IR-780 iodide modified lipid grafting material, drug delivery system and preparation method thereof
阅读说明：本技术 一种ir-780碘化物修饰脂质嫁接物、给药系统及其制备方法 (IR-780 iodide modified lipid grafting material, drug delivery system and preparation method thereof ) 是由 谭亚南 关新元 李咏梅 李珊珊 罗敏 于 2021-08-09 设计创作，主要内容包括：本发明公开了一种IR-780碘化物修饰脂质嫁接物、给药系统及其制备方法,其中,嫁接物的结构式为：通过采用本发明提供的IR-780碘化物修饰脂质嫁接物,不仅为制备热敏脂质给药系统以及NIR响应型仿生膜纳米囊泡奠定了基础,而且还实现了通过阻断PD-1/PD-L1通路,长期逆转肿瘤免疫抑制状态,协同发挥抗肿瘤免疫应答,抑制原发肿瘤及转移灶的生长,从而也有效地实现了提高肿瘤的治疗效果和降低肿瘤复发转移率的目的。(The invention discloses an IR-780 iodide modified lipid graft, a drug delivery system and a preparation method thereof, wherein the structural formula of the graft is as follows: by adopting the IR-780 iodide modified lipid graft provided by the invention, a foundation is laid for preparing a thermosensitive lipid drug delivery system and NIR response type bionic membrane nano vesicles, and the aims of blocking a PD-1/PD-L1 path, reversing the tumor immunosuppression state for a long time, cooperatively exerting anti-tumor immune response, inhibiting the growth of primary tumors and metastatic foci, and further effectively improving the tumor treatment effect and reducing the tumor recurrence and metastasis rate are fulfilled.)
1. An IR-780 iodide modified lipid graft is characterized in that the structural formula is as follows:
wherein the value range of n is 45-100.
2. A thermosensitive lipid delivery system comprising the IR 780-iodide modified lipid graft of claim 1.
3. The method for preparing the IR-780 iodide modified lipid graft of claim 1, comprising the steps of:
s1, weighing 5-15 mg of IR-780 iodide and 20-50 mg of NH2-PEG-DSPE dissolved in dichloromethane solution to obtain dichloromethane solution containing IR 780;
s2, weighing triethylamine serving as an acid-binding agent according to the molar ratio of the triethylamine to the IR-780 iodide of (1-3) to 1, adding the triethylamine into the dichloromethane solution containing the IR-780 iodide, and stirring at room temperature for reaction;
and S3, after the reaction is finished, adding the reaction solution into anhydrous ether, carrying out low temperature overnight, centrifuging, removing a by-product, and drying the precipitate to obtain the IR-780 iodide modified lipid graft.
4. The method for preparing an IR-780 iodide modified lipid graft as claimed in claim 3, wherein in S1, the NH is added2-PEG-DSPE is NH2-PEG1000-DSPE、NH2-PEG2000-DSPE、NH2-PEG3000-DSPE、NH2-at least one of PEG 4000-DSPE.
5. The method for preparing an IR-780 iodide modified lipid graft as claimed in claim 4, wherein in S3, the ratio of the volume of the anhydrous ethyl ether to the volume of the intermediate mixed solution is (8-12): 1.
6. the method for preparing the IR-780 iodide modified lipid graft as claimed in any one of claims 3 to 5, wherein the step S3 is performed overnight at 3-5 ℃; the rotating speed during centrifugation is 2500-3500 rpm, and the centrifugation time is 4-6 min.
7. A method of preparing a heat-sensitive lipid delivery system according to claim 2, comprising the steps of:
s1, dissolving a PD-1/PD-L1 inhibitor, an IR-780 iodide modified lipid graft, thermosensitive lipid DPPC and cholesterol in ethanol, and heating to obtain an ethanol phase solution; wherein the ethanol phase solution contains 5-20% by mass of PD-1/PD-L1 inhibitor, 10-20% by mass of IR-780 iodide modified lipid graft, 65-75% by mass of thermosensitive lipid DPPC and 3-6% by mass of cholesterol;
and S2, rapidly dispersing the ethanol phase solution into the F68 aqueous solution, uniformly stirring, and cooling to room temperature to prepare the thermosensitive lipid delivery system.
