Bionic nanoparticle suspension dosage form based on orthoester compounds and preparation method thereof

Abstract

The present invention belongs to the technical field of biomedicine, and discloses a bionic nanoparticle suspension dosage form based on orthoester compounds and a preparation method thereof. The bionic nanoparticle suspension dosage form includes liquid excipients, as well as a photosensitizer and bionic nanoparticles dispersed in the liquid excipients. In the present invention, an orthoester compound represented by Formula I is used as the liquid excipient, and the bionic nanoparticles and the photosensitizer are simultaneously introduced into OE to obtain a bionic nanoparticle suspension dosage form with multiple anti-tumor mechanisms. Among them, the bionic nanoparticles are nano-drugs encapsulated by cancer cell membrane vesicles, and through the cancer cell membrane bionic technology, the homologous targeting function provided by the cancer cell membrane is utilized to significantly improve the active targeting and cellular uptake of the nano-drugs. Formula I:

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a bionic nanoparticle suspension dosage form based on orthoester compounds and a preparation method thereof. Background Art

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

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

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

[0005] The bionic nanoparticle suspension dosage form based on orthoester compounds and the preparation method thereof of the present invention are realized through the following technical solutions:

[0006] The first object of the present invention is to provide a bionic nanoparticle suspension dosage form based on orthoester compounds, including liquid excipients, and a photosensitizer and bionic nanoparticles dispersed in the liquid excipients.

[0007] It should be noted that the liquid adjuvant used in the present invention has OE. For the convenience of description, the original acid ester compound shown in Formula I will be hereinafter simply referred to as OE. OE is a pH-sensitive modified original acid ester 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. Without adding any additional suspending agents, a stable suspension can be obtained, which is beneficial to realizing synergistic anti-tumor effects such as chemotherapy-photothermal. Moreover, OE can directly deliver the photosensitizer to the tumor area or the surgical wound surface. The lower specific heat capacity of OE makes the suspension heat up faster and can achieve the same photothermal therapy effect at a lower concentration. In addition, compared with aqueous solutions, OE can significantly improve the photothermal conversion ability of the photosensitizer, promote the photothermal therapy effect of the suspension, reduce the dosage of the photosensitizer at the same time, and reduce potential side effects. When the suspension is used for the wound surface after tumor resection, OE has appropriate fluidity and can cover target areas of any shape, thereby directly inhibiting tumor recurrence and expanding the application scenarios of this suspension formulation.

[0008]

[0009] Formula I.

[0010] It should also be noted that the biomimetic nanoparticles used in the present invention are based on nano-drugs and are obtained by wrapping cell membrane vesicles on their surfaces. The biomimetic nanoparticles are wrapped by cell membranes, endowing the nano-drugs with biological properties, including prolonging the circulation time, improving stability and cancer cell homologous targeting. As a result, the biomimetic nanoparticles used in the present invention can significantly improve the intracellular delivery efficiency of drugs through the active targeting function, further enhancing the anti-tumor effect, making the biomimetic nanoparticle suspension formulation of the present invention have active targeting performance and synergistic effects.

[0011] In some preferred embodiments of the present invention, the OE used in the present invention is prepared by the following steps:

[0012] S1, stirring reaction:

[0013] Using diglycerol and triethyl orthoacetate as raw materials, and p-toluenesulfonic acid as a catalyst, under a nitrogen atmosphere, disperse diglycerol and triethyl orthoacetate in an organic solvent at a molar ratio of 1:2 to 4, and then add the corresponding mass of p-toluenesulfonic acid according to the molar ratio of p-toluenesulfonic acid to diglycerol of 1:50 to 100. Subsequently, stir and react at room temperature for 8 h to 16 h, and its reaction equation is as shown in Reaction Formula 1:

[0014]

[0015] Reaction Formula 1.

[0016] S2, Post-treatment:

[0017] 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. Subsequently, it was extracted twice with saturated Na 2 CO 3 solution to obtain an extract. After the extract was dried by rotary evaporation, it was vacuum filtered to obtain the orthoester compound shown in Formula I, namely OE.

[0018] Through the above synthesis method, the present invention can obtain OE in one step under normal temperature and pressure, and there is only a carbon-oxygen bond in the structure of the prepared OE, which will not damage the drug structure. More preferably, when synthesizing OE, the organic solvent used is anhydrous dichloromethane, and the volume ratio of the organic solvent to diglycerol is 2 - 10:1.

[0019] More preferably, the addition amounts of both the photosensitizer and the biomimetic nanoparticles are therapeutically effective amounts, and they can be added according to the specific types of photosensitizer and biomimetic nanoparticles actually selected.

[0020] It should also be noted that in order to adjust the overall fluidity, degradation, and drug release rate of the biomimetic nanoparticle suspension dosage form, the present invention adjusts the content of the photosensitizer and the biomimetic nanoparticles in the biomimetic nanoparticle suspension dosage form by adjusting the addition amount of OE, so as to adjust the overall fluidity, degradation, and drug release rate of the biomimetic nanoparticle suspension dosage form. And preferably in the present invention, by adjusting the addition amount of OE, the photosensitizer accounts for 0.1 wt% - 20 wt% of the total mass of the biomimetic nanoparticle suspension dosage form; the biomimetic nanoparticles account for 0.1 wt% - 40 wt% of the total mass of the biomimetic nanoparticle suspension dosage form.

[0021] Preferably in the present invention, among the biomimetic nanoparticles, the cell membrane vesicles are selected from one cell membrane of immune cells, cancer cells, red blood cells, platelets, and exosomes or a cell membrane hybridized by multiple of them. 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.

[0022] Preferably in the present invention, the nano-drug is a pure drug nanoparticle or a drug-loaded nanoparticle. Among them, the pure drug nanoparticle is formed by self-assembly of small molecule drugs. The drug-loaded nanoparticle uses a nanoparticle matrix as a carrier and is obtained by loading small molecule drugs; the drug loading amount of the small molecule drug on the nanoparticle matrix is 1 wt% - 20 wt%. The nanoparticle matrix is a natural macromolecule nanoparticle or an inorganic nanoparticle.

[0023] Preferably, the small molecule drug of the present invention includes anti-tumor drugs. More preferably, the anti-tumor drug is selected from one or more of dasatinib, sunitinib, sorafenib, mitoxantrone, and doxorubicin. Further preferably, the anti-tumor drugs are dasatinib and sunitinib.

[0024] Preferably, the natural macromolecular nanoparticles of the present invention are selected from one or more of polysaccharide nanoparticles and protein nanoparticles. For example, they can be selected from any one or more of chitosan, carboxymethyl chitosan, hyaluronic acid, chondroitin sulfate, sodium alginate, collagen, gelatin, bromelain, hyaluronidase, catalase, cellulose, and hydroxypropyl methylcellulose nanoparticles. Further preferably, the natural macromolecular nanoparticles are carboxymethyl chitosan nanoparticles.

[0025] Preferably, the inorganic nanoparticles of the present invention are selected from any one or more of mesoporous silica nanoparticles, iron oxide nanoparticles, zinc sulfide nanoparticles, cuprous selenide nanoparticles, and cerium nanoparticles.

