37 °C for 30 min. After incubation, the solution was centrifuged at 10000 g, the supernatant was discarded, the bottom precipitate was collected, and the membrane was washed three times with PBS. Cell membranes were subjected to low-power, gentle sonication using an ultrasonic cell disruptor, followed by multiple extrusions using a liposome extruder to obtain Cy5-EMV, Cy5-cEMV(i) (containing 245 μg / mg MCPC), Cy5-cEMV(ii) (containing 720 μg / mg MCPC), and Cy5-cEMV(iii) (containing 1350 μg / mg MCPC), respectively. Fluorescently labeled vesicles were injected intravenously into the tail vein of healthy BALB / c mice. Approximately 0.1 mL of blood was collected via capillary periorbital sampling at 1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h post-injection, with the mice receiving adequate water intake during these times. The collected blood was immediately centrifuged at 800g for 5 min, and the supernatant plasma was collected, diluted tenfold with PBS, and the fluorescence value of Cy5 in the plasma was quantified using an ELISA reader at an excitation wavelength of 650 nm and an emission wavelength of 670 nm. Figure 8 In section a, the experimental results showed that as the anchoring amount of MCPC increased, the blood retention of cEMV increased. However, MCPC anchoring beyond a suitable amount was easily lost, and cEMV containing 1350 μg / mg MCPC was less effective than cEMV containing 720 μg / mg MCPC. Based on the results, the pharmacokinetic half-lives of Cy5-EMV and Cy5-cEMV(ii) (containing 720 μg / mg MCPC) were calculated to be 7.5 h and 13 h, respectively, with the 720 μg / mg MCPC anchoring amount showing the best stabilization effect.

[0077] II. Evaluation of Cy5-cEMV Tumor Targeting Efficacy

[0078] Mice bearing 4T1 orthotopic breast cancer were injected intravenously with fluorescently labeled vesicles: Cy5-EMV, Cy5-cEMV(i) (containing 245 μg / mg MCPC), Cy5-cEMV(ii) (containing 720 μg / mg MCPC), and Cy5-cEMV(iii) (containing 1350 μg / mg MCPC). After 24 hours, the mice were euthanized by cervical dislocation, and tumor tissue and tissues from the heart, liver, spleen, lungs, and kidneys were dissected. Cy5 fluorescence in various organs of the mice was detected using a small animal in vivo fluorescence imaging system (IVIS) at excitation at 650 nm and emission at 670 nm, and quantification was performed using the ROI function. Results are as follows: Figure 8 b and Figure 9 The results indicate that the Cy5 signal in the tumor of the Cy5-cEMV(ii) group was the strongest compared to that in the Cy5-EMV group, and was significantly higher than that in the Cy5-EMV group; the Cy5 fluorescence intensity in the tumor of the Cy5-cEMV(ii) group was three times that of the Cy5-EMV group. These results demonstrate that the optimal anchoring quantitative MCPC-mediated membrane stabilization enhancement promotes the accumulation of EMV in tumor tissue and has a stronger permeation and retention effect.

[0079] Example 3

[0080] cEMV delivery of DOX inhibits the growth of in situ TNBC tumors.

[0081] I. Preparation of cEMV-loaded DOX

[0082] 1.5 mg of DOX was dissolved in 1 mL of 0.25×PBS, and erythrocyte membranes containing 1 mg of membrane proteins were dispersed in the solution. The mixture was incubated at 4°C for 30 min. After incubation, a calculated amount of 10×PBS was added to raise the overall osmotic pressure of the solution to 1×PBS. After thorough mixing, the solution was transferred to 37°C and incubated for another 90 min. The solution was then centrifuged at 12000 g for 10 min, the supernatant was discarded, and the precipitated DOX-loaded erythrocyte membranes were collected. The cell membranes were gently sonicated at low power using an ultrasonic cell disruptor, and then extruded multiple times using a liposome extruder to obtain DOX / EMV. The DOX-loaded erythrocyte membranes obtained in the previous step were dispersed in 1 mL of PBS buffer containing 0.5 mg / mL, 1.5 mg / mL, and 3 mg / mL of MCPC, respectively, and incubated at 37°C for 30 min. The incubated solutions were centrifuged at 10000 g, the supernatant was discarded, the bottom precipitate was collected, and the solution was washed three times with PBS. The cell membrane was subjected to low-power, gentle sonication using an ultrasonic cell disruptor, followed by multiple extrusions using a liposome extruder to obtain DOX / cEMV (containing 245 μg / mg MCPC), DOX / cEMV (containing 720 μg / mg MCPC), and DOX / cEMV (containing 1350 μg / mg MCPC), respectively.

