Biomimetic nanoparticle suspension formulation based on orthoester compound and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a bionic nano-particle suspension formulation based on orthoester compounds and a preparation method of the bionic nano-particle suspension formulation. The bionic nanoparticle suspension formulation comprises a liquid auxiliary material, a photosensitizer and bionic nanoparticles, wherein the photosensitizer and the bionic nanoparticles are dispersed in the liquid auxiliary material. According to the present invention, an orthoester compound represented by a formula I is adopted as a liquid auxiliary material, bionic nanoparticles and a photosensitizer are simultaneously introduced into OE to obtain the bionic nanoparticle suspension formulation having multiple antitumor mechanisms, the bionic nanoparticles are cancer cell membrane vesicle-coated nano-drugs, and the cancer cell membrane vesicle-coated nano-drugs can be prepared through a cancer cell membrane biomimetic technology, such that the cancer cell membrane vesicle-coated nano-drugs can be prepared into the bionic nanoparticle suspension formulation having multiple antitumor mechanisms; the homologous targeting function provided by the cancer cell membrane is utilized, and the active targeting property and cellular uptake of the nano-drug are remarkably improved. Formula I: # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a bionic nanoparticle suspension formulation based on an orthoester compound and a preparation method thereof. Background Art

[0002] As a non-invasive treatment, phototherapy, including photodynamic therapy and photothermal therapy, has been identified as a diagnostic and therapeutic method for a variety of cancers by virtue of the effects of photosensitizers and photothermal converters. However, due to the hypoxic microenvironment of the tumor site and the limited penetration of light, the clinical efficacy of phototherapy is still far from satisfactory. Therefore, those skilled in the art have proposed that chemotherapy combined with phototherapy can produce a synergistic therapeutic effect and reduce the dose of small molecule chemotherapy drugs.

[0003] Nano drug delivery systems can effectively improve the targeting ability of drugs, reduce toxic side effects, and overcome problems such as drug resistance and metastasis through combined treatment of multiple drugs. Nevertheless, the potential toxicity, long-term safety, and complexity of biodistribution and clearance of nanomaterials may limit the scope and effectiveness of nano drug delivery systems in clinical applications. In contrast, bionic nanotechnology based on cancer cell membranes can not only improve the stability of nano drugs, but also increase active targeting capabilities. Although bionic technology provides a guarantee for the stability of nanoparticles, traditional nano preparations may still have problems such as premature drug release, insufficient delivery efficiency, and carrier toxicity when administered by intravenous injection. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a bionic nanoparticle suspension formulation based on an orthoester compound and a preparation method thereof.

[0005] The bionic nanoparticle suspension formulation based on an orthoester compound and the preparation method thereof of the present invention are achieved by the following technical scheme: The first object of the present invention is to provide a biomimetic nanoparticle suspension dosage form based on an orthoester compound, comprising a liquid auxiliary material, and a photosensitizer and biomimetic nanoparticles dispersed in the liquid auxiliary material.

[0006] It should be noted that the liquid excipient used in the present invention has OE, and for the sake of convenience, the present invention will refer to the orthoester compound shown in Formula I as OE below. OE is a pH-sensitive modified orthoester compound with a simple structure, a clear in vivo degradation mechanism, good biocompatibility, good fluidity and injectability, good solubility and / or dispersibility for various photosensitizers and biomimetic nanoparticles, and a stable suspension can be obtained without adding any additional suspending agent, which is conducive to achieving synergistic anti-tumor effects such as chemotherapy-photothermal. Moreover, OE can deliver photosensitizers directly to tumor areas or surgical wounds, and the lower specific heat capacity of OE makes the suspension heat up faster, and can achieve the same photothermal therapeutic effect at a lower concentration. In addition, compared with aqueous solutions, OE can significantly improve the photothermal conversion ability of photosensitizers, promote the photothermal therapeutic effect of suspensions, and reduce the amount of photosensitizers and potential side effects; when the suspension is used for wounds after tumor resection, OE has suitable fluidity and can cover target areas of any shape, thereby directly inhibiting tumor recurrence and expanding the application scenarios of the suspension formulation.

[0007]

[0008] Formula I.

[0009] It should also be noted that the bionic nanoparticles used in the present invention are based on nanodrugs as a matrix and are obtained by wrapping cell membrane vesicles on the surface thereof. The bionic nanoparticles are wrapped by cell membranes to give the nanodrugs biological properties, including extending the circulation time, improving stability and homologous targeting of cancer cells, so that the bionic nanoparticles used in the present invention can significantly improve the drug delivery efficiency in cancer cells through active targeting function, further enhance the anti-tumor effect, and make the bionic nanoparticle suspension dosage form of the present invention have active targeting performance and synergistic effect.

[0010] In some preferred embodiments of the present invention, the OE used in the present invention is prepared by the following steps: S1, stirring reaction: Using diglycerol and triethyl orthoacetate as raw materials and p-toluenesulfonic acid as a catalyst, diglycerol and triethyl orthoacetate are dispersed in an organic solvent at a molar ratio of 1:2-4 under a nitrogen atmosphere, and then p-toluenesulfonic acid is added in a corresponding mass according to a molar ratio of p-toluenesulfonic acid to diglycerol of 1:50-100, and then stirred at room temperature for reaction for 8h-16h, and the reaction equation is shown in Reaction Equation 1:

[0011] Reaction formula 1.

[0012] S2, post-processing: Triethylamine was added to the system after the above stirring reaction, and the pH of the solution system was adjusted to 8.0 to terminate the reaction, followed by two extractions with a saturated Na2CO3 solution to obtain an extract; the extract was dried by rotary evaporation and vacuum filtered to obtain the orthoester compound shown in Formula I, namely OE.

[0013] The present invention can prepare OE in one step at room temperature and pressure through the above-mentioned synthesis method, and the prepared OE has only carbon-oxygen bonds in its structure, which will not destroy the drug structure. More preferably, when synthesizing OE, the organic solvent used is anhydrous dichloromethane, and the volume ratio of the organic solvent used to diglycerol is 2 to 10:1.

[0014] More preferably, the amount of the photosensitizer added and the amount of the bionic nanoparticles added are both therapeutically effective amounts, and can be added according to the specific types of photosensitizer and bionic nanoparticles actually selected.

[0015] It should also be noted that in order to adjust the fluidity, degradation and drug release rate of the biomimetic nanoparticle suspension formulation as a whole, the present invention adjusts the content of the photosensitizer and the biomimetic nanoparticles in the biomimetic nanoparticle suspension formulation by adjusting the amount of OE added, thereby adjusting the fluidity, degradation and drug release rate of the biomimetic nanoparticle suspension formulation as a whole. And preferably, the present invention adjusts the amount of OE added so that the photosensitizer accounts for 0.1wt%~20wt% of the total mass of the biomimetic nanoparticle suspension formulation; the biomimetic nanoparticles account for 0.1wt%~40wt% of the total mass of the biomimetic nanoparticle suspension formulation.

[0016] Preferably, in the biomimetic nanoparticles, the cell membrane vesicles are selected from one cell membrane or multiple hybrid cell membranes of immune cells, cancer cells, red blood cells, platelets and exosomes. More preferably, the immune cells include granulocytes or macrophages. More preferably, the cancer cells are selected from any one of human liver cancer cells, breast cancer cells, lung cancer cells, mouse liver cancer cells and breast cancer cells. The cancer cells are more preferably human liver cancer cell line HepG2 or mouse liver cancer cell line H22.

[0017] Preferably, the nano drug is a pure drug nanoparticle or a drug-loaded nanoparticle. The pure drug nanoparticle is formed by self-assembly of a small molecule drug. The drug-loaded nanoparticle is obtained by loading a small molecule drug with a nanoparticle matrix as a carrier; the loading amount of the small molecule drug on the nanoparticle matrix is ​​1wt% to 20wt%. The nanoparticle matrix is ​​a natural macromolecular nanoparticle or an inorganic nanoparticle.