[0026] Preferably, the particle size of the nano-drug is 10 nm to 300 nm; the cell membrane vesicles are spherical and the diameter of the cell membrane vesicles is 20 nm to 1000 nm to adapt to the particle size of the nano-drug and achieve complete encapsulation of the nanoparticles.

[0027] Preferably, when the pure drug nanoparticles are used as the nano-drug, the drug loading of the nano-drug is 100%. To facilitate the understanding of the situation when the pure drug nanoparticles are used as the nano-drug, the present invention takes dasatinib and sunitinib as the small molecule drugs together as an example to illustrate the process of preparing pure drug nanoparticles by self-assembly of small molecule drugs:

[0028] Weigh the corresponding masses of dasatinib and sunitinib according to the mass ratio of 2.5 to 3.5:1 and set aside. Dissolve the weighed dasatinib and sunitinib in dimethyl sulfoxide respectively to obtain a dasatinib solution with a concentration of 350 μM to 450 μM and a sunitinib solution with a concentration of 350 μM to 450 μM. Mix the dasatinib solution and the sunitinib solution to obtain a mixed solution. Then, at room temperature, gradually add the mixed solution to deionized water and stir for 3 min to 10 min to enable dasatinib and sunitinib to self-assemble into a composite drug material. Then, centrifuge the solution at 7000 rpm to 9000 rpm for 5 min to 15 min, collect the precipitate and wash it at least 3 times to remove dimethyl sulfoxide. After drying the precipitate, pure drug nanoparticles are obtained.

[0029] Preferably, when the drug-loaded nanoparticles are used as nano-drugs, the drug-loaded nanoparticles are specifically 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, dropping the small molecule active drug aqueous solution into the nanoparticle matrix solution, and continuing to stir for 3 h to 5 h after the dropping 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 to 9000 rpm for 5 min to 15 min, the precipitate is collected, washed at least 3 times, and then freeze-dried to obtain the drug-loaded nanoparticles.

[0030] To facilitate the understanding of the situation when natural macromolecular nanoparticles are used as the nanoparticle matrix and the drug-loaded nanoparticles obtained by loading small molecule drugs are used as nano-drugs, the present invention takes carboxymethyl chitosan and mitoxantrone as examples to illustrate the process of preparing drug-loaded nanoparticles based on natural macromolecules:

[0031] (1) According to the dosage ratio of 5 g to 7 g of carboxymethyl chitosan powder to 6 L of water, 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 to 2.2:1, under stirring conditions, the corresponding volume of acetone is gradually dropped into the carboxymethyl chitosan aqueous solution, and a glutaraldehyde solution with a mass concentration of 25% and a volume of 9 vol% to 11 vol% of the water dosage is added. After mixing, the cross-linking reaction is carried out for 3 h to 5 h. Then, the reaction solution is centrifuged at 9000 rpm to 11000 rpm for 8 min to 15 min, and the obtained precipitate is carboxymethyl chitosan nanoparticles. The carboxymethyl chitosan nanoparticles are dispersed in water to obtain a carboxymethyl chitosan aqueous solution.

[0032] (2) According to the dosage ratio of 0.8 g to 1.2 g of mitoxantrone to 1 L of water, mitoxantrone is fully dispersed in water to obtain a mitoxantrone aqueous solution.

[0033] (3) According to the mass ratio of mitoxantrone in the mitoxantrone aqueous solution to carboxymethyl chitosan powder in the carboxymethyl chitosan aqueous solution of 1:2.5 to 3.5, the mitoxantrone aqueous solution is dropped into the carboxymethyl chitosan aqueous solution, and stirred for 3 h to 5 h, so that mitoxantrone is adsorbed on the surface of CMCS NPs through physical adsorption. The reaction solution is centrifuged at 7000 rpm to 9000 rpm for 5 min to 15 min, the precipitate is collected, washed at least 3 times, and then freeze-dried to obtain the drug-loaded nanoparticles.

[0034] Preferably, for the convenience of understanding the situation when inorganic nanoparticles are used as the nanoparticle matrix and the drug-loaded nanoparticles obtained by loading small molecule drugs are used as nanodrugs, the present invention uses mesoporous silica nanoparticles as the 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:

[0035] (1) Cetyltrimethylammonium bromide (CTAB) was dispersed in pure water with a volume ratio of 1.50 g: 700 - 800 mL to obtain a CTAB solution. (2) According to the volume ratio of 2.0 M aqueous sodium hydroxide solution to the above pure water of 5 - 5.5: 700 - 800, the corresponding volume of 2.0 M aqueous sodium hydroxide solution was added and mixed evenly, and the mixture was vigorously stirred at 75 °C - 85 °C. (3) After reacting for 0.4 h - 0.8 h, ethyl acetate with a volume of 9 vol% - 11 vol% of the pure water dosage was added, and the reaction was stirred for 2 h. After aging at room temperature for 6 h - 12 h, centrifugation was carried out at 7000 rpm - 9000 rpm for 10 min - 20 min to obtain unextracted mesoporous silica nanoparticles, denoted as MSN NPs. (4) It was washed three times with ultrapure water and ethanol and dried under vacuum. (5) According to the ratio of adding 1 mL of concentrated hydrochloric acid with a concentration of 37% and 100 mL of ethanol per gram of MSN NPs, the corresponding volume of concentrated hydrochloric acid and ethanol was added to the above-obtained MSN NPs, and after mixing evenly, the reaction was carried out at 70 °C for 24 h. Then, centrifugation, ethanol washing, and drying were carried out again to obtain mesoporous silica nanoparticles. (6) According to the dosage ratio of 8 mg - 12 mg: 5 mL, the mesoporous silica nanoparticle powder was dispersed in the corresponding volume of 0.1 M phosphate buffer solution with pH = 7.4. (7) According to the mass ratio of MIT to mesoporous silica nanoparticles of 1: 2.5 - 3.5, the corresponding volume of mitoxantrone solution was added, and the mixture was stirred in the dark for 3 h - 5 h. Then, centrifugation was carried out at 7000 rpm - 9000 rpm for 10 min, the supernatant was discarded, the precipitate was collected, and freeze-dried to obtain nanodrugs based on inorganic nanoparticles.

[0036] Preferably, the biomimetic nanoparticles are prepared by the following steps: using cell membrane vesicles as a coating agent and adopting the thin film extrusion method to wrap the cell membrane vesicles on the surface of the nanodrugs, that is, the biomimetic nanoparticles are obtained.

[0037] Preferably, the present invention wraps the cell membrane vesicles on the surface of the nanodrugs through the following steps to obtain biomimetic nanoparticles: after mixing the obtained cancer cell membrane vesicles with the nanoparticles, they are extruded back and forth through polycarbonate membranes with pore sizes of 400 nm and 200 nm, that is, the biomimetic nanoparticles are obtained. Among them, the mass ratio of cancer cell membrane vesicles to nanoparticles is 1 - 5: 1. The technology of the present invention using the thin film extrusion method to wrap cell membrane vesicles on the surface of nanomaterials belongs to the prior art in this field, so the present invention will not elaborate here.