8. The method of claim 7, wherein the PD-1/PD-L1 inhibitor is at least one of BMS-1, BMS-1001, BMS202 and BMS 1166 in S1.
9. The method of claim 8, wherein the heating to a temperature of 55-65 ℃ in S1 is performed.
10. The method of any one of claims 7-9, wherein the concentration of the aqueous solution of F68 in S2 is 0.5-1.5 mg/mL; the volume ratio of the F68 aqueous solution to the ethanol phase solution is (8-12) to 1.
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to an IR-780 iodide modified lipid graft, a drug delivery system and a preparation method thereof.
Background
The tumor immunotherapy is to introduce functional antigen-specific immune reaction to start the immune system of the organism and realize long-term immune memory, prevention and killing of tumor cells. However, at present, about 85% of tumor patients have poor response to immunotherapy, and researches find that the tumor microenvironment can protect tumor cells and immune escape is carried out through mechanisms such as immunosuppression.
Immune Checkpoint Blockade (ICB) therapy to reverse tumor Immune-suppressive microenvironment (TME) has become the primary modality of immunotherapy. Among them, ICB therapy directed to the PD-1/PD-L1 pathway has significant clinical efficacy in various types of cancer (e.g., melanoma, non-small cell lung cancer, and bladder cancer).
The use of PD-1/PD-L1 inhibitor alone is effective in only 10% to 30% of patients, which may be limited by the expression level of PD-L1 in Tumor tissues and the proportion of Tumor-infiltrating lymphocytes (TILs). The deep deficiency of TILs in most solid tumors, called "cold" tumors, is not sensitive to single anti-PD therapy and is a significant cause of ICB treatment failure.
It has been found that TILs are often confined to the stromal region of the tumor microenvironment rather than around tumor cells, where tumor-associated fibroblasts (CAFs) activate PD-L2 and FASL, leading to CD8+The T cells are exhausted, and the immune response effect of the cytotoxic T cells cannot be exerted.
Therefore, the combination with other effective treatment strategies, the tumor microenvironment is remodeled, the proportion of TILs at the deep part of the tumor is improved, and the activation of effector T cells to attack tumor cells is the key for enhancing the blocking treatment of PD-1/PD-L1, so that the tumor recurrence or metastasis is expected to be greatly inhibited.
At present, researchers combine the PD-1/PD-L1 blocking therapy with radiotherapy, phototherapy and other means by means of nanotechnology to synergistically stimulate a host immune system to generate an anti-tumor immune response to control the growth of metastatic tumors. Photothermal or photodynamic therapy has been found to induce Tumor Immunogenic Cell Death (ICD) by producing hyperthermia or reactive oxygen species, to release Tumor-associated antigens (TAA), and to activate immune responses in vivo. Photothermal therapy may be possible to increase the proportion of TILs deep in tumors and is beneficial to improve the immune suppressive tumor microenvironment, which can be an effective combination strategy for enhancing the blocking treatment of PD-1/PD-L1.
In the systemic circulation process, most of nano drug delivery systems have the problem of immunogenicity in different degrees, and the bionic drug delivery system coated by cell membranes can obviously reduce the immunogenicity, and simultaneously can simulate the functions of substances in vivo or pathogens with strong infectivity, replicate the physiological process and accurately deliver the drugs to target sites. The surface of the tumor cell membrane highly expresses specific proteins such as adhesion protein, integrin, adhesion kinase and the like, so that homologous tumor cells have stronger adhesion, the cells can highly self-identify, and the homologous tumor targeting can be realized.
The homologous tumor cell membrane can be used for constructing a bionic drug delivery system, and is expected to improve the targeting and treatment effects of the PD-1/PD-L1 blocking treatment on primary tumors and metastasis by combining photothermal therapy, and simultaneously can reduce off-target effect and realize the high-efficiency and low-toxicity immunotherapy target.