[0018] Preferably, the small molecule drug comprises an anti-tumor drug. More preferably, the anti-tumor drug is selected from one or more of dasatinib, sunitinib, sorafenib, mitoxantrone and doxorubicin. Further preferably, the anti-tumor drug is dasatinib and sunitinib.

[0019] Preferably, the natural macromolecular nanoparticles are selected from one or more of polysaccharide nanoparticles and protein nanoparticles, for example, any one or more of chitosan, carboxymethyl chitosan, hyaluronic acid, chondroitin sulfate, sodium alginate, collagen, gelatin, bromelain, hyaluronidase, catalase, cellulose and hydroxypropyl methylcellulose. Further preferably, the natural macromolecular nanoparticles are carboxymethyl chitosan nanoparticles.

[0020] Preferably, the inorganic nanoparticles are selected from any one or more of mesoporous silicon nanoparticles, ferrosoferric oxide nanoparticles, zinc sulfide nanoparticles, cuprous selenide nanoparticles and cerium nanoparticles.

[0021] Preferably, the particle size of the nanodrug is 10 nm to 300 nm; the cell membrane vesicle is spherical and the diameter of the cell membrane vesicle is 20 nm to 1000 nm, so as to adapt to the particle size of the nanodrug and achieve complete encapsulation of the nanoparticles.

[0022] Preferably, when pure drug nanoparticles are used as nanomedicines, the drug loading of the nanomedicine is 100%. In order to facilitate understanding of the situation when pure drug nanoparticles are used as nanomedicines, the present invention takes dasatinib and sunitinib as small molecule drugs as an example to illustrate the process of preparing small molecule drugs to self-assemble into pure drug nanoparticles: According to the mass ratio of 2.5~3.5:1, weigh the corresponding mass of dasatinib and sunitinib for use. Dissolve the weighed dasatinib and sunitinib in dimethyl sulfoxide to obtain a dasatinib solution with a concentration of 350μM~450μM and a sunitinib solution with a concentration of 350μM~450μM. Mix the dasatinib solution and the sunitinib solution to obtain a mixed solution, then gradually add the mixed solution to deionized water at room temperature and stir for 3 min~10min to allow dasatinib and sunitinib to self-assemble to form a composite drug material. Then, centrifuge the solution at 7000 rpm~9000 rpm for 5min~15min, collect the precipitate and wash it at least 3 times, remove dimethyl sulfoxide, and dry the precipitate to obtain pure drug nanoparticles.

[0023] Preferably, when the drug-loaded nanoparticles are used as nanomedicines, the drug-loaded nanoparticles are prepared by the following steps: dispersing the nanoparticle matrix in water to obtain a nanoparticle matrix solution; then dispersing the small molecule active drug in water to obtain a small molecule active drug aqueous solution; then, under stirring, dropwise adding the small molecule active drug aqueous solution to the nanoparticle matrix solution, and continuing to stir for 3h~5h after the dropwise addition is completed, so that the small molecule active drug is adsorbed on the surface of the nanoparticle matrix through physical adsorption. After the reaction is completed, the reaction solution is centrifuged at 7000 rpm~9000 rpm for 5min~15min, the precipitate is collected and washed at least 3 times, and then freeze-dried to obtain the drug-loaded nanoparticles.

[0024] In order to facilitate understanding of the situation when natural macromolecular nanoparticles are used as nanoparticle matrices and drug-loaded nanoparticles obtained by loading small molecule drugs are used as nanomedicines, the present invention takes carboxymethyl chitosan and mitoxantrone as examples to illustrate the process of preparing drug-loaded nanoparticles based on natural macromolecules: (1) According to the dosage ratio of carboxymethyl chitosan powder to water of 5g~7g:6L, the carboxymethyl chitosan powder is fully dissolved in the corresponding volume of water to obtain a carboxymethyl chitosan aqueous solution. According to the volume ratio of acetone to the water of 1.8~2.2:1, under stirring conditions, the corresponding volume of acetone is added dropwise to the carboxymethyl chitosan aqueous solution, and a glutaraldehyde solution with a mass concentration of 25% is added with a volume of 9vol%~11vol% of the amount of water, mixed and cross-linked for 3h~5h, and then the reaction solution is centrifuged at 9000 rpm~11000 rpm for 8 min~15min, and the precipitate obtained is collected as carboxymethyl chitosan nanoparticles. The carboxymethyl chitosan nanoparticles are dispersed in water to obtain a carboxymethyl chitosan aqueous solution.

[0025] (2) According to the dosage ratio of mitoxantrone to water of 0.8 g to 1.2 g: 1 L, mitoxantrone is fully dispersed in water to obtain a mitoxantrone aqueous solution.

[0026] (3) The mitoxantrone aqueous solution was added dropwise to the carboxymethyl chitosan aqueous solution in a ratio of 1:2.5-3.5, and the mixture was stirred for 3 h-5 h to allow the mitoxantrone to be adsorbed on the surface of CMCS NPs by physical adsorption. The reaction solution was centrifuged at 7000 rpm-9000 rpm for 5 min-15 min, and the precipitate was collected, washed at least 3 times, and then freeze-dried to obtain drug-loaded nanoparticles.

[0027] Preferably, in order to facilitate understanding of the situation when inorganic nanoparticles are used as nanoparticle matrix and the drug-loaded nanoparticles obtained by loading small molecule drugs are used as nanomedicines, the present invention uses mesoporous silica nanoparticles as nanoparticle matrix and mitoxantrone as an anti-tumor drug as an example to illustrate the process of preparing drug-loaded nanoparticles based on inorganic nanoparticles: (1) Disperse hexadecyltrimethylammonium bromide (CTAB) in a corresponding volume of pure water at a dosage ratio of 1.50 g:700~800 mL to obtain a CTAB solution. (2) Add a corresponding volume of 2.0 M sodium hydroxide aqueous solution in a volume ratio of 5~5.5:700~800 of the above pure water, mix well, and stir vigorously at 75°C~85°C. (3) After reacting for 0.4h~0.8h, add ethyl acetate in a volume of 9vol%~11vol% of the amount of pure water, stir and react for 2h, age at room temperature for 6h~12h, and centrifuge at 7000rpm~9000rpm for 10min~20min to obtain unextracted mesoporous silicon nanoparticles, which are referred to as MSN NPs. (4) Wash with ultrapure water and ethanol three times and dry in vacuum. (5) According to the ratio of 1 mL of 37% concentrated hydrochloric acid and 100 mL of ethanol per gram of MSN NPs, add the corresponding volume of concentrated hydrochloric acid and ethanol to the MSN NPs obtained above, mix well, react at 70 °C for 24 h, then centrifuge again, wash with ethanol, and dry to obtain mesoporous silicon nanoparticles. (6) According to the dosage ratio of 8mg~12mg:5mL, disperse the mesoporous silicon nanoparticle powder in the corresponding volume of 0.1 M phosphate buffer with pH=7.4. (7) According to the mass ratio of MIT to mesoporous silicon nanoparticles of 1:2.5~3.5, add the corresponding volume of mitoxantrone solution, stir in the dark for 3h~5h, then centrifuge at 7000rpm~9000rpm for 10 min, discard the supernatant, collect the precipitate, and freeze-dry to obtain nanomedicine based on inorganic nanoparticles.

[0028] Preferably, the bionic nanoparticles are prepared by the following steps: using cell membrane vesicles as coating agents and adopting a thin film extrusion method to wrap the cell membrane vesicles on the surface of the nano-drug, thereby obtaining the bionic nanoparticles.