[0038] Preferably, the photosensitizer of the present invention is selected from small molecule drugs for photodynamic therapy and photothermal therapy. For example, the photosensitizer is selected from one or more of chlorin e6, indocyanine green, and neoindocyanine green.

[0039] The second object of the present invention is to provide a method for preparing the above-mentioned biomimetic nanoparticle suspension formulation based on orthoester compounds, comprising the following steps: using the orthoester compound shown in Formula I as a liquid excipient, dispersing the photosensitizer and the biomimetic nanoparticles in the liquid excipient to obtain the biomimetic nanoparticle suspension formulation.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The biomimetic nanoparticle suspension formulation of the present invention comprises a liquid excipient, and a photosensitizer and biomimetic nanoparticles dispersed in the liquid excipient. The present invention uses OE as a liquid excipient, and simultaneously introduces biomimetic nanoparticles and a photosensitizer into OE to obtain a novel composite suspending agent with multiple anti-tumor mechanisms. Among them, the biomimetic nanoparticles change the drug uptake mechanism through cancer cell membrane biomimetic technology, utilize the homologous targeting function provided by the cancer cell membrane, change passive diffusion to active uptake, and significantly improve the active targeting and cellular uptake of nano-drugs. Compared with an aqueous solution, the present invention uses OE as a liquid excipient, which can significantly improve the photothermal conversion ability of the photosensitizer, promote the photothermal therapy effect of the suspending agent, reduce the drug dosage at the same time, and reduce potential side effects. And OE is a pH-sensitive liquid compound with a simple structure, a clear in vivo degradation mechanism, good biocompatibility, good fluidity and injectability, and can directly dissolve or disperse a variety of photosensitizers and nano-drugs to obtain a suspension formulation to facilitate the realization of synergistic anti-tumor effects such as chemotherapy-photothermal. Moreover, using OE as a liquid excipient can ensure the uniform stability of the components of the prepared biomimetic nanoparticle suspension formulation in the case of additional addition of a suspending agent. In addition, OE can also improve the photothermal conversion performance of the photosensitizer and significantly improve the photothermal therapy effect of tumors.

[0042] The biomimetic nanoparticle suspension formulation of the present invention can not only be used for in-situ injection of tumors, but also for the wound surface after tumor resection. OE has appropriate fluidity and can cover target areas of any shape, thereby directly inhibiting tumor recurrence and expanding the application scenarios of the suspension formulation. And the biomimetic nanoparticle suspension formulation of the present invention is an orthoester formulation loaded with biomimetic nanoparticles and a photosensitizer, and can achieve chemotherapy / photothermal combined therapy. Therefore, the orthoester suspension formulation loaded with biomimetic nano-drugs and a photosensitizer simultaneously has important significance in the field of realizing local drug delivery of tumors and inhibiting postoperative tumor recurrence. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 11H-NMR spectrum of the orthoester compound OE of the present invention 1 1H-NMR spectrum

[0044] Figure 2 13C-NMR spectrum of the orthoester compound OE of the present invention 13 13C-NMR spectrum

[0045] Figure 3 Particle size distribution diagrams of the nanoparticles NPs, cancer cell membrane vesicles CM, and the biomimetic nanoparticles CM@NPs prepared in Example 1 of the present invention Figure 3 Among them, Figure A is the particle size distribution diagram of the nanoparticles NPs prepared in the present invention, Figure B is the particle size distribution diagram of the cancer cell membrane vesicles CM prepared in the present invention, and Figure C is the particle size distribution diagram of the biomimetic nanoparticles CM@NPs prepared in Example 1

[0046] Figure 4 Physical pictures 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 Among them, Figure A is the physical picture of OE prepared in the present invention, Figure B is the physical picture of OE / IR of Comparative Example 1, Figure C is the physical picture of OE / CM@NPs of Comparative Example 3, and Figure D is the physical picture of OE / IR / CM@NPs of Example 1

[0047] Figure 5 Photothermal conversion test results of pure water, water / IR of Comparative Example 4, and OE / IR of Comparative Example 1 Figure 5 Among them, 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

[0048] Figure 6 Heating curves of water / IR of Comparative Example 4 and OE / IR of Comparative Example 1 under different power irradiation conditions Figure 6 Among them, Figure A is the heating curve of water / IR of Comparative Example 4 under different power irradiation conditions, and Figure B is the heating curve of OE / IR of Comparative Example 1 under different power irradiation conditions

[0049] Figure 7 Heating curves of water / IR of Comparative Example 4 and OE / IR of Comparative Example 1 and their dilutions to different concentrations under the irradiation condition of 1.0 W / cm 2 Power Figure 7 Among them, Figure A is the heating curve of Comparative Example 4 and its dilutions to different concentrations under the irradiation condition of 1.0 W / cm 2 Power 2 Power irradiation condition

[0050] Figure 8 In vitro drug release profiles of NPs, OE@NPs, and OE / CM@NPs at different pH values Figure 8 Among them, (a) shows the in vitro drug release profile of NPs at different pH values, (b) shows the in vitro drug release profile of OE@NPs at different pH values, and (c) shows the in vitro drug release profile of OE / CM@NPs at different pH values.

[0051] Figure 9 Cell viability of OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) at different concentrations

[0052] Figure 10 Changes in intracellular ROS levels induced by OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) under dark and light conditions

[0053] Figure 11 Live / dead cell staining results induced by OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) under dark and light conditions

[0054] Figure 12 In vivo photothermal conversion experimental results of the present invention Figure 12 Among them, Figure A shows the thermal imaging images after injecting OE / IR / CM@NPs with different IR820 dose contents of 0.8 mg / kg, 1.6 mg / kg, and 2.4 mg / kg of IR820 into the tumor region; Figure B shows the temperature counting results of the tumor region of each mouse in Figure A; Figure C shows the heating images of the tumor region of mice at 24 h, 72 h, and 120 h after injecting OR / IR / CM@NPs with an IR820 dose of 2.4 mg / kg into the tumor region; Figure D shows the temperature counting results of the tumor region of each mouse in Figure C.

[0055] Figure 13 Changes in in vivo tumor volume over time in H22 tumor-bearing mice in the Control group, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) groups

[0056] Figure 14 Changes in body weight over time in H22 tumor-bearing mice in the Control group, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) groups

[0057] Figure 15Changes in the in - vivo tumor mass over time of H22 - tumor - bearing mice in the Control group, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+).

[0058] Figure 16 Shows the change trends of the tumor volumes 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. Figure 16 Among them, Figure A shows the change trend of the tumor volumes of 6 mice in the normal saline group during the 50 - day observation period, Figure B shows the change trend of the tumor volumes of 6 mice in the OE / IR + group during the 50 - day observation period, Figure C shows the change trend of the tumor volumes of 6 mice in the OE / CM@NPs group during the 50 - day observation period, and Figure D shows the change trend of the tumor volumes of 6 mice in the OE / IR / CM@NPs + group during the 50 - day observation period.

