Tumor programmed drug permeation biomimetic mineralization nano-vesicle for enhancing macropinocytosis as well as preparation method and application of nano-vesicle

Abstract

The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and relates to a tumor programmed drug permeation biomimetic mineralization nano-vesicle capable of enhancing macropinocytosis as well as a preparation method and application of the tumor programmed drug permeation biomimetic mineralization nano-vesicle. The drug permeation biomimetic mineralization nano-vesicle is of a shell-core structure, chemotherapy drugs are co-loaded in the cell vesicle, and the exterior of the cell vesicle is encapsulated with a peptide fragment for specifically promoting the macropinocytosis of tumor cells through mineralization. The biomineralized cell membrane nano-vesicles which are good in stability and uniform in particle size are prepared, the calcium phosphate mineralized shell can specifically respond to tumor slightly acidic environment dissolution, and by enhancing the tumor macropinocytosis effect, uptake of apoptotic bodies by tumor cells is promoted, so that tumor apoptosis is promoted, and tumor apoptosis is inhibited. The apoptotic body-mediated drug has the advantages that the apoptotic body-mediated drug can be further improved in infiltration capacity in tumor tissues, programmed infiltration of the drug in the tumor tissues is realized, tumor growth is effectively inhibited, a great potential direction is provided for solving the tumor infiltration bottleneck of nano-drugs, and the apoptotic body-mediated drug has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and relates to a tumor-programmed drug-penetrating biomimetic mineralized nanovesicle capable of enhancing macropinocytosis, a preparation method and an application thereof. Background Art

[0002] With cancer incidence and mortality rates climbing year after year, it has gradually evolved into the most serious threat to human health. Against this backdrop, the application of nanomedicine technology in cancer treatment has attracted widespread attention. However, due to their relatively large size, systemically administered nanocarriers often accumulate around tumors and have difficulty penetrating the hypoxic regions of the tumor core. Cells deep within tumors are often more stubborn and have a higher propensity to metastasize. Consequently, improving the penetration of nanomedicines into tumors has become a challenge that must be addressed.

[0003] During cell apoptosis, the nucleus begins to aggregate, and the cell membrane shows signs of invagination and wrinkling. These changes ultimately trigger cytoplasmic fission, forming apoptotic bodies containing macromolecules, nuclear fragments, and organelles. Previous studies have demonstrated that apoptotic bodies, produced after tumor cells die from chemotherapy, can store free chemotherapeutic drugs and act as drug carriers to transfer remaining active drugs to neighboring tumor cells, continuously killing tumor cells and gradually penetrating deep into the tumor. However, ensuring the precise delivery of drug-containing apoptotic bodies to the tumor and their efficient uptake by tumor cells remains a challenge. Macropinocytosis, a common and conserved form of endocytosis, is known to be a key metabolic adaptation mechanism of tumor cells. It is driven by actin and does not require the involvement of clathrin. Specific promotion of macropinocytosis in tumor cells to enhance apoptotic body-mediated drug penetration deep into tumors has become a promising research direction. Summary of the Invention

[0004] The present invention aims to provide a tumor-programmed drug-penetrating biomimetic mineralized nanovesicle capable of enhancing macropinocytosis, as well as a preparation method and application thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A tumor-programmed drug-penetrating biomimetic mineralized nanovesicle that enhances macropinocytosis. The drug-penetrating biomimetic mineralized nanovesicle has a shell-core structure. Chemotherapy drugs are co-loaded inside the cell vesicle, and a peptide segment that specifically promotes tumor cell macropinocytosis is encapsulated on the outside through mineralization.

[0007] The chemotherapy drugs co-loaded inside the cell vesicles account for 70%-90% of the mass of the biomimetic mineralized nanovesicles, wherein the chemotherapy drugs are hypoxia-activated prodrugs and small molecule drugs with full tumor killing effects, and the mass ratio between the two is 0.7-1.0:1.0-1.6.

[0008] The cell vesicles are derived from mouse breast cancer (4T1) cells.

[0009] The peptide segment accounts for 11%-16% of the mass of the biomimetic mineralized nanovesicles; wherein the peptide segment is RFFE-SHAPAKPVSLSYR.

[0010] The two small molecule chemotherapy drugs with different spatial effects refer to the small molecule drug doxorubicin (DOX) that can comprehensively kill tumor cells and the hypoxia-activated prodrug anthraquinone (AQ4N) that exerts cytotoxicity only in the deep hypoxic areas inside the tumor; the cell membrane nanovesicles refer to nanoscale vesicles extracted from tumor cells by differential centrifugation; the biomineralized shell refers to a calcium phosphate shell composed of inorganic ions that can specifically respond to the decomposition of the micro-acidic environment of the tumor.

[0011] A method for enhancing macropinocytosis by tumor-programmed drug penetration biomimetic mineralized nanovesicles,

[0012] (1) Prepare complete culture medium and incubate cells;

[0013] (2) Isolation of cell membrane nanovesicles by centrifugation;

[0014] (3) mixing the chemotherapy drug solution and the cell membrane nanovesicle solution;

[0015] (4) ultrasonically treating the mixed solution;

[0016] (5) incubation to restore the cell membrane nanovesicle structure;

[0017] (6) centrifugation and resuspension to obtain drug-loaded cell membrane nanovesicles;

[0018] (7) mixing the drug-loaded cell membrane nanovesicles obtained above with calcium chloride solution, peptide solution and buffer solution;

[0019] (8) The mixture was stirred and centrifuged to obtain biomineralized cell membrane nanovesicles.

[0020] In the step (3), the hypoxia-activated prodrug, the small molecule drug with full tumor killing effect and the cell membrane nanovesicles are mixed in a mass ratio of 1-3:1-3:4-7.

[0021] In the step (7), the volume ratio of the drug-loaded cell membrane nanovesicles to the calcium chloride solution, the peptide solution and the buffer solution is 7-11:0.7-1.2:0.6-1.4:8-13; wherein the buffer solution is HEPES buffer solution.