[0029] Preferably, the present invention wraps the cell membrane vesicles on the surface of the nano drug by the following steps to obtain biomimetic nanoparticles: after mixing the obtained cancer cell membrane vesicles with the nanoparticles, the biomimetic nanoparticles are obtained by extruding them back and forth through a polycarbonate membrane with a pore size of 400 nm and 200 nm. The mass ratio of the cancer cell membrane vesicles to the nanoparticles is 1 to 5:1. The technology of wrapping the cell membrane vesicles on the surface of the nanomaterial by the thin film extrusion method of the present invention belongs to the prior art in the art, so the present invention will not be repeated here.

[0030] Preferably, the photosensitizer is selected from small molecule drugs for photodynamic therapy and photothermal therapy. For example, the photosensitizer is selected from one or more of dihydrochlorin e6, indocyanine green and neoindocyanine green.

[0031] The second object of the present invention is to provide a method for preparing the above-mentioned bionic nanoparticle suspension based on an orthoester compound, comprising the following steps: using an orthoester compound as shown in Formula I as a liquid excipient, dispersing a photosensitizer and bionic nanoparticles in the liquid excipient to obtain the bionic nanoparticle suspension.

[0032] Compared with the prior art, the present invention has the following beneficial effects: The biomimetic nanoparticle suspension dosage form of the present invention includes a liquid auxiliary material, and a photosensitizer and biomimetic nanoparticles dispersed in the liquid auxiliary material. The present invention uses OE as a liquid auxiliary material, and introduces biomimetic nanoparticles and photosensitizers into OE at the same time to obtain a new composite suspension with multiple anti-tumor mechanisms, wherein the biomimetic nanoparticles use the homologous targeting function provided by the cancer cell membrane through the biomimetic technology of the cancer cell membrane, change the drug uptake mechanism, and change passive diffusion to active uptake, significantly improving the active targeting and cellular uptake of nano drugs. Compared with aqueous solutions, the present invention uses OE as a liquid auxiliary material, which can significantly improve the photothermal conversion ability of the photosensitizer, promote the photothermal therapeutic effect of the suspension, and reduce the dosage of the drug at the same time, reducing potential side effects. And OE is a pH-sensitive liquid compound with a simple structure, a clear degradation mechanism in the body, good biocompatibility, good fluidity and injectability, and can directly dissolve or disperse a variety of photosensitizers and nano drugs to obtain a suspension dosage form to facilitate the realization of synergistic anti-tumor effects such as chemotherapy-photothermal. Moreover, using OE as a liquid excipient can ensure that the components of the biomimetic nanoparticle suspension formulation remain uniform and stable without the need for additional suspending agents. In addition, OE can also improve the photothermal conversion performance of photosensitizers and significantly improve the photothermal treatment effect of tumors.

[0033] The biomimetic nanoparticle suspension dosage form of the present invention can not only be used for in situ injection of tumors, but also for wounds after tumor resection. OE has suitable fluidity and can cover target areas of any shape, thereby directly inhibiting tumor recurrence and expanding the application scenarios of the suspension dosage form. And the biomimetic nanoparticle suspension dosage form of the present invention is an orthoester dosage form loaded with biomimetic nanoparticles and photosensitizers, which can achieve chemotherapy / photothermal combined therapy. Therefore, the orthoester suspension dosage form loaded with biomimetic nanomedicines and photosensitizers at the same time is of great significance in achieving local drug delivery to tumors and inhibiting postoperative tumor recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The orthoester compound OE of the present invention is 1H-NMR spectrum.

[0035] Figure 2 The orthoester compound OE of the present invention is 13 C-NMR spectrum.

[0036] Figure 3 The particle size distribution diagram of the nanoparticles NPs, cancer cell membrane vesicles CM prepared in the present invention and the biomimetic nanoparticles CM@NPs prepared in Example 1 is shown in FIG. Figure 3 In the figure, Figure A is the particle size distribution diagram of the nanoparticles NPs prepared by the present invention, Figure B is the particle size distribution diagram of the cancer cell membrane vesicles CM prepared by the present invention, and Figure C is the particle size distribution diagram of the bionic nanoparticles CM@NPs prepared in Example 1.

[0037] Figure 4 The physical pictures are of OE prepared in the present invention, OE / IR of Comparative Example 1, OE / CM@NPs of Comparative Example 3 and OE / IR / CM@NPs of Example 1. Figure 4 In the figure, Figure A is a physical picture of OE prepared by the present invention, Figure B is a physical picture of OE / IR of comparative example 1, Figure C is a physical picture of OE / CM@NPs of comparative example 3, and Figure D is a physical picture of OE / IR / CM@NPs of Example 1.

[0038] Figure 5 The light-to-heat conversion test results of pure water, water / IR of comparative example 4, and OE / IR of comparative example 1 are shown. Figure 5 In the figure, Figure A is the photothermal conversion test result of pure water, Figure B is the photothermal conversion test result of water / IR of comparative example 4, and Figure C is the photothermal conversion test result of OE / IR of comparative example 1.

[0039] Figure 6 The temperature rise curves of water / IR of comparative example 4 and OE / IR of comparative example 1 under different power irradiation conditions are shown in FIG. Figure 6 In the figure, Figure A is the temperature rise curve of water / IR of comparative example 4 under different power irradiation conditions, and Figure B is the temperature rise curve of OE / IR of comparative example 1 under different power irradiation conditions.

[0040] Figure 7 The water / IR of comparative example 4 and the OE / IR of comparative example 1 and their dilutions to different concentrations at 1.0 W / cm 2 The heating curve under power irradiation conditions, Figure 7 Figure A shows the comparative example 4 and its dilution to different concentrations at 1.0 W / cm 2 The temperature rise curve under power irradiation conditions, Figure B shows the comparative example 1 and its dilution to different concentrations at 1.0W / cm 2 Temperature rise curve under power irradiation conditions.

[0041] Figure 8 In vitro drug release diagrams of NPs, OE@NPs and OE / CM@NPs at different pH values. Figure 8 In the figure, (a) shows the in vitro drug release graph of NPs at different pH values, (b) shows the in vitro drug release graph of OE@NPs at different pH values, and (c) shows the in vitro drug release graph of OE / CM@NPs at different pH values.

[0042] Fig. 9 Cell viability at different concentrations of OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+).

[0043] Fig.10 Changes in intracellular ROS content caused by OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) under no light and light conditions, respectively.

[0044] Fig.11 These are the cell live and dead staining results of OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) under no light and light conditions, respectively.

[0045] Fig.12 The results of the in vivo photothermal conversion experiment of the present invention are as follows: Fig.12 Figure A is the thermal imaging image after OE / IR / CM@NPs with different IR820 dosage contents of 0.8 mg / kg, 1.6 mg / kg and 2.4 mg / kg were injected into the tumor area; Figure B is the temperature counting result of the tumor area of ​​each mouse in Figure A; Figure C is the temperature increase image of the tumor area of ​​the mouse 24h, 72h and 120h after OR / IR / CM@NPs with an IR820 dosage of 2.4 mg / kg were injected into the tumor area; Figure D is the temperature counting result of the tumor area of ​​each mouse in Figure C.

[0046] Fig.13 The changes in tumor volume over time in H22 tumor-bearing mice of the Control group, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+).

[0047] Fig.14 The changes in body weight of H22 tumor-bearing mice in the Control group, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) over time.

[0048] Fig.15The changes of tumor mass over time in H22 tumor-bearing mice of the Control group, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+).

[0049] Fig.16 Figure 2 shows the changing trends of tumor volumes of mice in the saline group, OE / IR+ group, OE / CM@NPs group, and OE / IR / CM@NPs+ group during the 50-day observation period. Fig.16 Figure A shows the changing trend of tumor volume of 6 mice in the normal saline group during the 50-day observation period, Figure B shows the changing trend of tumor volume of 6 mice in the OE / IR+ group during the 50-day observation period, Figure C shows the changing trend of tumor volume of 6 mice in the OE / CM@NPs group during the 50-day observation period, and Figure D shows the changing trend of tumor volume of 6 mice in the OE / IR / CM@NPs+ group during the 50-day observation period.