[0059] Figure 17 Is a graph showing the changes in the survival rates 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.

[0060] Figure 18 Shows the apoptosis experimental results of the Control group, MIT group, and OE / IR / CM@C / M NPs group against mouse hepatoma cells H22. Figure 18 Among them, (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.

[0061] Figure 19 Shows the live / dead double - staining experimental results of the Control group, MIT group, and OE / IR / CM@C / M NPs group against mouse hepatoma cells H22. Figure 19 Among them, (a) shows the live / dead double - staining experimental results caused by the Control group, (b) shows the live / dead double - staining experimental results caused by the MIT group, and (c) shows the live / dead double - staining experimental results caused by the OE / IR / CM@C / M NPs group.

[0062] Figure 20 Shows the apoptosis experimental results of the Control group, MIT group, and OE / IR / CM@M / M NPs group against mouse hepatoma cells H22. Figure 20Among them, (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.

[0063] Figure 21 The results of the live / dead double staining experiments of the Control group, the MIT group, and the OE / IR / CM@M / M NPs group against mouse hepatoma cells H22 are as follows. Figure 21 Among them, (a) shows the results of the live / dead double staining experiment caused by the Control group, (b) shows the results of the live / dead double staining experiment caused by the MIT group, and (c) shows the results of the live / dead double staining experiment caused by the OE / IR / CM@M / M NPs group. Specific implementation manners

[0064] The technical solutions in the embodiments of the present invention will be clearly and completely described below. For the convenience of description, the new indocyanine green is hereinafter represented as IR820 in the present invention. It should be noted that the H22 cancer cells and HepG2 cancer cells involved in the present invention are purchased from Jiangsu Kaygene Biotechnology Co., Ltd. and Shanghai Beyotime Biotechnology Co., Ltd., respectively.

[0065] It should be noted that all the Control groups in the present invention hereinafter represent the control groups. It should also be noted that in each of the following embodiments of the present invention, the liquid auxiliary material used is OE, and the OE used in the present invention is prepared through the following steps:

[0066] S1, Stirring reaction: Accurately weigh diglycerol, triethyl orthoacetate, and p-toluenesulfonic acid according to a molar ratio of 1:4:0.02; and weigh the corresponding volume of anhydrous dichloromethane according to a volume ratio of anhydrous dichloromethane to diglycerol of 5:1, and set aside; Disperse the weighed diglycerol, triethyl orthoacetate, and p-toluenesulfonic acid in anhydrous dichloromethane, and then stir and react at room temperature for 12 h under a nitrogen atmosphere, and its reaction equation is as shown in Reaction Formula 1:

[0067]

[0068] Reaction Formula 1.

[0069] S2, Post-treatment: Add triethylamine to the system after the above stirring reaction to adjust the pH of the reaction solution to 8.0 to terminate the reaction, and then perform two extractions with a saturated Na 2 CO 3 solution to obtain an extract; After drying the extract by rotary evaporation, perform vacuum filtration to obtain the orthoester compound shown in Formula I, that is, OE.

[0070] The present invention also uses proton nuclear magnetic resonance 1 1H-NMR and carbon nuclear magnetic resonance13 The structural chemistry of the OE prepared above was tested by 13C-NMR, and the test results are shown in Figure 1 and Figure 2 respectively.

[0071] Among them, Figure 1 is the 1H-NMR spectrum of the orthoester compound OE of the present invention, 1 and Figure 2 is the 13C-NMR spectrum of the orthoester compound OE of the present invention. From the test results of 13 and Figure 1 it can be seen that the OE shown in Formula 1 was successfully synthesized in the present invention. Figure 2 In each of the following examples of the present invention, the cell membrane vesicles used are cancer cell membrane vesicles, and they are obtained by the following steps: Using H22 cancer cells and HepG2 cancer cells together as the source of cell membrane vesicles, when the H22 cells and HepG2 cells reach 80% confluence respectively, the cells are collected by centrifugation. For every 1×10

[0072] cells, 500 μL of hypotonic lysis buffer and 5 μL of PMSF are added, and the cells are ultrasonically disrupted for 2 min. The ultrasonic parameters are: 6 s / time, 30 W. Then, it is centrifuged at a centrifugal force of 1000×g for 2 min at 4 °C, and the nuclear material in the precipitate part is discarded. The supernatant is collected and centrifuged at 14000 rpm for 40 min to obtain cell membrane fragments. The cell membrane fragments are repeatedly extruded through polycarbonate membranes with pore sizes of 800 nm and 400 nm to obtain H22 cell membrane vesicles and HepG2 cell membrane vesicles, abbreviated as CM. 6 Example 1

[0073] This example provides a suspension type of biomimetic nanoparticles based on an orthoester compound, and the suspension type of biomimetic nanoparticles based on an orthoester compound in this example is prepared by the following steps:

[0074] Step 1, preparing biomimetic nanoparticles:

[0075] Step 1, preparing biomimetic nanoparticles:

[0076] Step 1.1, Preparation of nano-drugs: The nano-drugs used in this example are pure drug nanoparticles formed by self-assembly of small molecule drugs. In this example, dasatinib and sunitinib with corresponding masses are weighed according to a mass ratio of 3:1 and reserved. 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 each. The dasatinib solution and the sunitinib solution are mixed to obtain a mixed solution. Subsequently, at room temperature, the mixed solution is gradually added to deionized water and stirred for 5 min to enable dasatinib and sunitinib to self-assemble into a composite drug material. Then, the solution is centrifuged at 8000 rpm for 10 min, and the precipitate is collected and washed 3 times to remove dimethyl sulfoxide. After drying the obtained precipitate, pure drug nanoparticles, abbreviated as NPs, are obtained.

[0077] Step 1.2, Coating with cell membrane vesicles: The cancer cell membrane vesicles CM extracted above and the nano-drugs NPs prepared in Step 1.1 are mixed by ultrasonic and vortex according to a mass ratio of 2:1, and then extruded back and forth through polycarbonate membranes with pore sizes of 400 nm and 200 nm to obtain the bionic nanoparticles, denoted as CM@NPs.

[0078] Step 2, Dispersion of bionic nanoparticles and photosensitizer:

[0079] Using IR820 as the photosensitizer, 1 mL of OE, 1 mg of IR820 and 1 mg of CM@NPs are mixed and stirred for 10 min to obtain a bionic nanoparticle suspension formulation, abbreviated as OR / IR / CM@NPs.

[0080] Example 2

[0081] The difference between this example and Example 1 is only that:

[0082] In this example, the mass ratio of cancer cell membrane vesicles to nano-drugs is 3:1.

[0083] Example 3

[0084] The difference between this example and Example 1 is only that:

[0085] In this example, the mass ratio of cancer cell membrane vesicles to nano-drugs is 4:1.

[0086] Example 4

[0087] The difference between this example and Example 1 is only that:

[0088] In this example, the mass ratio of cancer cell membrane vesicles to nano-drugs is 5:1.