[0083] The particle size, polydispersity index and drug loading of the prepared drug-loaded MV particles are shown in Table 1.

[0084] Table 1. Particle size, polydispersity index, and drug loading of DOX-loaded EMV

[0085]

[0086] II. cEMV delivery of DOX inhibits the growth of in situ TNBC tumors.

[0087] When the tumor volume in the 4T1 orthotopic breast cancer mouse model reached approximately 0.1 cm... 3 The antitumor efficacy of DOX / cEMV was investigated. The groups were G1 (saline), G2 (free DOX), G3 (DOX / EMV), G4 (DOX / cEMV containing 245 μg / mg MCPC), G5 (DOX / cEMV containing 720 μg / mg MCPC), and G6 (DOX / cEMV containing 1350 μg / mg MCPC). All groups were administered via tail vein injection, once every two days for five doses. The DOX dosage was 2 mg / kg (G2-G6), and the MCPC dosages were 5.44 mg / kg (G4), 15.98 mg / kg (G5), and 29.74 mg / kg (G6), respectively. The dosage for cell membrane protein content was 22 mg / kg (G3-G6). Tumor volume and body weight were measured in mice after administration, as shown below. Figure 8 c and Figure 8 After 14 days of drug administration, mice were euthanized by cervical dislocation, and intact tumor tissue was removed and photographed in groups as shown below. Figure 8 d. Weigh the tumor and calculate the tumor inhibition rate based on the tumor weight, as shown below. Figure 8 The tumor tissue was subjected to HE staining examination, such as... Figure 8 These results indicate that DOX / cEMV with different MCPC contents all produced higher tumor-suppressive efficacy than DOX / EMV, but the optimal anchoring concentration of DOX / cEMV at 720 μg / mg showed the highest anti-tumor efficacy.

[0088] Example 4

[0089] cEMV delivery of ICG for photothermal therapy of melanoma

[0090] I. Preparation of cEMV-loaded ICG

[0091] Dissolve 0.5 mg of ICG in 1 mL of 0.25× PBS, and disperse 1 mg of erythrocyte membrane containing membrane proteins in the solution. Incubate at 4 °C for 30 min. After incubation, add a calculated amount of 10× PBS to raise the overall osmotic pressure of the solution to 1× PBS, mix thoroughly, and incubate at 37 °C for another 90 min. Then, centrifuge the solution at 12000 g for 10 min, discard the supernatant, and collect the precipitated ICG-loaded erythrocyte membrane. Gently sonicate the cell membrane using an ultrasonic cell disruptor at low power, and then extrude it multiple times using a liposome extruder to obtain ICG / EMV. Disperse the ICG-loaded erythrocyte membrane obtained in the previous step in 1 mL of PBS buffer containing 0.5 mg / mL MCPC, and incubate at 37 °C for 30 min. Centrifuge the incubated solution at 10000 g, discard the supernatant, collect the bottom precipitate, and wash three times with PBS. The cell membrane was subjected to low-power, gentle sonication using an ultrasonic cell disruptor, followed by multiple extrusions using a liposome extruder to obtain ICG / cEMV, in which the MCPC anchoring quantity was 720 μg / mg.

[0092] The particle size, polydispersity index and drug loading of the prepared drug-loaded MV particles are shown in Table 2.