[0022] Further,

[0023] (1) Prepare complete culture medium and incubate cells; add double antibody and fetal bovine serum to RPMI-1640 cell culture medium to prepare complete culture medium, and incubate the cells in the complete culture medium for 48 hours.

[0024] (2) Isolation of cell membrane nanovesicles by centrifugation;

[0025] The adherent cells were digested and the cell suspension was centrifuged at 2000 rpm for 10 min to remove the nuclei. The supernatant was collected and centrifuged again at 5000 rpm for 10 min to remove cell debris and mitochondria. Finally, the precipitate was collected by centrifugation at 15000 rpm for 60 min and resuspended in PBS (pH 7.4) to obtain blank cell membrane nanovesicles.

[0026] (3) mixing the chemotherapy drug solution and the cell membrane nanovesicle solution;

[0027] Accurately take 40 μl of AQ4N solution (concentration: 5 mg / ml), 10 μl of DOX solution (concentration: 20 mg / ml), and 160 μl of cell membrane nanovesicle solution (concentration: 5 mg / ml). Mix these solutions with 840 μl of deionized water to ensure thorough mixing.

[0028] (4) ultrasonically treating the mixed solution;

[0029] The mixed solution was placed in an ice bath and sonicated using an ultrasonic probe with a power of 100 W. The working time was set to 2 s and the rest time to 4 s, and this process was repeated 20 times.

[0030] (5) Incubation to restore the cell membrane nanovesicle structure; after ultrasonic treatment, the mixed solution was incubated at a constant temperature of 37°C for 1 hour to promote the recovery of the cell membrane nanovesicle structure.

[0031] (6) Centrifugation and re-suspending to obtain drug-loaded cell membrane nanovesicles; the above-mentioned incubated cell membrane nanovesicle solution was centrifuged at a speed of 13500 r / min for 20 min, the supernatant was removed, and the precipitate was re-suspended using 10nM Tris-HCl buffer (pH 7.4) to prepare drug-loaded cell membrane nanovesicles AD-NVs.

[0032] (7) mixing the drug-loaded cell membrane nanovesicles obtained above with calcium chloride solution, peptide solution and buffer solution;

[0033] Take 1 ml of drug-loaded cell membrane nanovesicle AD-NVs solution (concentration of 1 mg / ml), slowly add 110 μl of calcium chloride solution (concentration of 3 mg / ml), 100 μl of RFFESHAPAKPVSLSYR solution (concentration of 1 mg / ml), and the same volume of HEPES buffer (containing Na3PO4, NaCl, pH 7.4) as the mixed solution of drug-loaded cell membrane nanovesicle AD-NVs solution, calcium chloride solution, and RFFESHAPAKPVSLSYR solution.

[0034] (8) stirring the mixture in the dark at room temperature and centrifuging to obtain biomineralized cell membrane nanovesicles;

[0035] The mixture was stirred at room temperature in the dark for 1 hour, then centrifuged at 13500 rpm for 20 minutes, the supernatant was discarded, and the precipitate was collected to obtain the biomineralized cell membrane nanovesicles.

[0036] An application of the tumor-programmed drug penetration biomimetic mineralized nanovesicles that enhance macropinocytosis, and an application of the biomineralized cell membrane nanovesicles in the field of drug delivery.

[0037] The application of the tumor-programmed drug-permeable biomineralized cell membrane nanovesicles with enhanced macropinocytosis in drug delivery systems. The application of the tumor-programmed drug-permeable biomineralized cell membrane nanovesicles with enhanced macropinocytosis in preventing tumor growth, metastasis, and postoperative recurrence.

[0038] The administration method of the tumor programmed drug permeating biomineralized cell membrane nanovesicles with enhanced macropinocytosis is injection administration, oral administration or local administration.

[0039] The present invention has the following advantages:

[0040] (1) Biomineralized cell membrane nanovesicles with good stability and uniform particle size were prepared. DOX and AQ4N were co-encapsulated in the vesicles, and RFFESHAPAKPVSLSYR was mineralized and modified outside the vesicles. The preparation method was simple and efficient.

[0041] (2) Biomineralized cell membrane nanovesicles were prepared, which can specifically respond to the slightly acidic environment of tumor tissue to dissolve the mineralized shell and release customized peptides and drug-loaded nanocores. The formulation characteristics, colloidal stability and storage stability of biomineralized cell membrane nanovesicles were investigated; the drug encapsulation efficiency of biomineralized cell membrane nanovesicles; the drug release characteristics of biomineralized cell membrane nanovesicles under different pH conditions; the cellular uptake and cytotoxicity of biomineralized cell membrane nanovesicles; the extraction and characterization of apoptotic bodies; the mechanism of intercellular drug delivery of biomineralized cell membrane nanovesicles; the penetration of biomineralized cell membrane nanovesicles in 3D tumor sphere models, the pharmacokinetic characteristics, and the in vivo distribution and pharmacodynamics of biomineralized cell membrane nanovesicles in mice were investigated. The results showed that the biomineralized cell membrane nanovesicles have uniform particle size distribution and good formulation stability. The calcium phosphate mineralized shell can specifically respond to the micro-acidic environment of the tumor and dissolve. By enhancing tumor macropinocytosis, it promotes the uptake of apoptotic bodies by tumor cells, thereby enhancing the apoptotic body-mediated drug penetration ability in tumor tissues, achieving programmed drug penetration in tumor tissues and effectively inhibiting tumor growth. This provides a highly promising direction for solving the bottleneck of nanodrug penetration in tumors and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is an investigation of the mineralization and pH response of the biomineralized cell membrane nanovesicles of Example 2 of the present invention.

[0043] A: Extraction of cell membrane nanovesicles (NVs) by differential centrifugation

[0044] B: Particle size and potential before and after mineralization

[0045] C: Transmission electron microscopy images of AD-NVs and AD-NVs@CPP

[0046] D: Investigation of peptide release under different pH conditions

[0047] E: Investigation of the cumulative release of DOX from AD-NVs@CPP under different pH conditions

[0048] F: Cumulative release of AQ4N from AD-NVs@CPP under different pH conditions

[0049] Figure 2 This is an investigation into the cellular uptake pathway of the biomineralized cell membrane nanovesicles in Example 3 of the present invention.