[0089] Example 5

[0090] The difference between this example and Example 1 is only that:

[0091] In this example, the mass ratio of cancer cell membrane vesicles to the nano-drug is 6:1.

[0092] Example 6

[0093] The difference between this example and Example 1 is only that:

[0094] In this example, the nano-drug is a drug-loaded nanoparticle prepared based on natural macromolecules, that is, the nano-drug used in this example is a drug-loaded nanoparticle, and the drug-loaded nanoparticle in this example uses natural macromolecule carboxymethyl chitosan nanoparticles as the nanoparticle matrix and mitoxantrone as the anti-tumor drug. The anti-tumor drug mitoxantrone is loaded on the natural macromolecule carboxymethyl chitosan nanoparticles through the following steps:

[0095] In this example, carboxymethyl chitosan (CMCS) and the chemotherapeutic drug mitoxantrone (MIT) are used as raw materials to prepare a nano-drug based on natural macromolecules, and its specific preparation process is as follows:

[0096] (1) Weigh 60 mg of CMCS powder and dissolve it fully in 6 mL of deionized water to make a 10 mg / mL CMCS aqueous solution. While stirring, gradually add 12 mL of acetone solution dropwise to the solution, and then add 60 μL of glutaraldehyde solution with a mass concentration of 25%, and crosslink for 4 h. Finally, centrifuge the reaction solution at a centrifugation rate of 10000 rpm for 10 min, collect the precipitate, and the obtained nanoparticles are denoted as CMCS NPs. Disperse CMCS NPs in water to obtain a 3 mg / mL CMCS NPs solution.

[0097] (2) Disperse MIT fully in water to obtain a 1 mg / mL MIT aqueous solution.

[0098] (3) According to the mass ratio of MIT to CMCS NPs of 1:3, gradually add the above-obtained MIT aqueous solution dropwise to the CMCS NPs solution, and stir for 4 h to enable mitoxantrone to be adsorbed on the surface of CMCS NPs through physical adsorption, and the drug-loaded nanoparticles are prepared, denoted as C / M NPs.

[0099] And the suspension dosage form of the biomimetic nanoparticles obtained in this example is simply referred to as OE / IR / CM@C / M NPs.

[0100] Example 7

[0101] The difference between this example and Example 1 is only that:

[0102] In this embodiment, the nano-drug is a drug-loaded nanoparticle prepared based on inorganic nanoparticles. That is, the nano-drug used in this embodiment is a drug-loaded nanoparticle, and the drug-loaded nanoparticle in this embodiment uses mesoporous silica nanoparticles as the nanoparticle matrix and mitoxantrone (MIT) as the anti-tumor drug. The anti-tumor drug mitoxantrone is loaded onto the mesoporous silica nanoparticles through the following steps:

[0103] (1) Disperse 1.50 g of cetyltrimethylammonium bromide (CTAB) in 750 mL of ultrapure water to obtain a CTAB solution. (2) Add 5.25 mL of 2.0 M aqueous sodium hydroxide solution to the CTAB solution and stir vigorously at 80 °C for 0.5 h. (3) Dropwise add 8.25 mL of tetraethyl orthosilicate to the system that has reacted for 0.5 h. After a white precipitate is formed, add 7.50 mL of ethyl acetate and stir for 2 h, then age at room temperature for 8 h. Centrifuge the aged reaction solution at 8000 rpm for 15 min to obtain unextracted mesoporous silica nanoparticles, denoted as MSN NPs. (4) Wash the MSN NPs three times with ultrapure water and ethanol, and dry them under vacuum to obtain dried MSN NPs. (5) According to the ratio of adding 1 mL of concentrated hydrochloric acid with a concentration of 37% and 100 mL of ethanol per gram of MSN NPs, add the corresponding volumes of concentrated hydrochloric acid and ethanol to the above dried MSN NPs, mix well, and react at 70 °C for 24 h. Then centrifuge again, wash with ethanol, and dry to obtain mesoporous silica nanoparticles. (6) Disperse 10 mg of mesoporous silica nanoparticle powder in 5 mL of phosphate buffer with a concentration of 0.1 M and pH = 7.4. According to the mass ratio of MIT to mesoporous silica nanoparticles of 1:3, continue to add the corresponding volume of 1 mg / mL mitoxantrone solution. Stir in the dark for 4 h, then centrifuge at 8000 rpm for 10 min, discard the supernatant, and collect the precipitate to obtain drug-loaded nanoparticles based on inorganic nanoparticles, denoted as M / M NPs.

[0104] And the suspension formulation of the biomimetic nanoparticles obtained in this embodiment is simply referred to as OE / IR / CM@M / M NPs.

[0105] Comparative Example 1

[0106] The difference between this comparative example and Example 1 is only that: in this comparative example, CM@NPs is not added, that is, the suspension formulation obtained in this comparative example is a formulation containing only the photosensitizer, simply referred to as OE / IR.

[0107] Comparative Example 2

[0108] The difference between this comparative example and Example 1 is only that: in this comparative example, NPs are directly dispersed into OE, that is, the suspension formulation of this comparative example is a formulation containing only nanoparticles, simply referred to as OE / NPs.

[0109] Comparative Example 3

[0110] The difference between this comparative example and Example 1 is only that: in this comparative example, photosensitizer IR820 is not added, that is, the suspension dosage form of this comparative example is a dosage form containing only biomimetic nanoparticles, simply referred to as OE / CM@NPs.

[0111] Comparative Example 4

[0112] The difference between this comparative example and Comparative Example 1 is only that: in this comparative example, OE is replaced with pure water of equal volume, that is, the dosage form obtained in this comparative example is an aqueous solution of photosensitizer, simply referred to as water / IR.

[0113] Experimental Section

[0114] (I) Detection of the particle size of biomimetic nano-drugs

[0115] Taking the nanoparticles NPs, cancer cell membrane vesicles CM prepared in the present invention, and the biomimetic nanoparticles CM@NPs prepared in Example 1 as examples, the hydrodynamic particle size thereof was tested by a dynamic light scattering instrument, and the test results are as Figure 3 shown, and Figure 3 in which, Figure A is the particle size distribution diagram of the nanoparticles NPs prepared in the present invention, Figure B is the particle size distribution diagram of the cancer cell membrane vesicles CM prepared in the present invention, and Figure C is the particle size distribution diagram of the biomimetic nanoparticles CM@NPs prepared in Example 1.

[0116] From Figure 3 the test results, it can be seen that the particle size of NPs is 161 nm, the PDI is 0.26, the particle size of CM is 220 nm, but after passing through the extruder, the cancer cell membrane prepared in the present invention is coated on the surface of NPs, making the particle size of CM@NPs 175 nm. It 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, and the membrane thickness is about 14 nm.

[0117] (II) Macroscopic morphology analysis of the suspension dosage form

[0118] Taking 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 as examples, their macroscopic morphologies were recorded as Figure 4 shown.