[0093] Table 2. Particle size, polydispersity index, and drug loading of ICG-loaded EMV

[0094]

[0095] II. ICG / cEMV in vitro photothermal conversion efficiency

[0096] The absorbance of ICG / cEMV in the 700-900 nm range was measured using a UV-Vis spectrophotometer, and the results are as follows: Figure 10 a. Compared to free ICG, the maximum absorption peak of ICG / cEMV shifts from 780nm to 800nm, demonstrating the integrity of the ICG encapsulation. Using an 808nm near-infrared laser at a power of 0.5W / cm², the absorption peak was further analyzed. 2 1W / cm 2 and 1.5W / cm 2 ICG / cEMV containing 50 μg / mL ICG was irradiated, and the temperature change was measured as follows: Figure 10 b. Utilizing 808nm, 1W / cm 2 Near-infrared laser was used to continuously irradiate free ICG or ICG / cEMV containing 50 μg / mLICG, and the temperature change was measured. The results are as follows: Figure 10 c. ICG / cEMV at concentrations of 0 μg / mL, 10 μg / mL, 30 μg / mL, and 50 μg / mL was applied at 808 nm and 1 W / cm². 2The near-infrared laser was continuously irradiated for 5 minutes, followed by 5 minutes without irradiation. The temperature change was measured as follows: Figure 10 d.

[0097] III. ICG / cEMV In vivo photothermal conversion efficiency

[0098] When the tumor size in the B16F10 tumor-bearing mouse was approximately 0.3 cm... 3 At that time, ICG / EMV or ICG / cEMV was administered via tail vein injection, with an equivalent ICG dose of 0.5 mg / kg. One day after administration, 808 nm, 1 W / cm² was applied topically to the tumor site in mice. 2 The mice were continuously irradiated with near-infrared laser light for 2.5 minutes. Thermal imaging was used to image the mice, and the results are as follows: Figure 11 a. The results of temperature measurement at the tumor site are as follows: Figure 11 In the b group, the local temperature at the tumor site was significantly higher than that in the ICG / cEMV group, reaching 56℃ after 1.5 minutes of irradiation and further increasing to 61℃ within 2.5 minutes. These experimental results indicate that ICG / cEMV has better tumor targeting and better photothermal conversion efficiency at the tumor site.

[0099] IV. Evaluation of the efficacy of ICG / cEMV photothermal therapy in inhibiting melanoma growth

[0100] When the tumor size in B16F10 melanoma-bearing mice was approximately 0.05 cm... 3 At that time, a single tail vein injection of the formulation was administered. One day after administration, 808nm, 1W / cm² was applied topically to the tumor site. 2 The tumors were irradiated with near-infrared laser for 1.5 minutes. Different treatment groups were: saline (G1), saline + laser (G2), ICG + laser (G3), ICG / EMV + laser (G4), ICG / cEMV (G5), and ICG / cEMV + laser (G6). The ICG dose was 0.5 mg / kg (G3-G6), the MCPC dose was 7.2 mg / kg (G5, G6), and the cell membrane protein dose was 10 mg / kg (G4-G6). Changes in tumor volume were recorded. Figure 12 ab. Fourteen days after the start of treatment, tissue or skin samples were collected from the tumor site for HE staining examination; the results were as follows. Figure 12 These results indicate that photothermal therapy following ICG / cEMV administration has the best inhibitory effect on melanoma growth, achieving complete tumor ablation with no tumor recurrence observed throughout the treatment period.

[0101] Example 5

[0102] EMV co-delivers MAPC and DOX to inhibit the growth of CSC-rich in situ TNBC tumors.