[0050] Figure 3 This is the characterization analysis of the in vitro cellular uptake, cytotoxicity and induced apoptotic bodies of the biomineralized cell membrane nanovesicles of Example 4 of the present invention.

[0051] A: Confocal fluorescence images of 4T1 cells treated with different preparations for 12 hours

[0052] B: Flow cytometry measurement of 4T1 cells treated with each preparation for 12 hours, ***p<0.001, ****p<0.0001

[0053] C: Survival rate of 4T1 cells treated with different preparations for 48 hours under normoxia

[0054] D: 4T1 cell survival rate after treatment with each preparation for 48 hours under hypoxia

[0055] E: Transmission electron microscopy image of apoptotic bodies induced by AD-NVs@CPP (pH 6.5)

[0056] F: Confocal imaging of apoptotic bodies and confocal imaging of apoptotic bodies co-incubated with 4T1 cells

[0057] G: Cytotoxicity of apoptotic bodies to 4T1 cells

[0058] Figure 4 This is an investigation of the adjacent effect mechanism of apoptotic bodies in Example 5 of the present invention.

[0059] A: Schematic diagram of investigation of ApoBDs-mediated intercellular programmed drug delivery

[0060] B: Confocal fluorescence images of programmed delivery at different stages

[0061] C: Molar ratio of bound to unbound AQ4N in cells iI, i-ii, and ii-I

[0062] D: Molar ratio of bound to unbound DXO in cells iI, i-ii, and ii-I

[0063] E: Molar ratio of AQ4N to DXO in cells iI, i-ii, and ii-I

[0064] F: Western blot analysis of apoptosis marker proteins in corresponding cells

[0065] G: The ratio of AQ4N and AQ4 in the corresponding cells

[0066] H: Fluorescence penetration of tumor spheres observed by confocal microscopy after 24 hours of treatment with each preparation group I: Fluorescence quantitative curve of AQ4N in tumor spheres

[0067] J: Fluorescence quantitative curve of DOX in tumor spheres

[0068] Figure 5 These are the pharmacokinetics, biodistribution and in vivo permeation investigation results of the biomineralized cell membrane nanovesicles of Example 6 of the present invention.

[0069] A: Concentration-time curves of DOX in mouse plasma after administration of different formulations

[0070] B: In vivo tissue distribution of AQ4N solution, AD-NVs, and AD-NVs@CPP in 4T1 tumor-bearing mice at 2, 4, 8, 12, and 24 hours

[0071] C: Fluorescence distribution of the main organs of mice (heart, liver, spleen, lung, kidney, and tumor) 24 hours after injection of different preparations

[0072] D: Fluorescence quantitative analysis of each organ

[0073] E: Fluorescence images of corresponding tumor tissue sections after administration of different preparations

[0074] Figure 6 These are the results of in vivo anti-tumor activity and biosafety investigation of the biomineralized cell membrane nanovesicles of Example 7 of the present invention.

[0075] A: Body weight change curves of mice in different treatment groups

[0076] B: Changes in tumor weight in different treatment groups

[0077] C: Tumor size images of different treatment groups

[0078] D: H&E, TUNEL, and Ki 67 staining of 4T1 tumor tissue sections of mice in different treatment groups

[0079] E: Body weight change curves of mice in different treatment groups

[0080] F: Liver function test of mice in different treatment groups

[0081] G: Renal function test of mice in different treatment groups

[0082] Figure 7 This is the H&E staining result of the main organs (heart, liver, spleen, lung, and kidney) of mice in different treatment groups in Example 8 of the present invention. DETAILED DESCRIPTION

[0083] The feasibility of the present invention is illustrated below by using embodiments. Those skilled in the art should understand that according to the teachings of the prior art, modifications or replacements of corresponding technical features still fall within the scope of protection claimed by the present invention.

[0084] The present invention presents programmed drug permeation biomineralized cell membrane nanovesicles that specifically enhance tumor cell macropinocytosis. The vesicles contain two small-molecule chemotherapy drugs, doxorubicin and the hypoxia-activated prodrug, banothramine, which activate under different spatial conditions. The vesicles are then biomineralized to encapsulate a peptide segment that specifically promotes tumor cell macropinocytosis. The biomineralized cell membrane nanovesicles prepared by the present invention exhibit excellent stability, effectively improving in vivo circulation time. By enhancing macropinocytosis, they demonstrate excellent programmed drug permeation in tumor tissues. Furthermore, they demonstrate a drug permeation mechanism mediated by apoptotic bodies. Overall, the present invention has great potential and prospects in the field of tumor treatment.

[0085] Example 1: Drug ratio screening

[0086] 4T1 cells were incubated in 500 mL of RPMI-1640 cell culture medium (1 wt% double-antibody and 10 wt% fetal bovine serum) to prepare complete culture medium for 48 hours. Adherent cells were digested, and the cell suspension was centrifuged at 2000 rpm for 10 minutes to remove nuclei. The supernatant was collected and centrifuged again at 5000 rpm for 10 minutes to remove cell debris and mitochondria. Finally, the pellet was collected by centrifugation at 15000 rpm for 60 minutes and resuspended in PBS (pH 7.4) to obtain blank cell membrane nanovesicles. Protein in the collected cell membrane nanovesicles was quantified by BCA assay, diluted to 5 mg / ml, and stored at -80°C for later use. Then, accurately take 40 μl of AQ4N solution (concentration of 5 mg / ml), 10 μl of DOX solution (concentration of 20 mg / ml), and 160 μl of cell membrane nanovesicle solution (concentration of 5 mg / ml). These solutions were mixed with 840 μl of deionized water to ensure thorough mixing. Next, the mixed solution was placed in an ice bath and sonicated using an ultrasonic probe with a power of 100 W, with a working time of 2 s and an interval of 4 s, and this process was repeated 20 times. After sonication, the mixed solution was incubated at a constant temperature of 37 ° C for 1 hour to promote the recovery of the cell membrane nanovesicle structure. Finally, centrifuge at a speed of 13500 r / min for 20 minutes, remove the supernatant, and resuspend the precipitate with 10 nM Tris-HCl buffer (pH 7.4) to prepare drug-loaded cell membrane nanovesicles AD-NVs (see Table 1).