[0050] Fig.17 This is a graph showing the changes in survival rate of mice in the normal saline group, OE / IR+ group, OE / CM@NPs group, and OE / IR / CM@NPs+ group during the 50-day observation period.

[0051] Fig.18 The apoptosis experimental results of the Control group, MIT group and OE / IR / CM@C / M NPs group on mouse liver cancer cell H22. Fig.18 In the figure, (a) shows the apoptosis experimental results caused by the Control group, (b) shows the apoptosis experimental results caused by the MIT group, and (c) shows the apoptosis experimental results caused by the OE / IR / CM@C / M NPs group.

[0052] Fig.19 Live / dead double staining results of the Control group, MIT group, and OE / IR / CM@C / M NPs group on mouse liver cancer cell H22. Fig.19 In the figure, (a) shows the live / dead double staining experiment results caused by the Control group, (b) shows the live / dead double staining experiment results caused by the MIT group, and (c) shows the live / dead double staining experiment results caused by the OE / IR / CM@C / M NPs group.

[0053] Fig. 20 The apoptosis experimental results of the Control group, MIT group and OE / IR / CM@M / M NPs group on mouse liver cancer cell H22. Fig. 20In the figure, (a) shows the apoptosis experimental results caused by the Control group, (b) shows the apoptosis experimental results caused by the MIT group, and (c) shows the apoptosis experimental results caused by the OE / IR / CM@M / M NPs group.

[0054] Fig.21 Live / dead double staining results of the Control group, MIT group, and OE / IR / CM@M / M NPs group on mouse liver cancer cell H22. Fig.21 In the figure, (a) shows the live / dead double staining experiment results caused by the Control group, (b) shows the live / dead double staining experiment results caused by the MIT group, and (c) shows the live / dead double staining experiment results caused by the OE / IR / CM@M / M NPs group. DETAILED DESCRIPTION

[0055] The technical solutions in the embodiments of the present invention are described clearly and completely below. For ease of description, the present invention will refer to the new indocyanine green as IR820 below. It should be noted that the H22 cancer cells and HepG2 cancer cells involved in the present invention below were purchased from Jiangsu Keygene Biotechnology Co., Ltd. and Shanghai Bio-Technology Co., Ltd., respectively.

[0056] It should be noted that all the Control groups in the present invention refer to the control group. It should also be noted that in the following embodiments of the present invention, the liquid auxiliary material used is OE, and the OE used in the present invention is prepared by the following steps: S1, stirring reaction: accurately weigh diglycerol, triethyl orthoacetate and p-toluenesulfonic acid according to a molar ratio of 1:4:0.02; and weigh a corresponding volume of anhydrous dichloromethane according to a volume ratio of anhydrous dichloromethane to the diglycerol of 5:1, and set aside; disperse the weighed diglycerol, triethyl orthoacetate and p-toluenesulfonic acid in anhydrous dichloromethane, and then stir the reaction at room temperature under a nitrogen atmosphere for 12 hours, and the reaction equation is shown in Reaction Formula 1:

[0057] Reaction formula 1.

[0058] S2, post-treatment: adding triethylamine to the system after the above stirring reaction to adjust the pH of the reaction solution to 8.0 to terminate the reaction, followed by two extractions with a saturated Na2CO3 solution to obtain an extract; the extract was dried by rotary evaporation and vacuum filtered to obtain an orthoester compound as shown in Formula I, namely OE.

[0059] The present invention also uses hydrogen spectrum 1 H-NMR and carbon spectroscopy 13 C-NMR was used to test the chemical structure of the OE prepared above, and the test results were as follows: Figure 1 and Figure 2 shown.

[0060] in, Figure 1 The orthoester compound OE of the present invention is 1 H-NMR spectrum, Figure 2 The orthoester compound OE of the present invention is 13 C-NMR spectrum. Figure 1 and Figure 2 It can be seen from the test results that the present invention successfully synthesized the OE shown in Formula 1.

[0061] In the following embodiments of the present invention, the cell membrane vesicles used are cancer cell membrane vesicles, and the cell membrane vesicles are extracted by the following steps: H22 cancer cells and HepG2 cancer cells are used as the source of cell membrane vesicles, and when the H22 cells and HepG2 cells grow to 80%, the cells are collected by centrifugation, and each 1×10 6 500 μL hypotonic lysis buffer and 5 μL PMSF were added to each cell, and the cells were ultrasonically disrupted for 2 min. The ultrasonic parameters were: 6 s / time, 30 W. Then, the cells were centrifuged at 1000×g for 2 min at 4°C, and the nuclear material in the precipitate was discarded. The supernatant was collected and centrifuged at 14000 rpm for 40 min to obtain cell membrane fragments. The cell membrane fragments were repeatedly squeezed through polycarbonate membranes with pore sizes of 800 nm and 400 nm in turn to obtain H22 cell membrane vesicles and HepG2 cell membrane vesicles, abbreviated as CM.

[0062] Example 1 This embodiment provides a biomimetic nanoparticle suspension formulation based on an orthoester compound, and the biomimetic nanoparticle suspension formulation based on an orthoester compound of this embodiment is prepared by the following steps: Step 1, preparation of biomimetic nanoparticles: Step 1.1, preparation of nanomedicine: The nanomedicine used in this embodiment is a pure drug nanoparticle formed by self-assembly of a small molecule drug, and in this embodiment, the corresponding mass of dasatinib and sunitinib are weighed according to a mass ratio of 3:1 for standby use. The weighed dasatinib and sunitinib are respectively dissolved in dimethyl sulfoxide to obtain a dasatinib solution and a sunitinib solution with a concentration of 400 μM. The dasatinib solution and the sunitinib solution are mixed to obtain a mixed solution, and then the mixed solution is gradually added to deionized water at room temperature and stirred for 5 min to allow dasatinib and sunitinib to self-assemble to form a composite drug material. Then, the solution is centrifuged at 8000 rpm for 10 min, the precipitate is collected and washed 3 times, dimethyl sulfoxide is removed, and the obtained precipitate is dried to obtain pure drug nanoparticles, referred to as NPs.

[0063] Step 1.2, coating cell membrane vesicles: the cancer cell membrane vesicles CM extracted above and the nanodrug NPs prepared in step 1.1 are mixed by ultrasound and vortex at a mass ratio of 2:1, and then squeezed back and forth through a polycarbonate membrane with a pore size of 400 nm and 200 nm to obtain the bionic nanoparticles, which are recorded as CM@NPs.

[0064] Step 2, dispersion of biomimetic nanoparticles and photosensitizer: Using IR820 as a photosensitizer, 1 mL of OE, 1 mg of IR820, and 1 mg of CM@NPs were mixed and stirred for 10 min to obtain a biomimetic nanoparticle suspension formulation, referred to as OR / IR / CM@NPs.

[0065] Example 2 The difference between this embodiment and embodiment 1 is only that: In this embodiment, the mass ratio of cancer cell membrane vesicles to nanomedicine is 3:1.

[0066] Example 3 The difference between this embodiment and embodiment 1 is only that: In this embodiment, the mass ratio of cancer cell membrane vesicles to nanomedicine is 4:1.

[0067] Example 4 The difference between this embodiment and embodiment 1 is only that: In this embodiment, the mass ratio of cancer cell membrane vesicles to nanomedicine is 5:1.

[0068] Example 5 The difference between this embodiment and embodiment 1 is only that: In this embodiment, the mass ratio of cancer cell membrane vesicles to nanomedicine is 6:1.