Disclosure of Invention
In view of the above, the application aims to provide an IR-780 iodide modified lipid graft, which effectively lays a foundation for preparing a drug delivery system and NIR response type bionic membrane nano vesicles capable of improving the treatment effect of PD-1/PD-L1 blocking treatment on primary tumors and metastatic foci.
It is a second object of the present invention to provide a heat-sensitive lipid delivery system.
The third purpose of the invention is to provide a preparation method of the IR-780 iodide modified lipid graft.
The fourth object of the present invention is to provide a method for preparing the thermosensitive lipid delivery system.
In order to achieve the purpose, the technical scheme of the invention is realized as follows: an IR-780 iodide modified lipid graft has the following structural formula:
wherein the value range of n is 45-100.
The second technical scheme of the invention is realized as follows: a thermosensitive lipid delivery system comprising the IR 780-iodide modified lipid graft of claim 1.
The third technical scheme of the invention is realized as follows: the preparation method of the IR-780 iodide modified lipid graft comprises the following steps:
s1, weighing 5-15 mg of IR-780 iodide and 20-50 mg of NH2-PEG2000-DSPE, precisely weighing, dissolving in dichloromethane solution to obtain dichloromethane solution containing IR 780;
s2, weighing triethylamine serving as an acid-binding agent according to the molar ratio of the triethylamine to the IR-780 iodide of (1-3) to 1, adding the triethylamine into the dichloromethane solution containing the IR-780 iodide, and stirring at room temperature for reaction;
and S3, after the reaction is finished, adding the reaction solution into anhydrous ether, carrying out low temperature overnight, centrifuging, removing a by-product, and drying the precipitate to obtain the IR-780 iodide modified lipid graft.
Preferably, in the S1, the NH2-PEG-DSPE is NH2-PEG1000-DSPE、NH2-PEG2000-DSPE、NH2-PEG3000-DSPE、NH2-at least one of PEG 4000-DSPE.
Preferably, in the step S2, the stirring reaction time is 8 to 30 hours.
Preferably, in the step S3, the ratio of the volume of the anhydrous ether to the volume of the intermediate mixed solution is (8-12): 1.
Preferably, in the S3, the reaction is carried out overnight at the temperature of 3-5 ℃; the rotating speed during centrifugation is 2500-3500 rpm, and the centrifugation time is 4-6 min.
The fourth technical scheme of the invention is realized as follows: a preparation method of the thermosensitive lipid delivery system comprises the following steps:
s1, dissolving a PD-1/PD-L1 inhibitor, an IR-780 iodide modified lipid graft, thermosensitive lipid DPPC and cholesterol in ethanol, and heating to obtain an ethanol phase solution; wherein the ethanol phase solution contains 5-20% by mass of PD-1/PD-L1 inhibitor, 10-20% by mass of IR-780 iodide modified lipid graft, 65-75% by mass of thermosensitive lipid DPPC and 3-6% by mass of cholesterol;
and S2, rapidly dispersing the ethanol phase solution into the F68 aqueous solution, uniformly stirring, and cooling to room temperature to prepare the thermosensitive lipid delivery system.
Preferably, in the S1, the PD-1/PD-L1 inhibitor is at least one of BMS-1, BMS-1001, BMS202 and BMS-1166.
Preferably, in the step S1, the heating temperature is 55-65 ℃.
Preferably, in the S2, the concentration of the F68 aqueous solution is 0.5-1.5 mg/mL; the volume ratio of the F68 aqueous solution to the ethanol phase solution is (8-12) to 1.
Compared with the prior art, the IR-780 iodide modified lipid graft provided by the invention not only lays a foundation for preparing a thermosensitive lipid drug delivery system and NIR response type bionic membrane nano vesicles, but also realizes long-term reversion of tumor immunosuppression state by blocking a PD-1/PD-L1 pathway, synergistically exerts antitumor immune response, inhibits growth of primary tumors and metastatic foci, and further effectively realizes the purposes of improving the tumor treatment effect and reducing the tumor recurrence and metastasis rate.