[0087] According to the above example process, the ratio of chemotherapy drugs was prepared as shown in Table 1 to obtain drug-loaded cell membrane nanovesicles AD-NVs with different drug loading amounts; the obtained drug-loaded cell membrane nanovesicles AD-NVs were measured, see Table 1.

[0088] Table 1

[0089]

[0090] As shown in Table 1, when the mass ratio of AQ4N to DOX is 1:1, the two drugs exhibit a high encapsulation efficiency, a particle size of approximately 150 nm, a uniform particle size distribution, and a measured Zeta potential of approximately -20 mV, which is conducive to the longer circulation of the nanoparticles in the body and the accumulation effect on tumors.

[0091] Example 2: Investigation of Mineralization and pH Response of Biomineralized Cell Membrane Nanovesicles

[0092] 1 wt% double antibody and 10 wt% fetal bovine serum were added to 500 mL RPMI-1640 cell culture medium to prepare complete culture medium, and 4T1 cells were incubated in complete culture medium for 48 h. The adherent cells were digested, and the cell suspension was centrifuged at 2000 r / min for 10 min to remove the cell nuclei. The supernatant was collected and centrifuged at 5000 r / min for 10 min to remove cell debris and mitochondria. Finally, the precipitate was collected by centrifugation at 15000 r / min for 60 min and resuspended in PBS with pH 7.4 to obtain blank cell membrane nanovesicles ( Figure 1 A).

[0093] To a 1ml AD-NVs solution (concentration: 1mg / ml), 110μl of calcium chloride solution (concentration: 3mg / ml) and 100μl of RFFESHAPAKPVSLSYR solution (concentration: 1mg / ml) were slowly added dropwise to obtain a mixture. An equal volume of HEPES buffer (containing Na3PO4 and NaCl, pH 7.4) was then added. The mixture was stirred at room temperature in the dark for 1h. Under stirring, the Na3PO4 in the mixture reacted with CaCl2 to form calcium phosphate, which then bound to the surface of the AD-NVs to form a calcium phosphate mineralized shell. The mixture was then centrifuged at 13,500 rpm for 20min, the supernatant discarded, and the precipitate collected to obtain the biomineralized cell membrane nanovesicles AD-NVs@CPP.

[0094] Transmission electron microscopy (TEM) imaging revealed that the mineralized drug-loaded nanovesicles transformed from a disc-like shape to a unique core-shell nanosphere structure ( Figure 1 C). Malvern particle sizer measurements showed that the particle size and zeta potential of AD-NVs@CPP increased compared to AD-NV ( Figure 1 B). The above results collectively confirm the successful modification of the calcium phosphate shell.

[0095] In addition, the release of peptides from AD-NVs@CPP under different pH conditions was further investigated. The results showed that at pH 6.5, approximately 50% of the peptides were released within 24 hours due to the acid-responsive dissolution of the CaP shell, which was significantly higher than the release observed at pH 7.4 ( Figure 1 D).

[0096] Furthermore, the release profiles of AQ4N and DOX from AD-NVs@CPP were evaluated under different pH conditions. Phosphate buffer solutions were prepared at pH values of 7.4, 6.5, and 5. 200 μl of the AD-NVs@CPP to be tested (containing 0.5 mg / ml AQ4N) was added to each of the three 10 ml phosphate buffer solutions at different pH values. At each experimental time point, 1 ml of the solution was collected and centrifuged at 13,500 rpm for 20 minutes, and the supernatant was separated.

[0097] The results showed that only a small amount of DOX and AQ4N was released at neutral pH 7.4. However, under acidic conditions (pH 6.5 and pH 5), the release amount and rate of DOX and AQ4N increased significantly, and the increase was more obvious at pH 5 compared with pH 6.5 ( Figure 1 E and F).

[0098] Example 3: Investigation of the main pathways of cellular uptake of biomineralized cell membrane nanovesicles

[0099] AD-NVs were co-incubated with 4T1 cells and used as a control group. Three groups of endocytosis inhibitors, chlorpromazine (100 μL, 1 mol / mL), filipin (100 μL, 1 mol / mL), and EIPA (100 μL, 1 mol / mL), were added as experimental groups. After incubation in a cell culture incubator for 12 h, the cells were washed three times with cold PBS, digested with trypsin, and collected from each group. After filtration, the cells were detected by flow cytometry to explore the endocytosis mechanism of AD-NVs in 4T1 cells (see Figure 2 We investigated the mechanism and main pathway of cellular uptake of nanoformulations by applying specific cellular uptake inhibitors. The results showed that the process mainly occurred through the macropinocytosis pathway and was energy-dependent.

[0100] Example 4: Characterization and analysis of in vitro cellular uptake, cytotoxicity, and induced apoptotic bodies of biomineralized cell membrane nanovesicles

[0101] 4T1 cells (5×10 4cells / well) were inoculated into a 24-well plate containing a circular cell crawler and cultured overnight in a constant temperature cell culture incubator (37°C) to promote cell adherence growth. Subsequently, the old culture medium was discarded, and culture medium containing DOX, AQ4N, AD-NVs prepared in the steps of Example 1 above, AD-NVs@CPP and AD-NVs@CPP (pH 6.5) prepared in Example 2 above were added, respectively, wherein the equivalent concentrations of the above-mentioned substances DOX and AQ4N were both 10 μg / ml, and the cells were incubated for another 12 hours. After the incubation, the cells were washed three times with cold PBS (pH 7.4) to remove free drugs. Subsequently, they were fixed with 4% paraformaldehyde for 10 minutes and washed again three times with PBS to remove residual paraformaldehyde. Finally, the cells were stained with Hoechst 33342 for 10 minutes. After washing, the fluorescence signal in the cells was observed using CLSM.