[0069] Example 6 The difference between this embodiment and embodiment 1 is only that: In this embodiment, the nano drug is a drug-loaded nanoparticle prepared based on natural macromolecules, that is, the nano drug used in this embodiment is a drug-loaded nanoparticle, and the drug-loaded nanoparticle of this embodiment uses natural macromolecular carboxymethyl chitosan nanoparticles as the nanoparticle matrix, and uses mitoxantrone as the anti-tumor drug, and the anti-tumor drug mitoxantrone is loaded on the natural macromolecular carboxymethyl chitosan nanoparticles by the following steps: In this embodiment, carboxymethyl chitosan CMCS and chemotherapy drug mitoxantrone MIT are used as raw materials to prepare nanomedicine based on natural macromolecules, and the specific preparation process is as follows: (1) Weigh 60 mg of CMCS powder and fully dissolve it in 6 mL of deionized water to prepare a 10 mg / mL CMCS aqueous solution. Add 12 mL of acetone solution dropwise to the solution while stirring, then add 60 μL of 25% glutaraldehyde solution and crosslink for 4 hours. Finally, centrifuge the reaction solution at 10,000 rpm for 10 min, collect the precipitate, and the obtained nanoparticles are recorded as CMCS NPs. Disperse CMCS NPs in water to obtain a 3 mg / mL CMCS NPs solution.

[0070] (2) Fully disperse MIT in water to obtain a 1 mg / mL MIT aqueous solution.

[0071] (3) The MIT aqueous solution obtained above was added dropwise into the CMCS NPs solution in a mass ratio of 1:3 between MIT and CMCS NPs and stirred for 4 h to allow mitoxantrone to be adsorbed on the surface of CMCS NPs by physical adsorption to obtain drug-loaded nanoparticles, which were recorded as C / M NPs.

[0072] The biomimetic nanoparticle suspension formulation obtained in this example is referred to as OE / IR / CM@C / M NPs.

[0073] Example 7 The difference between this embodiment and embodiment 1 is only that: In this embodiment, the nanomedicine is a drug-loaded nanoparticle prepared based on inorganic nanoparticles, that is, the nanomedicine used in this embodiment is a drug-loaded nanoparticle, and the drug-loaded nanoparticle in this embodiment uses mesoporous silicon nanoparticles as the nanoparticle matrix and mitoxantrone MIT as the anti-tumor drug, and the anti-tumor drug mitoxantrone is loaded on the mesoporous silicon nanoparticles by the following steps: (1) Disperse 1.50 g of hexadecyltrimethylammonium bromide (CTAB) in 750 mL of ultrapure water to obtain a CTAB solution. (2) Add 5.25 mL of 2.0 M sodium hydroxide aqueous solution to the CTAB solution and stir vigorously at 80°C for 0.5 h. (3) Add 8.25 mL of ethyl orthosilicate dropwise to the above system after 0.5 h of reaction. After a white precipitate is produced, add 7.50 mL of ethyl acetate dropwise, stir for 2 h, and age at room temperature for 8 h. The reaction solution obtained after aging is centrifuged at 8000 rpm for 15 min to obtain unextracted mesoporous silicon nanoparticles, which are referred to as MSN NPs. (4) Wash the MSN NPs with ultrapure water and ethanol three times and vacuum dry to obtain dry MSN NPs. (5) According to the ratio of 1 mL of 37% concentrated hydrochloric acid and 100 mL of ethanol per gram of MSN NPs, add the corresponding volume of concentrated hydrochloric acid and ethanol to the above dried MSN NPs, mix well, react at 70 °C for 24 h, and then centrifuge again, wash with ethanol, and dry to obtain mesoporous silicon nanoparticles. (6) Disperse 10 mg of mesoporous silicon nanoparticle powder in 5 mL of 0.1 M phosphate buffer with pH = 7.4, and continue to add the corresponding volume of 1 mg / mL mitoxantrone solution according to the mass ratio of MIT to mesoporous silicon nanoparticles of 1:3. Stir in the dark for 4 h, centrifuge at 8000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain drug-loaded nanoparticles based on inorganic nanoparticles, which are recorded as M / M NPs.

[0074] The biomimetic nanoparticle suspension formulation obtained in this example is referred to as OE / IR / CM@M / M NPs.

[0075] Comparative Example 1 The difference between this comparative example and Example 1 is that in this comparative example, CM@NPs are not added, that is, the suspension formulation obtained in this comparative example is a formulation containing only a photosensitizer, referred to as OE / IR.

[0076] Comparative Example 2 The only difference between this comparative example and Example 1 is that in this comparative example, NPs are directly dispersed into OE, that is, the suspension dosage form of this comparative example is a dosage form containing only nanoparticles, referred to as OE / NPs.

[0077] Comparative Example 3 The difference between this comparative example and Example 1 is that in this comparative example, no photosensitizer IR820 is added, that is, the suspension formulation of this comparative example is a formulation containing only biomimetic nanoparticles, referred to as OE / CM@NPs.

[0078] Comparative Example 4 The only difference between this comparative example and comparative example 1 is that in this comparative example, OE is replaced by an equal volume of pure water, that is, the dosage form obtained in this comparative example is an aqueous solution of photosensitizer, referred to as water / IR.

[0079] Experimental Section 1. Particle size detection of biomimetic nanomedicine The present invention takes the nanoparticles NPs prepared by the present invention, the cancer cell membrane vesicles CM and the biomimetic nanoparticles CM@NPs prepared in Example 1 as examples, and uses a dynamic light scattering instrument to test their hydrated particle sizes, and the test results are as follows: Figure 3 shown, and Figure 3 In the figure, Figure A is the particle size distribution diagram of the nanoparticles NPs prepared by the present invention, Figure B is the particle size distribution diagram of the cancer cell membrane vesicles CM prepared by the present invention, and Figure C is the particle size distribution diagram of the bionic nanoparticles CM@NPs prepared in Example 1.

[0080] Depend on Figure 3 The test results show that the particle size of NPs is 161 nm, the PDI is 0.26, and the particle size of CM is 220 nm. However, after passing through the extruder, the cancer cell membrane prepared by the present invention is coated on the surface of NPs, so that the particle size of CM@NPs is 175 nm. This shows that after NPs and CM pass through the extruder, CM is successfully wrapped on the surface of NPs to obtain cancer cell membrane biomimetic nanoparticles CM@NPs, in which the membrane thickness is about 14 nm.

[0081] 2. Macroscopic morphology analysis of suspension dosage forms The present invention takes the OE prepared by the present invention, the OE / IR of comparative example 1, the OE / CM@NPs of comparative example 3 and the OE / IR / CM@NPs of embodiment 1 as examples, and records their macroscopic morphologies as follows: Figure 4 shown.

[0082] Figure 4 In the figure, Figure A is a physical picture of OE prepared by the present invention, Figure B is a physical picture of OE / IR of Comparative Example 1, Figure C is a physical picture of OE / CM@NPs of Comparative Example 3, and Figure D is a physical picture of OE / IR / CM@NPs of Example 1. Figure 4 It can be seen that the liquid excipient OE is a clear and transparent liquid, the green photosensitizer IR820 is successfully dispersed in OE to obtain OE / IR, the light yellow biomimetic nanoparticles CM@NPs are also smoothly dispersed in OE to obtain OE / CM@NPs, and the photosensitizer IR820 and biomimetic nanoparticles are simultaneously dispersed in OE to obtain the final dosage form OE / IR / CM@NPs. In addition, the prepared suspension has good stability, and no particle enlargement or aggregation and stratification occurs after standing for 48 hours.

[0083] (III) Photothermal conversion performance test The present invention uses pure water as a blank control, and takes the water / IR of Comparative Example 4 and the OE / IR of Comparative Example 1 as examples, and adds them into the cuvette respectively, at a power of 1 W / cm 2 The samples were irradiated with 808 nm laser for 10 min, and the thermal image of the tested samples was recorded every 60 s using the infrared thermal imager Testo 865. The test results are summarized as follows: Figure 5 shown, and Figure 5 In the figure, Figure A shows the light-to-heat conversion test results of pure water, Figure B shows the light-to-heat conversion test results of water / IR of Comparative Example 4, and Figure C shows the light-to-heat conversion test results of OE / IR of Comparative Example 1. Figure 5 It can be seen that pure water has almost no temperature change under laser irradiation, the temperature of water / IR solution rises to about 60°C within 10 minutes, and the temperature of OE / IR solution rises rapidly to 80°C, which preliminarily proves that OE can significantly improve the photothermal conversion efficiency and heating capacity of IR820.