[0119] Figure 4 in which, Figure A is the physical picture of OE prepared in the present invention, Figure B is the physical picture of OE / IR of Comparative Example 1, Figure C is the physical picture of OE / CM@NPs of Comparative Example 3, and Figure D is the physical picture of OE / IR / CM@NPs of Example 1. From Figure 4It can be seen that the liquid auxiliary OE is a clear and transparent liquid. The green photosensitizer IR820 is successfully dispersed in OE to obtain OE / IR, and the light yellow biomimetic nanoparticles CM@NPs are also smoothly dispersed in OE to obtain OE / CM@NPs. The photosensitizer IR820 and the biomimetic nanoparticles are simultaneously dispersed in OE to obtain the final dosage form OE / IR / CM@NPs. Moreover, the prepared suspension has good stability, and no particle enlargement or aggregation and stratification phenomena occur after standing for 48 h.

[0120] (III) Photothermal conversion performance test

[0121] In the present invention, pure water is used as a blank control. Taking water / IR of Comparative Example 4 and OE / IR of Comparative Example 1 as examples, they are respectively added into cuvettes and irradiated under an 808 nm laser with a power of 1 W / cm 2 for 10 min. An infrared thermal imager of model Testo 865 is used to record the thermal images of the tested samples every 60 s, and the test results are sorted out as Figure 5 shown. And Figure 5 in it, 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. It can be seen from Figure 5 this that the temperature of pure water hardly changes under laser irradiation, the temperature of the water / IR solution rises to about 60 °C within 10 minutes, while the temperature of the OE / IR solution rises rapidly to 80 °C, preliminarily proving that OE can significantly improve the photothermal conversion efficiency and heating capacity of IR820.

[0122] In the present invention, taking water / IR and OE / IR as examples, they are respectively irradiated under 808 nm lasers with different powers of 0.5 W / cm 2 , 0.75 W / cm 2 , 1.0 W / cm 2 , 1.25 W / cm 2 , 1.75 W / cm 2 , 2.0 W / cm 2 , 2.5 W / cm 2 and 3.0 W / cm 2 for 10 min. An infrared thermal imager of model Testo 865 is used to record the temperature changes of the samples and draw the heating curves as Figure 6 shown. And Figure 6 in it, Figure A is the heating curve of water / IR of Comparative Example 4 under different power irradiation conditions, and Figure B is the heating curve of OE / IR of Comparative Example 1 under different power irradiation conditions. It can be seen from the test results of Figure 6 this that when the power of the 808 nm laser is 3 W / cm 2At this time, the water / IR solution at 1 mg / mL can reach 79.7 °C after 10 min, while OE / IR can reach an astonishing 126 °C. This shows that under the same power and concentration, OE significantly improves the photothermal conversion efficiency of IR820.

[0123] In the present invention, water / IR and OE / IR of Comparative Example 1 and their dilutions to different concentrations of 800 μg / mL, 600 μg / mL, 400 μg / mL, 400 μg / mL, 50 μg / mL and pure water were respectively irradiated under an 808 nm laser with a power of 1.0 W / cm 2 for 10 min, and a thermal imager of infrared rays with the model of Testo 865 was used to record the temperature change of the samples, and the obtained results were plotted as the Figure 7 heating curves as shown. Figure 7 In the figure, Figure A is the heating curve of Comparative Example 4 and its dilutions to different concentrations under the irradiation condition of 1.0 W / cm 2 power, and Figure B is the heating curve of Comparative Example 1 and its dilutions to different concentrations under the irradiation condition of 1.0 W / cm 2 power.

[0124] It can be seen from Figure 7 that when the power of the laser is fixed at 1 W / cm 2 , if the solution temperature is to reach 50 °C, the concentration of IR820 in water / IR is 800 μg / mL, while the concentration of IR820 in OE / IR is only 200 μg / mL. This indicates that under the same effect requirement, OE can significantly reduce the dosage of photosensitizer and achieve the purpose of reducing toxicity and increasing efficacy.

[0125] (IV) In vitro drug release performance test

[0126] Taking the prepared NPs, OE@NPs and OE / CM@NPs of the present invention as examples, the drug release experiments of each dosage form at different pH values were explored by detecting the content of DAS, and the specific process is as follows:

[0127] (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. Hereinafter, the phosphate buffer solution will be simply referred to as PBS for ease of expression. (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 a dialysis bag with a MWCO of 3500 Da. Tie the dialysis bag and place it in a 50 mL EP tube. (3) Add 5 mL of PBS with the corresponding pH to the tube and place it in the dark at 37 °C and shake. (4) Replace the fresh phosphate buffer solution at each predetermined time point of 1 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h of shaking. After shaking for 72 h, take 1 mL from all the collected PBS, freeze-dry it, add 1 mL of dimethyl sulfoxide to dissolve it, and detect the cumulative release rate of DAS by a multifunctional microplate reader. The detection results are as Figure 8 shown.

[0128] Figure 8 The in vitro drug release diagrams of NPs in Example 1, OE / NPs in Comparative Example 2, and OE / CM@NPS in Comparative Example 3 at different pH values are shown. It can be seen 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.

[0129] (V) Cytotoxicity assessment

[0130] In the present invention, the MTT method is used to evaluate the in vitro cytotoxicity of the biomimetic nanoparticles. The specific method is as follows:

[0131] (1) Prepare OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) samples:

[0132] Prepare OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) as test samples containing dasatinib concentrations of 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM, and IR820 concentrations of 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively, for standby. Among them, OE / IR(+) represents the sample of OE / IR after being irradiated with a 1 W / cm 2 laser at 808 nm for 5 min. Similarly, OE / IR / CM@NPs(+) represents the sample of OE / IR / CM@NPs after being irradiated with a 1 W / cm 2 laser at 808 nm for 5 min.

[0133] (2) Add the H22 cell suspension to a 96-well plate with 5000 cells per well. Add 20 μL of the prepared test sample to each well. After incubating for 48 h, add 20 μL of a thiazolyl blue aqueous solution with a concentration of 5 mg / mL to each well. After 4 h, add 100 μL of a trypan blue solution to each well and mix thoroughly by shaking. Finally, measure the absorbance of each well at a wavelength of 490 nm using a multifunctional microplate reader. Calculate the corresponding cell viability based on the absorbance and organize it into a graph as shown in Figure 9 shown below.

[0134] Figure 9 The cell viabilities of OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) at different concentrations are shown. It can be seen that OE does not show obvious cytotoxicity, indicating that OE is safe and non-toxic as a liquid pharmaceutical excipient; both OE / IR(+) and OE / CM@NPs exhibit concentration-dependent cytotoxicity, indicating that the OE-based dosage forms can produce good photothermal therapy and chemotherapy effects, while OE / IR / CM@NPs(+) shows the strongest cytotoxicity, indicating that the dosage form obtained by integrating biomimetic nanoparticles and photosensitizers in the present invention can produce a chemo / photothermal / photodynamic synergistic antitumor effect.