[0102] In addition, flow cytometry was used to semi-quantitatively analyze the cellular uptake of biomineralized nanovesicles. 5 Cells were uniformly seeded in 12-well plates at a density of 10 cells / well and cultured in a cell culture incubator for 12 hours to allow adherent growth. Subsequently, the old culture medium was discarded and culture medium containing DOX, AQ4N, the AD-NVs prepared in Example 1, and AD-NVs@CPP and AD-NVs@CPP (pH 6.5) prepared in Example 2 were added, respectively. The equivalent concentrations of DOX and AQ4N were 10 μg / ml. After incubation in a cell culture incubator for 12 hours, the drug-containing culture medium was discarded and the cells were washed three times with cold PBS (pH 7.4). After trypsinization, the cells were harvested and centrifuged (1500 rpm, 4°C, 7 min). The cells were resuspended in PBS (pH 7.4), filtered through a filter, and collected into flow cytometers. Cell uptake was measured by flow cytometry.

[0103] At 12 h, the uptake of drugs in each group by cells was observed by confocal microscopy ( Figure 3 A) and determined by flow cytometry ( Figure 3 B) Results show that DOX and AQ4N solutions exhibited lower cellular uptake efficiencies, while AD-NVs exhibited significantly higher cellular uptake, demonstrating its targeted targeting of homologous tumor cells. Furthermore, due to the acid-responsive dissolution properties of the biological calcium phosphate mineralized shell, AD-NVs@CPP exhibited relatively low cellular uptake efficiencies at pH 7.4. However, in the acidic environment of pH 6.5, the calcium phosphate mineralized shell dissolved, releasing the encapsulated peptides, specifically promoting tumor cell macropinocytosis and resulting in the highest cellular uptake efficiency.

[0104] The cytotoxic effects of biomineralized cell membrane nanovesicles on 4T1 cells under different oxygen concentration conditions were evaluated by the MTT standard method. First, 2000 4T1 cells were seeded per well in a 96-well plate and cultured in a cell culture incubator for 12 hours to promote cell attachment. After attachment was completed, the cells were cultured for another 12 hours in an incubator or hypoxic culture box. Then, the culture medium was replaced and cell culture medium containing DOX, AQ4N, AD-NVs prepared in the steps of Example 1, AD-NVs@CPP and AD-NVs@CPP (pH 6.5) prepared in Example 2 were added, with the concentrations gradually increasing (20 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, 1000 ng / mL). The samples were incubated in a culture box or incubator under hypoxic conditions for 48 hours, with untreated cells used as a reference. After 48 hours of incubation, 50 μl of MTT solution (concentration of 5 mg / ml) was added to each well and incubated at 37°C for another 4 hours. Subsequently, the liquid in the wells was removed, and 200 μl of DMSO was added to each well of the 96-well plate, and then shaken for 10 minutes in a dark environment. Finally, the absorbance was measured at a wavelength of 570 nm using a microplate reader to calculate the IC 50 The cell survival rate curve was drawn based on the values. Figure 3 C, Figure 3 D). The results showed that the DOX solution group exerted a strong tumor-killing effect and possessed relatively strong cytotoxicity both in normoxia and hypoxia. The hypoxia-activated prodrug AQ4N had low cytotoxicity under normoxia but exhibited relatively strong cytotoxicity in hypoxia. The mixed solution group of DOX and AQ4N exhibited strong cytotoxicity in both normoxia and hypoxia. Compared with the mixed solution, AD-NVs exhibited relatively low cytotoxicity due to the sustained drug release characteristics of the nanoformulation, even with good cellular uptake efficiency. In addition, we also found that AD-NVs@CPP exhibited stronger cytotoxicity in a slightly acidic environment of pH 6.5, accompanied by strong cellular uptake, compared to a neutral environment.

[0105] First, 4T1 cells were seeded into a 12-well plate at a density of 100,000 per well and cultured in an incubator for 12 hours to promote cell adhesion growth. The old culture medium was removed, and then RPMI-1640 cell culture medium without fetal bovine serum containing AD-NVs@CPP (concentration of 300μg / ml) was added and cultured for 24 hours in a normoxic environment with a pH of 6.5. The concentrations of DOX and AQ4N were about 10μg / ml. After the culture was completed, the supernatant was collected and centrifuged (4°C, 800g / min, 10min) to remove cell debris, and then centrifuged at 1500g / min for 30min to separate and obtain ApoBDs and observe their morphological structure under TEM. The results are as follows Figure 3 As shown in E, the particle size is observed to be around 1 μm, and the morphology is a spherical and regular structure.

[0106] Next, the drug fluorescence distribution in ApoBDs was observed under a confocal microscope. At the same time, 4T1 cells were cultured with cell culture medium containing ApoBDs, and then the cell nuclei were stained with Hoechst 33342 stain, and the fluorescence distribution in the cells was observed using a confocal microscope. The results are as follows: Figure 3 As shown in Figure F, the red fluorescence representing AQ4N and the green fluorescence representing DOX are distributed in the ApoBDs and overlap again, indicating that the remaining DOX and AQ4N in the cells are indeed stored in the ApoBDs. The above ApoBDs were added to 4T1 cells for co-culture, and then the dual-color fluorescence of AQ4N and DOX was observed under a confocal microscope. This result proves that the drug-containing ApoBDs can be efficiently taken up by tumor cells. In addition, the protein content in ApoBDs was determined by the BCA protein quantification kit. After setting different concentrations of ApoBDs to co-incubate with cells, the MTT experimental method was used to evaluate the toxic effect of ApoBDs on cells. The results are shown in Figure 3. Figure 3 As shown in G, its IC is calculated 50 The value was 1.12 μg / ml, showing strong tumor cell killing ability.