[0084] The present invention also takes water / IR and OE / IR as examples, and respectively 2 , 0.75W / cm 2 , 1.0W / cm 2 , 1.25W / cm 2 , 1.75W / cm 2 , 2.0W / cm 2 , 2.5W / cm 2 and 3.0 W / cm 2 The samples were irradiated with 808 nm laser of different powers for 10 min, and the temperature changes of the samples were recorded by infrared thermal imager of model Testo 865 and plotted as follows: Figure 6 The heating curve shown in Figure 6 In the figure, Figure A is the temperature rise curve of water / IR of Comparative Example 4 under different power irradiation conditions, and Figure B is the temperature rise curve of OE / IR of Comparative Example 1 under different power irradiation conditions. Figure 6 The test results show that the power of the 808 nm laser is 3W / cm 2 When 1 mg / mL water / IR solution was used, the temperature could reach 79.7 ℃ after 10 min, while OE / IR could reach an astonishing 126 ℃. This indicates that under the same power and concentration, OE significantly improves the photothermal conversion efficiency of IR820.

[0085] The present invention also used water / IR and OE / IR of comparative example 1 and diluted them to different concentrations of 800 μg / mL, 600 μg / mL, 400 μg / mL, 400 μg / mL, 50 μg / mL and pure water, and respectively irradiated them with 1.0 W / cm 2The samples were irradiated with an 808 nm laser of 1000 nm power for 10 min, and the temperature changes of the samples were recorded using an infrared thermal imager of model Testo 865 and plotted as follows: Figure 7 The heating curve shown is Figure 7 Figure A shows the comparative example 4 and its dilution to different concentrations at 1.0 W / cm 2 The temperature rise curve under power irradiation conditions, Figure B shows the comparative example 1 and its dilution to different concentrations at 1.0W / cm 2 Temperature rise curve under power irradiation conditions.

[0086] Depend on Figure 7 It can be seen that the power of the laser is fixed at 1W / cm 2 To make the solution temperature reach 50°C, the IR820 concentration in water / IR is 800 μg / mL, while the IR820 concentration in OE / IR is only 200 μg / mL. This indicates that under the same effect requirements, OE can significantly reduce the amount of photosensitizer and achieve the purpose of reducing toxicity and increasing efficacy.

[0087] 4. In vitro drug release performance test The present invention takes the prepared NPs, OE@NPs and OE / CM@NPs as examples to explore the drug release experiment of each dosage form at different pH values ​​and detect the content of DAS. The specific process is as follows: (1) Preheat 0.05 M phosphate buffer solutions with pH values ​​of 7.4, 6.8 and 5.0 in a shaker at 37°C. The phosphate buffer solution will be referred to as PBS below for ease of description. (2) Take 1 mL of NPs in Example 1, OE / NPs in Comparative Example 2 and OE / CM@NPS in Comparative Example 3 and place them in dialysis bags with MWCO 3500 Da, tie the dialysis bags and place them in 50 mL EP tubes. (3) Add 5 mL of PBS of corresponding pH to the tubes and shake them at 37°C in the dark. (4) Replace the phosphate buffer solution with fresh one at each predetermined time point of shaking for 1h, 3h, 6h, 12h, 24h, 36h, 48h and 72h. After shaking for 72h, take out 1 mL of all the collected PBS, freeze-dry them, add 1 mL of dimethyl sulfoxide to dissolve them, and detect the cumulative release rate of DAS using a multifunctional microplate reader. The test results are shown in the figure. Figure 8 shown.

[0088] Figure 8 The in vitro drug release diagrams of NPs of Example 1, OE / NPs of Comparative Example 2, and OE / CM@NPS of Comparative Example 3 at different pH values ​​show that OE / NPs show a release behavior similar to that of NPs, while the release amount of OE / CM@NPs is significantly reduced, indicating that cell membrane encapsulation can effectively reduce the burst release behavior of drugs.

[0089] 5. Cytotoxicity assessment The present invention uses the MTT method to evaluate the in vitro cytotoxicity of biomimetic nanoparticles, and the specific method is as follows: (1) Preparation of OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) samples: OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) were prepared into test samples containing 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM of dasatinib and 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL and 20 μg / mL of IR820, respectively, for later use. OE / IR(+) means that OE / IR was exposed to 1 W / cm 2 Samples after 5 minutes of laser irradiation. Similarly, OE / IR / CM@NPs(+) means OE / IR / CM@NPs after 1 W / cm2 of NIR808 nm. 2 Samples after 5 minutes of laser irradiation.

[0090] (2) Add H22 cell suspension to a 96-well plate, with 5000 cells in each well. Add 20 μL of the prepared test sample to each well. After incubation for 48 hours, add 20 μL of 5 mg / mL thiazolyl blue aqueous solution to each well. After 4 hours, add 100 μL of trimethoprim solution to each well and shake to mix thoroughly. Finally, use a multifunctional microplate reader to measure the absorbance of each well at a wavelength of 490 nm. Calculate the corresponding cell survival rate based on the absorbance and organize it into a graph as shown below. Fig. 9 shown.

[0091] Fig. 9 The cell survival rates of OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) at different concentrations show that OE does not show obvious cytotoxicity, indicating that OE is safe and non-toxic as a liquid pharmaceutical excipient; OE / IR(+) and OE / CM@NPs both show concentration-dependent cytotoxicity, indicating that the OE-based dosage form can produce good photothermal therapy and chemotherapy effects, and OE / IR / CM@NPs(+) shows the strongest cytotoxicity, indicating that the dosage form obtained by integrating bionic nanoparticles and photosensitizers in the present invention can produce chemotherapy / photothermal / photodynamic synergistic anti-tumor effects.

[0092] 6. Cell live-death staining experiment The present invention uses a reactive oxygen species detection kit to detect the level of intracellular reactive oxygen species (ROS): (1) H22 cells were plated at 1×10 per well. 5 The cells were seeded in a 6-well plate at a density of 100 cells / cm2. OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) were added to each well and incubated for 4 h. OE / IR(+) means that OE / IR was exposed to 1 W / cm2 of NIR 808 nm. 2 Samples after 5 minutes of laser irradiation. Similarly, OE / IR / CM@NPs(+) means OE / IR / CM@NPs after 1 W / cm2 of NIR 808nm. 2 Samples after 5 minutes of laser irradiation. The experimental group with pure culture medium added was used as the control group.

[0093] (2) The DAS concentration in each sample was set to 20 μg / mL, and the IR820 concentration was set to 10 μg / mL. Then, according to the requirements of the kit, DCFH-DA was diluted with pure MEM at a ratio of 1:1000 to obtain a DCFH-DA solution. Subsequently, 1 mL of DCFH-DA solution was added to each well, incubated for 30 min, washed 3 times, fixed with paraformaldehyde for 10 min, and the fluorescence of each sample was observed and recorded by CLSM. Among them, DCFH-DA is 2,7-dichlorofluorescein diacetate, also known as the reactive oxygen species (ROS) fluorescent probe. ‌

[0094] Cell viability was further detected by live-dead fluorescence staining experiment. H22 cells were plated at 1×10 5 The cells were seeded in a 6-well plate at a density of 100 cells / mL. OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) were added to each well and incubated for 4 h. The DAS concentration in NPs was set to 20 μg / mL, and the IR820 concentration was set to 10 μg / mL. “+” indicates that the cells were exposed to NIR 808 nm at 1 W / cm 2 After laser irradiation for 5 min, the cells were washed with fresh PBS, 4 μM calcein and 4 μM propidium iodide were added to each well, and incubated at 37 °C in the dark for 30 min. The cell staining was then observed under an inverted fluorescence microscope.