Drawings
FIG. 1 is a synthetic route of IR-780 iodide modified lipid graft IR 780-PEG-DSPE;
FIG. 2 shows IR780 and DSPE-PEG-NH2And the 1H NMR spectrum of IR 780-PEG-DSPE;
FIG. 3 is a graph of the in vivo distribution of membrane nanovesicles (M/IR NPs) of different mass ratios in 4T1 tumor-bearing mice, wherein the dashed circles represent tumors;
FIG. 4 is a schematic diagram showing the comparison of particle sizes of IR NPs, M and M/IR NPs;
FIG. 5 is a Zeta potential comparison diagram of different materials;
FIG. 6 is a transmission electron micrograph of NIR responsive biomimetic cell membrane nanovesicles (M/IR NPs);
FIG. 7 is a graph showing the results of membrane protein analysis of NIR-responsive biomimetic cell membrane nanovesicles (M/IR NPs), wherein,
FIG. 7A is a Coomassie blue stained image of SDS-PAGE separated proteins; wherein, I: 4T1 cell lysate, II: 4T1 cell membrane solution, III: M/IR NPs;
FIG. 7B is a diagram showing the result of Western blot analysis of cell membrane specific proteins (EpCAM, N-cadherin and galectin-3); wherein, I: 4T1 cell lysate, II: 4T1 cell membrane solution, III: M/IR NPs;
fig. 8 is a schematic diagram of in vivo distribution of NIR-responsive biomimetic cell membrane nanovesicles (M/IR NPs), wherein:
FIG. 8A is an in vivo profile of 4T1 tumor in a mouse model after 2h, 8h, 24h, 48h and 72h tail vein injection with IR NPs or M/IR NPs;
fig. 8B is a graph showing the mean fluorescence intensity of tumor sites after 2h, 8h, 24h, 48h and 72h tail vein injection of IR NPs or M/IR NPs in a 4T1 tumor mouse model, wherein n is 3;
FIG. 9 is a graph of the ultraviolet absorption spectrum of NIR responsive biomimetic cell membrane nanovesicles M/IR NPs;
FIG. 10 shows M/IR NPs irradiated with near-infrared laser (808nm, 1W/cm)2)5min temperature change curve graph;
FIG. 11 shows that NIR response type bionic cell membrane nanovesicles M/IR NPs are irradiated by laser in vitro (808nm, 1W/cm)23min) before and after drug release profile;
FIG. 12 is a transmission electron micrograph of IR NPs and M/IR NPs at different temperatures;
FIG. 13 is a schematic diagram of the therapeutic effect of NIR-responsive biomimetic cell membrane nanovesicles M/IR NPs on in vivo anti-tumor, which is a graph showing the tumor growth of 4T1 tumor-bearing mice within 21 days;
FIG. 14 is a graph comparing the weights of tumor tissues in different groups after the administration;
FIG. 15 shows the therapeutic effect of NIR-responsive biomimetic cell membrane nanovesicles M/IR NPs on in vivo anti-tumor metastasis,
FIG. 15A is a photograph of lung tissue from each treatment group;
FIG. 15B shows the number of lung metastases for each treatment group; wherein n is 5, p <0.01, p < 0.001);
FIG. 16 is a flow cytometer measuring CD8 in tumor tissue of various groups of mice after drug administration+Schematic representation of the T cell infiltration ratio;
FIG. 17 is a graph showing IFN-. gamma.secretion levels in tumor tissues.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The materials, cells, etc. used in the following examples can be obtained by purchase, self-made, or extraction.
The embodiment of the invention provides an IR-780 iodide modified lipid graft, which has the following structural formula:
wherein the value range of n is 45-100.
The embodiment of the invention also provides a thermosensitive lipid delivery system, which comprises the IR-780 iodide modified lipid graft.