[0107] Example 5: Investigation of the Proximity Effect Mechanism of Apoptotic Bodies

[0108] First, a cell slide was placed in a 6-well plate and labeled as Plate i. 4T1 cells were evenly seeded at a density of 400,000 per well and incubated in an incubator for 12 hours. After the cells adhered, the original cell culture medium was removed and replaced with a cell culture medium containing DOX solution, AQ4N solution, a mixed solution of AQ4N and DOX, the AD-NVs prepared in Example 1, and AD-NVs@CPP and AD-NVs@CPP (pH 6.5) prepared in Example 2. The cells were incubated under normoxia for another 12 hours (Plate iI). Both DOX and AQ4N concentrations were 10 μg / ml. After the scheduled time, the culture medium from each drug-containing group was aspirated and washed three times with cold PBS. The cells were then incubated in a blank medium without the drug for an additional 12 hours (Plate iA). The drug-containing ApoBDs were then extracted from the supernatants by differential centrifugation and labeled as i-ii.

[0109] Take another 6-well plate with a slide and mark it as plate ii. After evenly inoculating 400,000 4T1 cells into each well, place it in an incubator and culture it for 12 hours. In the subsequent experimental steps, the drug-containing ApoBDs were separated and added to the RPMI-1640 cell culture medium containing 10% fetal bovine serum and co-cultured with the 4T1 cells for 12 hours (ii-I). Subsequently, the cell culture medium containing ApoBDs was removed, washed three times with cold PBS, and continued to be cultured in blank culture medium for 12 hours. The drug-containing ApoBDs (ii-iii) were then collected by differential centrifugation and co-cultured with the cells in the third 6-well plate (marked as iii) for 12 hours ( Figure 4 A). Finally, the cells in plates i, ii, and iii were fixed and stained with Hoechst33342 for nuclear staining (blue). The fluorescence distribution of the drug in the cells was observed using a confocal microscope ( Figure 4 B), the results showed that AD-NVs@CPP (pH 6.5) and AD-NVs-treated ApoBDs were able to effectively deliver drugs to neighboring cells. Strong drug fluorescence was still maintained in the phase I cells in plate iii, which also verified that the cells treated with the nanoformulation had a strong neighboring effect. In contrast, although the DOX solution was able to effectively induce cell apoptosis and generate ApoBDs, it failed to demonstrate the ability to deliver drugs to neighboring cells, so almost no drug fluorescence signal was detected in plate ii. Furthermore, as the drug transferred between cells, the green fluorescence signal labeled DOX gradually decayed, while the red fluorescence signal labeled AQ4N was relatively enhanced. This phenomenon reveals the programmed delivery process of the drug. Compared with AQ4N, DOX was less transferred to neighboring cells through ApoBDs.

[0110] Unbound drug was separated by 3KDa ultrafiltration tubes, and the state and content of the drug in different phases were analyzed by HPLC. We found that under normal oxygen conditions, the specific interaction between DOX and the intracellular target resulted in about 1 / 3 of DOX being in a bound state in phase I of plate i. On this basis, the binding rate of DOX in ApoBDs produced in plate i further increased, reaching a peak in phase I of plate ii, as shown in Figure 2. Figure 4 D. This phenomenon reveals that the transfer of active DOX to neighboring cells through ApoBDs is limited. At the same time, AQ4N maintains a relatively stable free state ratio during the intercellular transfer process, as shown in Figure 4 As shown in C, most AQ4N was well preserved in ApoBDs and effectively transferred to neighboring cells. As the relative content of AQ4N increased, we observed a gradual increase in the intensity of the red fluorescence signal. Figure 4 E, which shows the process of programmed drug delivery. In addition, the apoptosis results of the cells in plates (i) and (ii) were confirmed by the high expression of apoptosis-related proteins γH2AX and cleaved caspase-3, as shown in Figure 5. Figure 4 As shown in F. In apoptotic cells, the proportion of AQ4N reduced to AQ4 with cell-killing ability is relatively low, as shown in Figure 4 As shown in G, it was shown that the depletion of DOX was the main factor leading to cell apoptosis.

[0111] 1×10 4 4T1 cancer cells were evenly distributed in 20μl complete cell culture medium containing 0.24% methylcellulose. Then, the mixture was added dropwise to a round-bottomed 96-well plate cover, and it was placed upside down in a cell culture incubator and incubated for 24 hours. Next, 200μl of cell culture medium was injected into each well, and centrifuged at 2800rpm for 3 minutes to allow the tumor spheres to settle to the bottom of the wells. Thereafter, the culture was continued and the cell culture medium was replaced every 48 hours. On the sixth day, the supernatant in the well was removed, and cell culture medium containing different reagents was added, in which the concentrations of DOX and AQ4N were 10μg / mL. After 24 hours, the cell culture medium was removed and the tumor spheres were washed with PBS. Finally, the distribution of the drug in the tumor spheres was examined using a confocal microscope, and the penetration effect of the drug was evaluated by setting a Z-axis scan at 40μm intervals. The results are shown in Figure 2. Figure 4As shown in Figure 3, although the DOX solution has a strong tumor-killing effect, it does not have an adjacent effect, so the drug fluorescence distribution is only observed in the outer layer of the tumor sphere, and it cannot penetrate into the interior of the tumor sphere. In the DOX and AQ4N mixed solution experimental group, it was observed that AQ4N can penetrate deeper into the tumor interior than DOX, showing a "relay race" type of programmed penetration characteristics. In particular, AD-NVs@CPP (pH 6.5) showed the strongest penetration ability, and strong fluorescence signals can be observed even in the center of the tumor. In contrast, due to the lack of customized peptides that promote tumor cell macropinocytosis, the AQ4N red fluorescence of the AD-NVs and mixed solution treatment groups still cannot penetrate deep into the tumor sphere. ImageJ software was used to quantitatively analyze the changes in fluorescence intensity with depth, which is consistent with the observations under the confocal microscope ( Figure 4 I, Figure 4 J) The above results demonstrate that enhancing macropinocytosis can significantly improve ApoBDs-mediated deep tumor drug delivery.