[0095] The above test results are organized as follows Fig.10 and Fig.11 As shown, Fig.10 The figures show the changes in intracellular ROS content caused by OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) under no light and light conditions, respectively. It can be seen that under light, a large amount of ROS was produced in the cells treated with OR / IR and OE / IR / CM@NPs, indicating that this dosage form can produce a PDT effect.

[0096] Fig.11 The live and dead staining results of cells caused by OE, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) under no light and light conditions, respectively. It can be seen that OR / IR / CM@NPs caused the most cell death, which is consistent with the above-mentioned MTT experimental results.

[0097] 7. In vivo photothermal conversion experiments The present invention uses mouse liver cancer cell H22 to establish a mouse subcutaneous tumor model and conduct subsequent anti-tumor research. The specific experimental method is as follows: After the H22 cells are in the logarithmic growth phase, the cells are collected and centrifuged, and diluted with physiological saline to a suitable concentration. Finally, 100 μL of the diluted H22 cell suspension is injected into the left armpit of the mouse, with 1×10 6 When the tumor volume of the mouse reaches 150 mm 3 Subsequent experiments were conducted. 100 μL of OE / IR / CM@NPs with different IR820 dosages of 0.8 mg / kg, 1.6 mg / kg, and 2.4 mg / kg were injected into the tumor area via peritumoral injection. 2 The temperature rise of the tumor site during the whole irradiation process was recorded with an infrared thermal imager and the temperature rise curve was drawn. In order to explore the retention and photothermal stability of OE / IR / CM@NPs in the tumor area, the dose of IR820 was selected as 2.4 mg / kg, and the irradiation was performed and photographed 24h, 72h and 120h after injection. The results are shown in Fig.12 shown.

[0098] Fig.12 Figure A shows the thermal imaging of OE / IR / CM@NPs with different IR820 dosage contents of 0.8 mg / kg, 1.6 mg / kg and 2.4 mg / kg after being injected into the tumor area under 808 nm near-infrared laser irradiation. It can be seen that the temperature of the tumor area located in the left upper limb of the mouse increases with time, and the temperature increase trend is dose-dependent. The higher the IR820 concentration, the faster the temperature rises and the higher the temperature.

[0099] Fig.12Figure B is the temperature counting result of the tumor area of ​​each mouse in Figure A. It can be seen that when the IR820 dose is 0.8 mg / kg, the temperature of the tumor area reaches 50°C after 5 minutes, while the temperature of the tumor area of ​​mice with IR820 doses of 1.6 mg / kg and 2.4 mg / kg reaches 55°C and 60°C, respectively, further proving the results of Figure A, that is, the in vivo photothermal effect of OR / IR / CM@NPs is dose-dependent.

[0100] Fig.12 Figure C shows the temperature rise of the mouse tumor area under the same 808 nm near-infrared light irradiation condition 24h, 72h and 120h after the injection of OR / IR / CM@NPs with a dose of IR820 of 2.4 mg / kg. It should be noted that the ordinate in Figure C indicates the time when the sample was injected into the mouse body; the abscissa indicates the irradiation time. It can be seen that the temperature of the tumor area can still rise to above 50°C 1 day, 3 days and 5 days after the injection.

[0101] Fig.12 Figure D in Figure C shows the temperature counting results of the tumor area of ​​each mouse. It can be seen that even 5 days after injection, the temperature of the tumor area can still reach 50°C, which meets the requirements of PTT treatment. The above results show that the OE dosage form can remain in the tumor area for a long time, thereby prolonging the PTT treatment effect and reducing the frequency of administration. The OE dosage form shows a dose-dependent temperature increase ability, and the tumor area of ​​the mouse can reach 50°C, which meets the requirements of PTT treatment. In addition, 5 days after peritumoral injection, the temperature of the tumor area can still reach 50°C under laser irradiation, indicating that the OE dosage form can remain in the tumor area for a long time, which is very beneficial to extend the treatment cycle of a single dose, reduce the frequency of administration, and relieve patient discomfort.

[0102] 8. In vivo anti-tumor ability assessment The present invention uses H22 tumor-bearing mice to carry out direct in vivo anti-tumor research, and the specific test method is as follows: H22 tumor-bearing mice were randomly divided into 4 groups, with 6 mice in each group. The initial tumor volume was about 200 mm 3 . The tumor was injected in situ with 100 μL saline as the control group, 100 μLOE / IR(+), 100 μLOE / CM@NPs and 100 μLOE / IR / CM@NPs(+). The doses of DAS and IR820 were 20 mg / kg and 1.7 mg / kg, respectively. "(+)" means that the groups containing IR820 were treated with light on the 1st, 3rd and 5th days, and the light treatment parameters were 1 W / cm 2 , 5 min. The weight of the mice and the short and long diameters of the tumors were recorded every two days, and the volume of the tumor was calculated using the formula: ; In the formula, V represents the volume of the tumor, R represents the long diameter of the tumor, r Represents the short diameter of the tumor.

[0103] Direct antitumor results in vivo Figure 13~Figure 15 As shown, Fig.13 The changes of tumor volume over time in H22 tumor-bearing mice of the Control group, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) Fig.14 The body weight changes over time of H22 tumor-bearing mice in the Control group, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+) Fig.15 The changes of tumor mass over time in H22 tumor-bearing mice of the Control group, OE / IR(+), OE / CM@NPs and OE / IR / CM@NPs(+).

[0104] Depend on Figure 13~Figure 16 The test results show that OE / IR / CM@NPs showed the strongest anti-tumor effect, with the smallest average tumor volume and the lowest tumor mass, indicating that the suspension dosage form achieved a strong anti-tumor effect by producing a synergistic effect of chemotherapy / photothermal / photodynamic therapy.

[0105] 9. Evaluation of the ability to inhibit tumor recurrence The ability of the suspension formulation to inhibit tumor recurrence was evaluated using the H22 tumor surgical resection model. The specific experimental method was as follows: H22 tumor-bearing mice were randomly divided into 4 groups, namely, normal saline group, OE / IR+ group, OE / CM@NPs group and OE / IR / CM@NPs+ group, with 6 mice in each group. Under anesthesia, the mouse tumor was removed, leaving about 5% of residual tumor tissue to simulate the micro-tumor remaining in the operating bed. Then 100 μL of the drug was applied to the resection site and the wound was sutured. The doses of DAS and IR820 were 20 mg / kg and 1.7 mg / kg, respectively. The groups containing IR820 were all treated with light on the 1st, 3rd and 5th days, and the light treatment parameters were 1W / cm 2 During the experiment, the tumor size, weight change and survival status of these mice were regularly tested and sorted as follows: Fig.16 and Fig.17 shown.

[0106] Fig.16Figure A shows the changing trend of tumor volume of 6 mice in the normal saline group during the 50-day observation period, Figure B shows the changing trend of tumor volume of 6 mice in the OE / IR+ group during the 50-day observation period, Figure C shows the changing trend of tumor volume of 6 mice in the OE / CM@NPs group during the 50-day observation period, and Figure D shows the changing trend of tumor volume of 6 mice in the OE / IR / CM@NPs+ group during the 50-day observation period.

[0107] Depend on Fig.16 It can be seen that normal saline is completely unable to inhibit tumor growth. Within 15 days, tumors in all 6 mice recurred and all mice died; OE / IR+ and OE / CM@NPs can only inhibit tumor growth in the early stage of the experiment. After 20 days, the tumor recurrence rates reached 5 / 6 and 6 / 6, respectively; mice treated with OE / IR / CM@NPs(+) did not show any tumor recurrence after 20 days, and 67% of the mice still had no tumor recurrence after 50 days.