The embodiment of the invention also provides a preparation method of the IR-780 iodide modified lipid graft (IR780-PEG-DSPE), which comprises the following steps:
s1, weighing 5-15 mg of IR-780 iodide (IR780) and 20-50 mg of NH2-PEG-DSPE, precisely weighing, dissolving in dichloromethane solution to obtain dichloromethane solution containing IR-780 iodide; wherein, the NH2-PEG-DSPE is NH2-PEG1000-DSPE、NH2-PEG2000-DSPE、NH2-PEG3000-DSPE、NH2-at least one of PEG 4000-DSPE; preferably NH2-PEG2000-DSPE；
S2, taking Triethylamine (TEA) as an acid-binding agent, weighing the Triethylamine (TEA) according to the molar ratio of the Triethylamine (TEA) to the IR780 of (1-3) to 1, adding the weighed Triethylamine (TEA) into the dichloromethane solution containing the IR-780 iodide, and stirring and reacting for 8-30 hours at room temperature;
s3, after the reaction is finished, adding the reaction solution into anhydrous ether with the volume 8-12 times that of the reaction solution, carrying out overnight at the temperature of 3-5 ℃, centrifuging for 4-6min at the rotating speed of 2500-3500 rpm, removing byproducts, and drying the precipitate to obtain the IR-780 iodide modified lipid graft (IR 780-PEG-DSPE).
The embodiment of the invention also provides a preparation method of the thermosensitive lipid delivery system, which comprises the following steps:
s1, dissolving a PD-1/PD-L1 inhibitor, an IR-780 iodide modified lipid graft, thermosensitive lipid DPPC and cholesterol in ethanol, and heating to obtain an ethanol phase solution;
wherein the ethanol phase solution contains 5-20% by mass of PD-1/PD-L1 inhibitor, 10-20% by mass of IR-780 iodide modified lipid graft, 65-75% by mass of thermosensitive lipid DPPC and 3-6% by mass of cholesterol; the PD-1/PD-L1 inhibitor is at least one of BMS-1, BMS-1001, BMS202 and BMS-1166;
s2, weighing an F68 aqueous solution with the concentration of 0.5-1.5 mg/mL according to the volume ratio of the F68 aqueous solution to the ethanol phase solution of (8-12) to 1, rapidly dispersing the ethanol phase solution into the F68 aqueous solution at the temperature of 55-65 ℃, uniformly stirring, and cooling to room temperature to prepare the thermosensitive lipid delivery system.
After the scheme is adopted, the IR-780 iodide modified lipid graft prepared by the method provided by the embodiment of the invention not only lays a foundation for preparing a thermosensitive lipid drug delivery system and an NIR response type bionic membrane nano vesicle, but also realizes long-term reversion of tumor immunosuppression state by blocking a PD-1/PD-L1 pathway, synergistically exerts antitumor immune response, and inhibits the growth of primary tumors and metastatic foci, thereby effectively realizing the purposes of improving the tumor treatment effect and reducing the tumor recurrence and metastasis rate.
The following are specific examples
Example 1
The IR-780 iodide modified lipid graft provided by the embodiment 1 of the invention is obtained by the following steps:
10mg of IR-780 iodide and 40mg of NH were weighed out2-PEG2000-DSPE, precisely weighing, dissolving in dichloromethane solution to obtain dichloromethane solution containing IR 780; triethylamine (TEA) is used as an acid-binding agent, the Triethylamine (TEA) is weighed according to the molar ratio of the Triethylamine (TEA) to the IR780 of 2: 1, added into the dichloromethane solution containing the IR-780 iodide, and stirred and reacted for 24 hours at room temperature; after the reaction, the reaction solution was added to 10 times the volume of anhydrous ether, and the mixture was left overnight at 4 ℃ and at a rotating speedCentrifuging at 3000rpm for 5min to remove byproduct, and drying the precipitate to obtain IR-780 iodide modified lipid graft (IR780-PEG 2000-DSPE).
The specific synthetic route of the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE) in example 1 is shown in fig. 1 (note that n in fig. 1 ranges from 45 to 100, and when PEG2000 is selected as polyethylene glycol, n in fig. 1 is 46). The chemical structure of the IR780-PEG2000-DSPE graft was confirmed by 1H NMR, as shown in FIG. 2.