[0112] Example 6: Pharmacokinetics, biodistribution and in vivo permeation of biomineralized cell membrane nanovesicles

[0113] Nine male SD rats weighing between 180 and 220 g were randomly divided into three groups of three rats in each group. The DOX solution, the AD-NVs prepared in Example 1, and the AD-NVs@CPP prepared in Example 2 were injected into the corresponding groups of rats through the tail vein (the equivalent dose of DOX in each group was 2 mg / kg). Blood samples from the rat ocular vein were collected at nine preset time points of 0.03 h, 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 12 h in anticoagulation EP tubes pre-coated with heparin. The plasma samples collected were centrifuged (13000 r / min, 5 min) and stored in a -80 ° C refrigerator for later use. Then, UPLC-MS-MS was used to determine the DOX content in plasma. The results are shown in the figure below. Figure 5 As shown in A, DOX solution is rapidly cleared from the blood, and its half-life (t 1 / 2 ) is only about 1.2h. Compared with DOX solution, nanovesicles can significantly prolong the half-life of drugs in the blood circulation. In addition, compared with DOX solution, AD-NVs and AD-NVs@CPP significantly increased the area under the drug-time curve (AUC 0-24h ), indicating that nanovesicles can increase the stability of drugs in the blood and improve their circulation time in the body, which is beneficial for the accumulation of drugs in tumor sites and enhances the anti-tumor effect.

[0114] 100 μl of the prepared 4T1 cell suspension (5×10 6cells) to establish a 4T1 orthotopic tumor mouse model. 3 The mice were randomly divided into three groups with three mice in each group and injected with AQ4N solution, AD-NVs prepared in Example 1, and AD-NVs@CPP prepared in Example 2 (the equivalent dose of AQ4N in each group was 2 mg / kg) through the tail vein. The fluorescence signal of AQ4N in the mice was observed using a small animal in vivo imaging device at 2 h, 4 h, 8 h, 12 h, and 24 h after administration ( Figure 5 B). Mice were sacrificed 24 hours later, and their hearts, livers, spleens, lungs, kidneys, and tumors were isolated, and the fluorescence signals of these organs were semi-quantitatively analyzed ( Figure 5 C). The results showed that AQ4N in the group injected with AQ4N solution was rapidly cleared from the body, and its distribution in the tumor and other organs was not specific. In contrast, AD-NVs prepared in Example 1 and AD-NVs@CPP prepared in Example 2 showed significant fluorescence accumulation in the tumor area, which gradually increased over time, reaching a peak at 12 hours. After 24 hours, the fluorescence in the tumor area was still clearly visible, indicating its specific aggregation and persistent accumulation in the tumor site. In addition, the fluorescence distribution in the organs was observed by the in vivo imaging system and combined with the semi-quantitative analysis results ( Figure 6 D) It can be seen that after 24 hours, the AQ4N solution showed little drug distribution in the tumor, with the drug primarily distributed in the liver and lungs. In contrast, AD-NVs and AD-NVs@CPP maintained significant fluorescence accumulation in tumor tissue. These results demonstrate that nanovesicles can enhance drug accumulation in the tumor region and reduce toxic side effects during systemic circulation.

[0115] Observe the staining of 4T1 mouse model tumor sections in each group ( Figure 5 E), the results showed that the AD-NVs prepared in Example 1 and the AD-NVs@CPP treatment group prepared in Example 2 were able to effectively penetrate into the tumor and were accompanied by programmed drug penetration. The red fluorescence representing AQ4N penetrated deeper into the tumor tissue than the green fluorescence representing DOX. The AD-NVs prepared in Example 1 were limited in their ability to penetrate deep into the tumor due to the customized peptide segment that specifically promoted macropinocytosis, and they mainly accumulated at the edge of the tumor. The mixed solution of DOX and AQ4N was unable to cross multiple physiological barriers in the body and was rapidly cleared during the circulation process, resulting in its inability to accumulate in the tumor tissue and the absence of basically no drug fluorescence signal. The above results demonstrate the importance of considering the design of drug delivery systems in tumor treatment.

[0116] Example 7: In vivo anti-tumor activity and biosafety investigation of biomineralized cell membrane nanovesicles

[0117] 100 μl of the prepared 4T1 cell suspension (5×10 6 cells) to establish a 4T1 mouse orthotopic tumor model. When the tumor volume reached approximately 100 mm 3 The mice were randomly divided into 6 experimental groups with 5 mice in each group. Each group received the following treatments: intravenous injection of normal saline, DOX solution, AQ4N solution, a mixed solution of DOX and AQ4N, AD-NVs prepared in Example 1 above, and AD-NVs@CPP prepared in Example 2 above (the equivalent dose of DOX and AQ4N in each group was 2 mg / kg). Treatment started on day 0 and was administered once every other day for a total of 5 times. The tumor volume of the mice was recorded daily during the treatment period, and their body weight was monitored. The calculation formula for tumor volume is as follows. Based on this data, we plotted a graph showing the changes in tumor volume and mouse body weight over time ( Figure 6 A, Figure 6 B) to evaluate the efficacy of different treatment options.

[0118] Tumor volume = long diameter × (short diameter) 2 ×1 / 2

[0119] After the efficacy evaluation phase was completed, the tumor-bearing mice were killed, their tumors were removed, weighed, and photographed. Figure 6 C). Afterwards, these tumor samples were placed in 4% paraformaldehyde and sent to Sevier Biotechnology Co., Ltd. for sectioning. TUNEL apoptosis assay and Ki67 cell proliferation assay were performed and stained. The cell proliferation and apoptosis status were evaluated by observing the tumor sections. In addition, H&E staining was further performed to observe the pathological changes of the tumor tissue ( Figure 6 D).

[0120] The results showed that compared with other treatment groups, AD-NVs@CPP significantly inhibited tumor growth and exhibited the most significant anti-tumor activity, effectively controlling the tumor volume to approximately 100 mm. 3 , essentially preventing further tumor growth. This remarkable effect is attributed to the specific promoting effect of the SDF1 mimetic peptide, which significantly enhances the drug's local penetration into the tumor, enabling the combined use of DOX and AQ4N to effectively act on both normoxic and hypoxic areas of the tumor, thereby achieving comprehensive tumor inhibition. Furthermore, histological analysis using H&E staining, TUNEL apoptosis staining, and Ki67 staining revealed that AD-NVs@CPP has the strongest ability to induce tumor cell apoptosis and significantly reduce tumor cell proliferation, demonstrating the superiority of biomineralized cell membrane nanovesicles in tumor treatment.