[0103] I. Preparation of MAPC and DOX co-loaded by EMV

[0104] 1.5 mg of DOX was dissolved in 1 mL of 0.25×PBS, and erythrocyte membranes containing 1 mg of membrane proteins were dispersed in the solution. The solution was incubated at 4°C for 30 min. After incubation, a calculated amount of 10×PBS was added to raise the overall osmotic pressure of the solution to 1×PBS. After thorough mixing, the solution was transferred to 37°C and incubated for another 90 min. Then, the solution was centrifuged at 12000 g for 10 min, the supernatant was discarded, and the precipitated DOX-loaded erythrocyte membranes were collected. The DOX / EMV preparation method was the same as in Example 3. The DOX-loaded erythrocyte membranes obtained in the previous step were dispersed in 1 mL of PBS buffer containing 0.5 mg / mL, 1.5 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL of MAPC, respectively, and incubated at 37°C for 30 min. The incubated solutions were centrifuged at 10000 g, the supernatant was discarded, the bottom precipitate was collected, and the solution was washed three times with PBS. Cell membranes were gently sonicated at low power using an ultrasonic cell disruptor, followed by multiple extrusions using a liposome extruder to obtain DOX / aEMV with MAPC:DOX molar ratios of 1:1, 2:1, 3:1, 4:1, and 5:1. Separately, erythrocyte membranes containing 1 mg of membrane protein were dispersed in 1 mL of PBS buffer containing 4 mg / mL MAPC and incubated at 37°C for 30 min. The incubated solution was centrifuged at 10000 g, the supernatant was discarded, and the bottom precipitate was collected and washed three times with PBS. The cell membranes were then gently sonicated at low power using an ultrasonic cell disruptor, followed by multiple extrusions using a liposome extruder to obtain aEMV.

[0105] The particle size, polydispersity index and drug loading of the prepared drug-loaded MV particles are shown in Table 3.

[0106] Table 3. Particle size, polydispersity index, and drug loading of EMV co-loaded with MAPC and DOX

[0107]

[0108] II. In vitro evaluation of DOX / aEMV inhibition of CSC-rich 4T1 cells

[0109] 1. Killing effect of DOX / aEMV on CSC-rich 4T1 cells

[0110] After digestion, 4T1 cells in the logarithmic growth phase were dispersed in DMEM / F12 medium containing 1×B27 supplement, 1% penicillin-streptomycin, 5 μg / mL recombinant human insulin, 20 ng / mL EGF, 20 ng / mL bFGF, and 4% BSA. The cell-containing medium was then adjusted at a ratio of 1×10⁻⁶ cells / mL. 5Cells were seeded at a density of 10 cells / well in six-well ultra-low adhesion cell culture plates. After seeding, the six-well plates were incubated in a tri-gas incubator at 37°C and 5% CO2 for 6-8 days to obtain CSC-rich 4T1 cells for in vitro culture. After digesting the cell spheroids to a single-cell state, they were seeded at a density of 5 × 10⁻⁶ cells / well. 3 Cells were seeded at a density of 100 cells / well in 96-well ultra-low adhesion plates. The blank control group received an equal volume of sterile PBS, the control group received an equal volume of sterile PBS after cell seeding, and the experimental groups received different concentrations of DOX / aEMV containing an equal amount of erythrocyte membranes and MAPC:DOX molar ratios of 1:1, 2:1, 3:1, 4:1, and 5:1. All cells were incubated for 96 hours after drug administration. Cell viability was determined by CCK-8 assay, and the results are shown below. Figure 13 Experimental results showed that the MAPC to DOX ratio of 4:1 and 5:1 had the best killing effect on CSC-rich 4T1 cells.

[0111] 2. DOX / aEMV inhibits dryness-related biomarkers

[0112] 4T1 cell spheres rich in CSCs were digested into a single-cell state and then digested at a concentration of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density per well in six-well ultra-low adhesion cell culture plates. The control group received the same volume of sterile PBS, while the treatment groups received DOX / EMV, aEMV, and DOX / aEMV (DOX:MAPC molar ratio 1:4) in the wells of the culture plates and were incubated for 48 h at concentrations of 0.5 μM DOX and 2 μM MAPC, respectively. After incubation, the cells were collected, digested with trypsin to a single-cell state, washed three times with PBS, and then seeded using ALDEFLUOR. TM The kit, FITC-Sca-1 antibody, and Nanog antibody were used for treatment, and cell-related markers were detected using flow cytometry. The experimental results are as follows: Figure 14 The experimental results showed that both aEMV and DOX / aEMV significantly reduced the expression of three stem cell markers, and the addition of MAPC acted as a prodrug to promote tumor stem cell differentiation.