[0108] Fig.17 The figure is a graph showing the survival rate of mice in the saline group, OE / IR+ group, OE / CM@NPs group and OE / IR / CM@NPs+ group during the 50-day observation period. It can be seen that the tumors of mice treated with saline, OE / IR+ and OE / CM@NPs quickly recurred and eventually died, while 4 mice treated with OE / IR / CM@NPs(+) eventually survived for more than 120 days. This shows that the OE suspension provided by the present invention can effectively prevent tumor recurrence after surgery and completely relieve tumor treatment.

[0109] (X) In vitro cell experiment of OE / IR / CM@C / M NPs of Example 6 The in vitro antitumor activity of OE / IR / CM@C / M NPs in Example 6 was verified by cell apoptosis experiment: H22 cells were plated at 1×10 5 The cells were seeded at a density of 100 cells / mL in a 6-well plate, and MIT and OE / IR / CM@C / M NPs were added to each well and incubated for 4 h. The concentration of MIT was set to 20 μg / mL, and the concentration of IR820 was set to 10 μg / mL. After the incubation, the cells were washed with fresh PBS, and 2 μL Annexin V-FITC and 2 μL propidium iodide PI were added to each well and incubated at 4 °C in the dark for 10 min. Then, the cell viability was detected by flow cytometry. Physiological saline was used as the control group.

[0110] Cell viability was further detected by live-dead fluorescence staining experiment. H22 cells were plated at 1×10 5The cells were seeded at a density of 100 cells / mL in a 6-well plate, and MIT and OE / IR / CM@C / M NPs were added to each well and incubated for 4 h. The concentration of MIT was set to 20 μg / mL, and the concentration of IR820 was set to 10 μg / mL. After the incubation, the cells were washed with fresh PBS, and 4 μM calcein and 4 μM propidium iodide were added to each well, and incubated at 37 °C in the dark for 30 min. The cell staining was then observed under an inverted fluorescence microscope, and the above test results were organized as follows: Fig.18 and Fig.19 shown.

[0111] Fig.18 The apoptosis experimental results of the Control group, MIT group and OE / IR / CM@C / M NPs group on mouse liver cancer cell H22 are shown in Figure 2. Fig.18 It can be seen that the apoptosis rate of cells after treatment in the Control group was 5.49%, the apoptosis rate of cells after treatment in the MIT group was 45.44%, and the apoptosis rate of cells treated with OE / IR / CM@C / M NPs was the highest, reaching 71.7%.

[0112] Fig.19 The live / dead double staining results of the control group, MIT group and OE / IR / CM@C / M NPs group on mouse liver cancer cell H22, where the green signal is live cells and the red signal is dead cells. Fig.19 It can be seen that the cells treated with OE / IR / CM@C / MNPs all showed red signals of death, indicating that OE / IR / CM@C / M NPs caused the most cell death, which is consistent with the above-mentioned apoptosis experimental results.

[0113] (XI) In vitro cell experiment of OE / IR / CM@M / M NPs of Example 7 The in vitro antitumor activity of OE / IR / CM@C / M NPs in Example 7 was verified by cell apoptosis experiment: H22 cells were plated at 1×10 5 The cells were seeded at a density of 100 cells / mL in a 6-well plate, and MIT and OE / IR / CM@M / M NPs were added to each well and incubated for 4 h. The concentration of MIT was set to 20 μg / mL, and the concentration of IR820 was set to 10 μg / mL. After the incubation, the cells were washed with fresh PBS, and 2 μL Annexin V-FITC and 2 μL propidium iodide PI were added to each well and incubated at 4 °C in the dark for 10 min. Then, the cell viability was detected by flow cytometry. Physiological saline was used as the control group. Among them, the Chinese name of Annexin V-FITC is extramembrane phosphatidylserine binding protein V.

[0114] Cell viability was further detected by live-death fluorescence staining experiment. H22 cells were plated at 1×10 5 The cells were seeded at a density of 100 cells / mL in a 6-well plate, and MIT and OE / IR / CM@M / M NPs were added to each well and incubated for 4 h. The concentration of MIT was set to 20 μg / mL, and the concentration of IR820 was set to 10 μg / mL. After the incubation, the cells were washed with fresh PBS, and 4 μM calcein and 4 μM propidium iodide were added to each well, and incubated at 37 °C in the dark for 30 min. The cell staining was then observed under an inverted fluorescence microscope. The above test results are organized as follows: Fig. 20 and Fig.21 shown.

[0115] Fig. 20 The results of apoptosis experiments on mouse liver cancer cell H22 in the Control group, MIT group and OE / IR / CM@M / M NPs group show that the apoptosis rate of cells after treatment in the Control group is 4.61%, the apoptosis rate of cells after treatment in the MIT group is 40.79%, and the apoptosis rate of cells treated with OE / IR / CM@M / M NPs is the highest, reaching 86.63%.

[0116] Fig.21 The live / dead double staining results of the control group, MIT group, and OE / IR / CM@M / M NPs group on mouse liver cancer cell H22, where the green signal is live cells and the red signal is dead cells. Fig. 20 It can be seen that the cells treated with OE / IR / CM@M / MNPs all showed red signals of death, indicating that OE / IR / CM@M / M NPs caused the most cell death, which is consistent with the above-mentioned apoptosis experimental results.

[0117] Obviously, the above embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A biomimetic nanoparticle suspension formulation based on an orthoester compound, characterized in that: It comprises a liquid auxiliary material, and a photosensitizer and biomimetic nanoparticles dispersed in the liquid auxiliary material; Wherein, the liquid auxiliary material is an orthoester compound having the formula I: Formula I; The bionic nanoparticles use nanomedicine as a matrix and are obtained by wrapping cell membrane vesicles on the surface of the nanoparticles.

2. The biomimetic nanoparticle suspension formulation based on an orthoester compound according to claim 1, characterized in that: The photosensitizer accounts for 0.1wt% to 20wt% of the total mass of the biomimetic nanoparticle suspension formulation; The bionic nanoparticles account for 0.1wt% to 40wt% of the total mass of the bionic nanoparticle suspension formulation.

3. The biomimetic nanoparticle suspension formulation based on orthoester compounds according to claim 1, characterized in that: The nanomedicine is pure drug nanoparticles or drug-loaded nanoparticles; Wherein, the pure drug nanoparticles are formed by self-assembly of small molecule drugs; The drug-loaded nanoparticles are obtained by loading small molecule drugs using nanoparticle matrix as a carrier.

4. The biomimetic nanoparticle suspension formulation based on orthoester compounds according to claim 3, characterized in that: The loading amount of the small molecule drug on the nanoparticle matrix is ​​1 wt% to 20 wt%.

5. The biomimetic nanoparticle suspension formulation based on orthoester compounds according to claim 3, characterized in that: The small molecule drugs include anti-tumor drugs.

6. The biomimetic nanoparticle suspension formulation based on orthoester compounds according to claim 3, characterized in that: The nanoparticle matrix is ​​natural macromolecular nanoparticles or inorganic nanoparticles.

7. The biomimetic nanoparticle suspension formulation based on orthoester compounds according to claim 3, characterized in that: The particle size of the nano drug is 10nm~300nm.

8. The biomimetic nanoparticle suspension formulation based on orthoester compounds according to claim 1, characterized in that: The cell membrane vesicles are spherical, and the diameter of the cell membrane vesicles is 20nm~1000nm.

9. The biomimetic nanoparticle suspension formulation based on orthoester compounds according to claim 1, characterized in that: The photosensitizer is selected from small molecule drugs for photodynamic therapy or photothermal therapy.

10. A method for preparing a biomimetic nanoparticle suspension formulation based on an orthoester compound according to any one of claims 1 to 9, characterized in that: The following steps are involved: The orthoester compound shown in Formula I is used as a liquid auxiliary material, and a photosensitizer and biomimetic nanoparticles are dispersed in the liquid auxiliary material to obtain the biomimetic nanoparticle suspension dosage form.

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