[0121] Before dissecting each group of mice, blood samples were obtained by removing the mouse eyeballs and collected in anti-coagulation vacuum blood collection tubes. After the sample was centrifuged at 4000r / min for 10 minutes, the supernatant was collected and sent to Sevier Biotechnology Co., Ltd. for liver and kidney function parameter testing. After recording the weight changes of tumor-bearing mice, it was found that the weight of mice in the AD-NVs@CPP treatment group remained stable, proving that it has good safety. In contrast, DOX has a known cardiotoxicity, especially at cumulative doses, and the risk of chronic toxicity to the heart, resulting in a significant weight loss in mice in the DOX and mixed solution groups ( Figure 6 E). In addition, there was no significant difference in liver and kidney function test results between AD-NVs@CPP and the control group (normal saline), indicating that it had no adverse effects on the functions of these major organs ( Figure 6 F, Figure 6 G).

[0122] Example 8:

[0123] H&E staining of the main organs (heart, liver, spleen, lung, and kidney) of the mice in the different treatment groups in Example 7 was performed. After the staining experiment, the heart, liver, spleen, lung, and kidney tissues of the mice were collected and fixed with 4% paraformaldehyde. These tissue samples were then sent to Sevier Biotechnology Co., Ltd. for H&E staining to evaluate the pathological changes of the organs. The results are shown below. Figure 7 As shown, H&E staining of major organs such as the heart, liver, spleen, lungs, and kidneys showed no obvious lesions, further confirming the biosafety of biomineralized cell membrane nanovesicles.

[0124] In summary, the present invention obtains biomineralized cell membrane nanovesicles that can specifically promote the macropinocytosis of tumor cells and have programmed drug permeation properties. Specifically, the full tumor-killing small molecule chemotherapy drug doxorubicin and the hypoxia-activated prodrug banoxanthraquinone are combined and co-loaded in the nanovesicles. Through the mineralization strategy, the peptide segment RFFE-SHAPAKPVSLSYR that can specifically promote the macropinocytosis of tumor cells is encapsulated outside the drug-loaded nanovesicles to construct biomineralized cell membrane nanovesicles. Due to the acid-responsive dissolution characteristics of the calcium phosphate mineralized shell, the drug mineralized shell can dissolve and release the peptide RFFE-SHAPAKPVSLSYR in the slightly acidic environment of the tumor, promoting the macropinocytosis of tumor cells, thereby increasing the rate of tumor cell uptake of the nanovesicle core, so that more nanovesicles can enter tumor cells, thereby killing tumor cells and inducing the production of drug-containing apoptotic bodies. Next, through the programmed drug delivery process, in a "relay race" mode, the banothramine is delivered to the deep hypoxic area of the tumor tissue and exerts its killing effect on tumor cells, achieving a comprehensive killing effect on tumor tissue. The present invention has a good application prospect in solving the problem of nano drug delivery and is also very potential in clinical applications.

Claims

1. A tumor-programmed drug-penetrating biomimetic mineralized nanovesicle with enhanced macropinocytosis, characterized in that: The drug-penetrating biomimetic mineralized nanovesicles have a shell-core structure. Chemotherapy drugs are co-loaded inside the cell vesicles, and peptides that specifically promote tumor cell macropinocytosis are encapsulated on the outside through mineralization.

2. The tumor-programmed drug-penetrating biomimetic mineralized nanovesicles with enhanced macropinocytosis according to claim 1, characterized in that: The chemotherapy drugs co-loaded inside the cell vesicles account for 70%-90% of the mass of the biomimetic mineralized nanovesicles, wherein the chemotherapy drugs are hypoxia-activated prodrugs and small molecule drugs with full tumor killing effects, and the mass ratio between the two is 0.7-1.0:1.0-1.

6.

3. The tumor-programmed drug-penetrating biomimetic mineralized nanovesicles with enhanced macropinocytosis according to claim 1 or 2, characterized in that: The cell vesicles are derived from mouse breast cancer (4T1) cells.

4. The tumor-programmed drug-penetrating biomimetic mineralized nanovesicles with enhanced macropinocytosis according to claim 1, characterized in that: The peptide segment accounts for 11%-16% of the mass of the biomimetic mineralized nanovesicles; wherein the peptide segment is RFFE-SHAPAKPVSLSYR.

5. A method for enhancing macropinocytosis-induced tumor-programmed drug penetration of biomimetic mineralized nanovesicles according to claim 1, characterized in that: (1) Prepare complete culture medium and incubate cells; (2) Isolation of cell membrane nanovesicles by centrifugation; (3) mixing the chemotherapy drug solution and the cell membrane nanovesicle solution; (4) ultrasonically treating the mixed solution; (5) incubation to restore the cell membrane nanovesicle structure; (6) centrifugation and resuspension to obtain drug-loaded cell membrane nanovesicles; (7) mixing the drug-loaded cell membrane nanovesicles obtained above with calcium chloride solution, peptide solution and buffer solution; (8) The mixture was stirred and centrifuged to obtain biomineralized cell membrane nanovesicles.

6. The method of enhancing macropinocytosis by tumor-programmed drug penetration biomimetic mineralized nanovesicles according to claim 5, characterized in that: In the step (3), the hypoxia-activated prodrug, the small molecule drug with full tumor killing effect and the cell membrane nanovesicles are mixed in a mass ratio of 1-3:1-3:4-7.

7. The method of enhancing macropinocytosis by tumor-programmed drug penetration biomimetic mineralized nanovesicles according to claim 5, characterized in that: In step (7), the volume ratio of the drug-loaded cell membrane nanovesicles to the calcium chloride solution, the peptide solution and the buffer solution is 7-11: 0.7-1.2:0.6-1.4:8-13; wherein, the buffer solution is HEPES buffer solution.

8. A use of the tumor-programmed drug penetration biomimetic mineralized nanovesicles for enhancing macropinocytosis according to claim 1, characterized in that: Application of the biomineralized cell membrane nanovesicles in the field of drug delivery.

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