[0113] 3. DOX / aEMV inhibits the self-renewal ability of tumor stem cells and the destruction of tumor spheroids.

[0114] 4T1 cells rich in CSCs were digested to a single-cell state and seeded into 96-well ultra-low adhesion plates at densities of 160, 80, 40, 20, 10, and 5 cells / well, with 24 replicates per group. Tumor stem cell culture medium was added to each well, while sterile PBS was added to the control group. Simultaneously, each group was incubated for 48 h with DOX / EMV, aEMV, and DOX / aEMV (DOX:MAPC = 1:4, mol:mol), at concentrations of 0.5 μM DOX and 2 μM MAPC, respectively. After 8 days of hypoxic culture, the formation of tumor cell spheroids was observed using an inverted fluorescence microscope. The presence of tumor spheroids in the wells was considered a positive result; otherwise, a negative result was indicated. The experimental results are shown below. Figure 15 In step a, >50 μm 4T1 tumor cell spheroids were seeded at a density of 30 spheroids / well. The control group received sterile PBS, while the treatment groups received DOX / EMV, aEMV, and DOX / aEMV (DOX:MAPC = 1:4, mol:mol), respectively, and were incubated for 48 h at concentrations of 0.5 μM DOX, 2 μM MAPC, and so on. The number of >50 μm tumor cell spheroids in each well was recorded every two days. The experimental results are shown below. Figure 15 b. These two experiments showed that, compared with other control groups, DOX / aEMV treatment most significantly reduced the self-renewal capacity of 4T1-TCs and destroyed 4T1 tumor spheroids.

[0115] III. Evaluation of the efficacy of DOX / aEMV in inhibiting the growth of CSC-rich TNBC tumors

[0116] When mice were inoculated with CSC-rich 4T1 cells, the tumor volume reached approximately 0.1 cm³. 3 Subsequently, mice were administered saline (G1), aEMV (G2), DOX / EMV (G3), DOX / aEMV (G4, DOX / MAPC molar ratio 1:3), DOX / aEMV (G5, DOX / MAPC molar ratio 1:4), and DOX / aEMV (G6, DOX / MAPC molar ratio 1:5) via tail vein injection, respectively, every two days for five administrations. The DOX dosage was 2 mg / kg (G2-G6), and the MAPC dosages were 7.66 mg / kg (G2), 5.75 mg / kg (G4), 7.66 mg / kg (G5), and 9.58 mg / kg (G6), respectively. The dosage based on erythrocyte membrane protein content was 22 mg / kg (G2-G6). Changes in tumor volume in vivo were measured in mice. Figure 16 In the first administration, 14 days later, tumor tissue was removed, weighed, and photographed in groups. The tumor inhibition rate of each treatment group was calculated, and the results were as follows: Figure 16 The tumor tissue was examined by HE staining, and the expression of Nanog within the tumor was examined by IHC staining. The results are as follows: Figure 16 China and Figure 17 DOX / aEMV demonstrated a significant advantage in inhibiting tumor growth, with the optimal in vivo therapeutic effect achieved at a MAPC:DOX molar ratio of 4:1. Following DOX / aEMV treatment, tumors exhibited extensive cell death, and Nanog expression was significantly reduced.

[0117] Example 6

[0118] PMV co-delivers MAPC and DOX to suppress CSC-rich TNBC lung metastases.