[0135] (6) Cell viability and cytotoxicity staining experiment

[0136] The present invention uses a reactive oxygen species detection kit to detect the level of intracellular reactive oxygen species (ROS):

[0137] (1) Seed H22 cells in a 6-well plate at a density of 1×10 5 cells per well. Add OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) to each well and incubate for 4 h. Among them, OE / IR(+) represents the sample after OE / IR is irradiated with a 1 W / cm 2 laser at 808 nm for 5 min. Similarly, OE / IR / CM@NPs(+) represents the sample after OE / IR / CM@ is irradiated with a 1 W / cm 2 laser at 808 nm for 5 min. Use the experimental group with pure culture medium added as the Control group.

[0138] (2)The DAS concentration in each sample was set at 20 μg / mL, and the IR820 concentration was set at 10 μg / mL. Then, according to the kit requirements, DCFH-DA was diluted with pure MEM at a ratio of 1:1000 to obtain a DCFH-DA solution. Subsequently, 1 mL of the DCFH-DA solution was added to each well. After incubation for 30 min, the wells were washed 3 times and fixed with paraformaldehyde for 10 min. 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 fluorescence probe.

[0139] Cell viability was further detected by live / dead fluorescence staining experiment. H22 cells were seeded in 6-well plates at a density of 1×10 5 cells per well. After adding OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) to the wells respectively, they were co-incubated for 4 h. The DAS concentration in NPs was set at 20 μg / mL, and the IR820 concentration was set at 10 μg / mL. "+" indicates 5 min under laser irradiation at 808 nm with a power of 1 W / cm 2 2. After incubation, 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. Then the cell staining was observed under an inverted fluorescence microscope.

[0140] The above test results were sorted out as shown in Figure 10 and Figure 11 shown. Figure 10 Figure shows the changes in intracellular ROS content caused by OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) under dark and light conditions respectively. It can be seen that under light, a large amount of ROS was generated in the cells treated with OR / IR and OE / IR / CM@NPs, indicating that this dosage form can produce a PDT effect.

[0141] Figure 11 Figure shows the live / dead cell staining results of OE, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) under dark and light conditions respectively. It can be seen that OR / IR / CM@NPs caused the most cell deaths, which is consistent with the results of the above MTT experiment.

[0142] (VII) In vivo photothermal conversion experiment

[0143] This invention established a subcutaneous tumor model in mice using mouse liver cancer cells H22 and conducted subsequent anti-tumor research. The specific experimental methods are as follows: After the H22 cells were in the logarithmic growth phase, the cells were collected and centrifuged, and then diluted with physiological saline to a suitable concentration. Finally, 100 μL of the diluted H22 cell suspension was injected into the left axilla of the mice, with 1×10 6 cells per mouse. When the tumor volume of the mice reached about 150 mm 3 , subsequent experiments were carried out. Different IR820 dose contents of OE / IR / CM@NPs with IR820 doses of 0.8 mg / kg, 1.6 mg / kg, and 2.4 mg / kg were injected into the tumor area by peritumoral injection at 100 μL each. The tumor area was irradiated at a power of 1 W / cm 2 for 5 min, and an infrared thermal imager was used to record the temperature rise of the tumor area during the entire irradiation process and draw a temperature rise curve. To explore the retention and photothermal stability of OE / IR / CM@NPs in the tumor area, an IR820 dose of 2.4 mg / kg was selected, and photos were taken and recorded after irradiation at 24 h, 72 h, and 120 h respectively. The results are as Figure 12 shown.

[0144] Figure 12 Figure A in

[0145] Figure 12 is the thermal imaging diagram of different IR820 dose contents of OE / IR / CM@NPs with IR820 doses of 0.8 mg / kg, 1.6 mg / kg, and 2.4 mg / kg after injection into the tumor area and 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 rises with time, and the trend of temperature rise is dose-dependent. The higher the IR820 concentration, the faster the temperature rises and the higher the temperature.

[0146] Figure 12Figure C in it shows the heating images of the tumor region of mice after injecting OR / IR / CM@NPs with an IR820 dose of 2.4 mg / kg into the tumor region for 24 h, 72 h, and 120 h, under the same 808 nm near-infrared light irradiation conditions. It should be noted that the ordinate in Figure C represents the time when the sample is injected into the mouse body; the abscissa represents the time of light irradiation. It can be seen that 1 day, 3 days, and 5 days after injection, the temperature in the tumor region can still rise above 50 °C.

[0147] Figure 12 Figure D in it shows the temperature counting results of the tumor region of each mouse in Figure C. It can be seen that even 5 days after injection, the temperature in the tumor region can still reach 50 °C, meeting the requirements of PTT treatment. The above results indicate that the OE dosage form can remain in the tumor region for a long time, thereby prolonging the PTT treatment effect and reducing the dosing frequency. The OE dosage form shows a dose-dependent heating ability, and the temperature in the tumor region of mice can reach 50 °C, meeting the requirements of PTT treatment. In addition, 5 days after peritumoral injection, the temperature in the tumor region can still reach 50 °C under laser irradiation, indicating that the OE dosage form can remain in the tumor region for a long time, which is very beneficial for prolonging the treatment cycle of a single dose, reducing the dosing frequency, and alleviating the discomfort of patients.

[0148] (VIII) Evaluation of anti-tumor ability in vivo

[0149] In this invention, in vivo direct anti-tumor research was carried out on H22 tumor-bearing mice, and the specific test method is as follows:

[0150] The H22 tumor-bearing mice were randomly divided into 4 groups, with 6 mice in each group, and the initial tumor volume was about 200 mm 3 . 100 μL of normal saline was respectively injected into the tumor in situ as the Control group, 100 μL of OE / IR(+), 100 μL of OE / CM@NPs, and 100 μL of OE / IR / CM@NPs(+). Among them, the doses of DAS and IR820 were 20 mg / kg and 1.7 mg / kg respectively. "(+)" indicates that the groups containing IR820 were subjected to light treatment on the 1st, 3rd, and 5th days, and the light treatment parameters were 1 W / cm 2 , 5 min. The body weight, short diameter, and long diameter of the tumor of the mice were recorded every two days, and the volume of the tumor was calculated through the formula. The calculation formula for the tumor volume is: ; 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.

[0151] The results of in vivo direct anti-tumor are as Figures 13 - 15 shown, among which, Figure 13Changes in in vivo tumor volume over time in H22 tumor-bearing mice in the Control group, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) groups, Figure 14 Changes in body weight over time in H22 tumor-bearing mice in the Control group, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) groups, Figure 15 Changes in in vivo tumor mass over time in H22 tumor-bearing mice in the Control group, OE / IR(+), OE / CM@NPs, and OE / IR / CM@NPs(+) groups.

[0152] From Figures 13 - 16 the test results, it can be seen that OE / IR / CM@NPs showed the most powerful anti-tumor effect, with the smallest average tumor volume and the lowest tumor mass, indicating that this suspension formulation achieved a powerful anti-tumor effect through the generation of chemotherapy / photothermal / photodynamic synergistic effects.

[0153] (IX) Evaluation of the ability to inhibit tumor recurrence

[0154] 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 the 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 tumors of the mice were resected, leaving about 5% of the residual tumor tissue to simulate the microtumors remaining in the surgical 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 subjected to light treatment on the 1st, 3rd, and 5th days, and the light treatment parameters were 1 W / cm 2 , 5 min. During the experiment, the tumor size, body weight changes, and survival status of these mice were regularly detected and sorted as shown in Figure 16 and Figure 17 respectively.