[0119] I. Preparation of MV co-loaded MAPC and DOX

[0120] The preparation method is the same as in Example 5. 1.5 mg of DOX was dissolved in 1 mL of 0.25×PBS, and a platelet membrane containing 1 mg of membrane protein was dispersed in it. The solution was incubated at 4°C for 30 min. After incubation, a calculated amount of 10×PBS was added to raise the overall osmotic pressure of the solution to 1×PBS. After thorough mixing, the solution was transferred to 37°C and incubated for another 90 min. Then, the solution was centrifuged at 12000g for 10 min, the supernatant was discarded, and the precipitated DOX-loaded platelet membrane was dispersed in 1 mL of PBS buffer containing 4 mg / mL MAPC and incubated at 37°C for 30 min. The incubated solution was centrifuged at 10000g, the supernatant was discarded, and the bottom precipitate was collected and washed three times with PBS. The cell membrane was gently sonicated at low power using an ultrasonic cell disruptor, and then extruded multiple times using a liposome extruder to obtain DOX / PMV. The DOX-loaded platelet membrane obtained in the previous step was dispersed in 1 mL of PBS buffer containing 4 mg / mL MAPC and incubated at 37°C for 30 min. The incubated solution was centrifuged at 10000 g, the supernatant was discarded, and the bottom precipitate was collected. The membrane was washed three times with PBS. The cell membrane was gently sonicated at low power using an ultrasonic cell disruptor, and then extruded multiple times using a liposome extruder to obtain DOX / aPMV with a MAPC:DOX molar ratio of 4:1. Separately, platelet membranes were dispersed in 1 mL of PBS buffer containing 4 mg / mL MAPC and incubated at 37°C for 30 min. The incubated solution was centrifuged at 10000 g, the supernatant was discarded, and the bottom precipitate was collected. The membrane was washed three times with PBS. The cell membrane was gently sonicated at low power using an ultrasonic cell disruptor, and then extruded multiple times using a liposome extruder to obtain aPMV with a MAPC:DOX molar ratio of 4:1.

[0121] The particle size, polydispersity index and drug loading of the prepared drug-loaded MV particles are shown in Table 4.

[0122] Table 4. Particle size, polydispersity index, and drug loading of MV co-loaded with MAPC and DOX

[0123]

[0124] II. DOX / aPMV inhibits CSC-rich TNBC lung metastases

[0125] Collect 4T1 breast cancer cell spheres transfected with fluorescent enzyme and enriched with CSCs, digest them to disperse them into single cells at a density of approximately 1 × 10⁻⁶ cells / cells. 5 One cell per mouse was injected via tail vein into BALB / c mice. Four hours after modeling, the following formulations were injected via tail vein: saline (G1), DOX / aEMV (G2), DOX / PMV (G3), aPMV (G4), and DOX / aPMV (G5). Administered every two days for a total of five doses. The dosage of DOX was 2 mg / kg (G2-G5), the dosage of MAPC was 7.66 mg / kg (G2, G4, G5), the dosage of erythrocyte membrane protein content was 22 mg / kg (G2), and the dosage of platelet membrane protein content was 18 mg / kg (G4, G5). Following the first administration, on days 0, 4, 9, 11, and 14, mice were intraperitoneally injected with 150 mg / kg of fluorescein sodium. Fifteen minutes later, autofluorescence in the mice was monitored and captured using an in vivo fluorescence imaging system (IVIS). Software was used to directly quantify the whole-body in vivo fluorescence values. Figure 18 On day 14 after drug administration, mice were euthanized by cervical dislocation, and their intact lungs were dissected and immersed in Bouin's solution for 12 hours. The stained lungs were then photographed, and the number of visible tumor metastases in the lung tissue was counted, as shown below. Figure 18 In this study, lung tumor tissue was examined using HE staining, and Nanog expression within the tumor was detected by IHC staining. The results were as follows: Figure 18 f and Figure 19 These experimental results demonstrate that MAPC stabilizes the platelet membrane drug delivery system in the formulation, leveraging the platelet membrane's targeting ability for circulating tumor cells and metastatic lesions, thereby increasing the delivery and killing effect of DOX on tumor metastases. The combined delivery of MAPC and DOX can exert a synergistic effect by inducing differentiation of highly stem tumor cells. Following DOX / aPMV treatment, mouse lung tumors exhibited the smallest tumor volume and the most extensive cell death, while Nanog expression was also significantly reduced.