[0155] Figure 16 In, Figure A shows the change trend of the tumor volume of 6 mice in the normal saline group during the 50-day observation period, Figure B shows the change trend of the tumor volume of 6 mice in the OE / IR+ group during the 50-day observation period, Figure C shows the change trend of the tumor volume of 6 mice in the OE / CM@NPs group during the 50-day observation period, and Figure D shows the change trend of the tumor volume of 6 mice in the OE / IR / CM@NPs+ group during the 50-day observation period.

[0156] From Figure 16It can be seen that normal saline was completely unable to inhibit tumor growth. Within 15 days, tumors recurred in all 6 mice, and all the mice died; OE / IR+ and OE / CM@NPs could 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; in the mice treated with OE / IR / CM@NPs(+), no tumor recurrence occurred within 20 days, and 67% of the mice still had no tumor recurrence after 50 days.

[0157] Figure 17 It is a graph showing the changes in the survival rates 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. It can be seen that the tumors in the mice treated with normal saline, OE / IR+, and OE / CM@NPs recurred rapidly, ultimately leading to the death of the mice, while 4 mice treated with OE / IR / CM@NPs(+) finally survived for more than 120 days. This indicates that the OE suspension provided by the present invention can effectively prevent tumor recurrence after surgery and completely relieve tumor treatment.

[0158] (X) In vitro cell experiments of OE / IR / CM@C / M NPs in Example 6

[0159] Verify the in vitro anti-tumor activity of OE / IR / CM@C / M NPs in Example 6 through apoptosis experiments: Seed H22 cells in a 6-well plate at a density of 1×10 5 cells per well, and add MIT and OE / IR / CM@C / M NPs to each well and incubate for 4 h. The concentration of MIT is set at 20 μg / mL, and the concentration of IR820 is set at 10 μg / mL. After incubation, wash the cells with fresh PBS, add 2 μL of Annexin V-FITC and 2 μL of propidium iodide PI to each well, and incubate in the dark at 4 °C for 10 min. Then detect cell viability using a flow cytometer. Use normal saline as the Control group.

[0160] Further detect cell viability through live-dead fluorescence staining experiments. Seed H22 cells in a 6-well plate at a density of 1×10 5 cells per well, and add MIT and OE / IR / CM@C / M NPs to each well and incubate for 4 h. The concentration of MIT is set at 20 μg / mL, and the concentration of IR820 is set at 10 μg / mL. After incubation, wash the cells with fresh PBS, add 4 μM of calcein and 4 μM of iodide propidium to each well, and incubate in the dark at 37 °C for 30 min. Then observe the cell staining situation under an inverted fluorescence microscope, and organize the above test results as shown in Figure 18 and Figure 19 shown.

[0161] Figure 18 The apoptosis experimental results of the Control group, MIT group, and OE / IR / CM@C / M NPs group against mouse hepatoma cell line H22. From Figure 18 It can be seen that the apoptosis ratio of cells treated with the Control group is 5.49%, that of the MIT group is 45.44%, and the apoptosis ratio of cells treated with OE / IR / CM@C / M NPs is the highest, reaching 71.7%.

[0162] Figure 19 The live / dead double staining experimental results of the Control group, MIT group, and OE / IR / CM@C / M NPs group against mouse hepatoma cell line H22, where the green signal represents live cells and the red signal represents dead cells. From Figure 19 It can be seen that the cells treated with OE / IR / CM@C / M NPs all show the red signal of death, indicating that OE / IR / CM@C / M NPs cause the most cell death, which is consistent with the above apoptosis experimental results.

[0163] (XI)In vitro cell experiments of OE / IR / CM@M / M NPs in Example 7

[0164] Verify the in vitro anti-tumor activity of OE / IR / CM@C / M NPs in Example 7 through apoptosis experiments: Seed H22 cells in a 6-well plate at a density of 1×10 5 cells per well, and add MIT and OE / IR / CM@M / M NPs to each well and incubate for 4 h. The concentration of MIT is set at 20 μg / mL, and the concentration of IR820 is set at 10 μg / mL. After incubation, wash the cells with fresh PBS, add 2 μL of Annexin V-FITC and 2 μL of propidium iodide PI to each well, and incubate in the dark at 4 °C for 10 min. Then detect cell viability using a flow cytometer. Use normal saline as the Control group. Among them, the Chinese name of Annexin V-FITC is extracellular phosphatidylserine-binding protein V.

[0165] Further detect cell viability through live / dead fluorescence staining experiments. Seed H22 cells in a 6-well plate at a density of 1×10 5Cells were seeded at a density of [number of cells] in 6-well plates. MIT, OE / IR / CM@M / M NPs were added to each well and incubated for 4 h. The concentration of MIT was set at 20 μg / mL and the concentration of IR820 was set at 10 μg / mL. After incubation, 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. Then, the cell staining was observed under an inverted fluorescence microscope. The above test results were organized as shown in Figure 20 and Figure 21 shown below.

[0166] Figure 20 Figure [figure number] shows the apoptosis assay results of the Control group, MIT group, and OE / IR / CM@M / M NPs group against mouse hepatoma cells H22. It can be seen that the apoptosis rate of cells treated with the Control group was 4.61%, the apoptosis rate of cells treated with the MIT group was 40.79%, and the apoptosis rate of cells treated with OE / IR / CM@M / M NPs was the highest, reaching 86.63%.

[0167] Figure 21 Figure [figure number] shows the live / dead dual staining assay results of the Control group, MIT group, and OE / IR / CM@M / M NPs group against mouse hepatoma cells H22. The green signal represents live cells and the red signal represents dead cells. As Figure 20 can be seen, cells treated with OE / IR / CM@M / M NPs all showed red signals of dead cells, indicating that OE / IR / CM@M / M NPs caused the most cell deaths, which was consistent with the above apoptosis assay results.

[0168] Obviously, the above embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A biomimetic nanoparticle suspension formulation based on an orthoester compound, characterized in that: It consists of 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 are obtained by using nanomedicine as a matrix and wrapping cell membrane vesicles on the surface thereof; 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, and the small molecule drugs are dasatinib and sunitinib; the drug-loaded nanoparticle is obtained by loading mitoxantrone with carboxymethyl chitosan nanoparticles or mesoporous silicon nanoparticles as a nanoparticle matrix; The cell membrane vesicles are H22 cell membrane vesicles and HepG2 cell membrane vesicles; The photosensitizer is new indocyanine green.

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 loading amount of the small molecule drug on the nanoparticle matrix is ​​1 wt% to 20 wt%.

4. The biomimetic nanoparticle suspension formulation based on orthoester compounds according to claim 1, characterized in that: The particle size of the nano drug is 10nm~300nm.

5. 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.

6. A method for preparing a biomimetic nanoparticle suspension formulation based on an orthoester compound according to any one of claims 1 to 5, 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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