[0126] Example 8

[0127] In vivo safety evaluation

[0128] Healthy mice were administered DOX / cEMV (DOX dose 2 mg / kg, MCPC dose 15.98 mg / kg, erythrocyte membrane protein dose 22 mg / kg), DOX / aEMV (DOX dose 2 mg / kg, MAPC dose 7.66 mg / kg, erythrocyte membrane protein dose 22 mg / kg, MAPC:DOX molar ratio 4:1), and DOX / aPMV (DOX dose 2 mg / kg, MAPC dose 7.66 mg / kg, platelet membrane protein dose 18 mg / kg, MAPC:DOX molar ratio 4:1) via tail vein injection, five times over two days. Healthy mice were also administered ICG / cEMV via tail vein injection, with an ICG dose of 0.5 mg / kg, MCPC dose of 7.2 mg / kg, and erythrocyte membrane protein dose of 10 mg / kg, in a single injection. Blood samples were collected and analyzed using a blood analyzer to count red blood cells (RBCs), platelets (PLTs), and white blood cells (WBCs). Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were measured using diagnostic kits. The experimental results are as follows: Figure 20 Compared with the saline group, the levels of DOX / cEMV, ICG / cEMV, DOX / aEMV, and DOX / aPMV in the Chinese ag group showed no significant differences, indicating that the in vivo safety of the Chinese ag group was satisfactory.

Claims

1. A lipid-anchored cell-derived vesicle, characterized in that, The monoacylphosphatidylcholine is made from glycerophosphocholine and a hydrophobic molecule by ester bond, wherein the hydrophobic molecule is a molecule with rigid ring structure. The hydrophobic molecule is selected from cholesterol, 10-(benzyloxy)-10-oxodecanoic acid, all-trans retinoic acid, abiraterone, belinostat or vorinostat.

2. The lipid-anchored cell-derived vesicle according to claim 1, characterized in that, The cell membrane is red blood cell membrane, platelet membrane, white blood cell membrane, tumor cell membrane or mesenchymal stem cell membrane.

3. The lipid-anchored cell-derived vesicle of claim 1, wherein, The lipid-anchored cell-derived vesicle further encapsulates an active drug, wherein the active drug is a water-soluble drug.

4. The lipid-anchored cell-derived vesicle according to any one of claims 1 to 3, characterized in that, The molar ratio of the monoacylphosphatidylcholine to the active drug is 0.5:1-10:

1.

5. The lipid-anchored cell-derived vesicle of claim 4, wherein, The cell membrane is added to the aqueous solution of the monoacylphosphatidylcholine, and after incubation, centrifugation is performed to collect the precipitate, which is then extruded by a liposome extruder to obtain the lipid-anchored cell-derived vesicle.

6. The method of producing lipid-anchored cell-derived vesicles according to claim 1, characterized in that, The aqueous solution is phosphate buffer.

7. The production method according to claim 6, wherein The active drug is first encapsulated in the cell membrane, and then the cell membrane encapsulating the active drug is added to the aqueous solution of the monoacylphosphatidylcholine, and after incubation, centrifugation is performed to collect the precipitate, which is then extruded by a liposome extruder to obtain the lipid-anchored cell-derived vesicle.

8. The production method according to claim 6 or 7, characterized by, 9. Use of the lipid-anchored cell-derived vesicle of any one of claims 1-5 in the preparation of a tumor treatment drug. The tumor is a common tumor or a tumor stem cell-like cell related drug-resistant tumor.

10. Use according to claim 9, characterized in that, The tumor is a common tumor or a tumor stem cell-like cell related drug-resistant tumor.

Images