As can be seen from the NMR spectrum of FIG. 2, characteristic peaks at 0.96 and 1.29ppm were assigned to NH2-CH in PEG2000-DSPE3and-CH2-upper proton peak. The characteristic peak near 8.33ppm is attributed to the proton peak of benzene ring on IR 780; these results show that IR780-PEG2000-DSPE has been successfully prepared.
Example 2
The IR-780 iodide modified lipid graft provided by the embodiment 2 of the invention is obtained by the following steps:
5mg of IR-780 iodide and 20mg of NH were weighed out2-PEG2000-DSPE, precisely weighing, dissolving in dichloromethane solution to obtain dichloromethane solution containing IR-780 iodide; triethylamine (TEA) is used as an acid-binding agent, the Triethylamine (TEA) is weighed according to the molar ratio of the Triethylamine (TEA) to the IR780 of 1: 1, added into the dichloromethane solution containing the IR-780 iodide, and stirred and reacted for 8 hours at room temperature; after the reaction is finished, adding the reaction solution into 8 times volume of anhydrous ether, standing overnight at 3 ℃, centrifuging for 4min at the rotation speed of 2500rpm, removing byproducts, and drying the precipitate to obtain the IR-780 iodide modified lipid graft (IR780-PEG 2000-DSPE).
Example 3
The IR-780 iodide modified lipid graft provided by the embodiment 3 of the invention is obtained by the following steps:
15mg of IR-780 iodide and 50mg of NH were weighed out2-PEG2000-DSPE, precisely weighing, dissolving in dichloromethane solution to obtain dichloromethane solution containing IR-780 iodide; triethylamine (TEA) is used as an acid-binding agent, and iodine is added into the mixture according to the weight ratio of the Triethylamine (TEA) to IR-780Weighing Triethylamine (TEA) according to the compound molar ratio of 3: 1, adding the TEA into the dichloromethane solution containing the IR-780 iodide, and stirring and reacting for 30 hours at room temperature; after the reaction is finished, adding the reaction solution into anhydrous ether with the volume being 12 times that of the reaction solution, standing overnight at the temperature of 5 ℃, centrifuging for 6min at the rotation speed of 3500rpm, removing byproducts, and drying precipitates to obtain the IR-780 iodide modified lipid graft (IR780-PEG 2000-DSPE).
Example 4
The IR-780 iodide modified lipid graft provided by the embodiment 4 of the invention is obtained by the following steps:
5mg of IR-780 iodide and 50mg of NH were weighed out2-PEG2000-DSPE, precisely weighing, dissolving in dichloromethane solution to obtain dichloromethane solution containing IR-780 iodide; triethylamine (TEA) is used as an acid-binding agent, the Triethylamine (TEA) is weighed according to the molar ratio of the Triethylamine (TEA) to the IR-780 iodide of 3: 1, added into the dichloromethane solution containing the IR-780 iodide, and stirred and reacted for 8 hours at room temperature; after the reaction is finished, adding the reaction solution into anhydrous ether with the volume being 12 times that of the reaction solution, standing overnight at the temperature of 3 ℃, centrifuging for 4min at the rotation speed of 3500rpm, removing byproducts, and drying precipitates to obtain the IR780 modified lipid graft (IR780-PEG 2000-DSPE).
Example 5
The IR-780 iodide modified lipid graft provided by the embodiment 5 of the invention is obtained by the following steps:
15mg of IR-780 iodide and 20mg of NH were weighed out2-PEG2000-DSPE, precisely weighing, dissolving in dichloromethane solution to obtain dichloromethane solution containing IR-780 iodide; triethylamine (TEA) is used as an acid-binding agent, the Triethylamine (TEA) is weighed according to the molar ratio of the Triethylamine (TEA) to the IR-780 iodide of 1: 1, added into the dichloromethane solution containing the IR-780 iodide, and stirred and reacted for 30 hours at room temperature; after the reaction is finished, adding the reaction solution into 8 times volume of anhydrous ether, standing overnight at 5 ℃, centrifuging for 6min at the rotation speed of 2500rpm, removing byproducts, and drying the precipitate to obtain the IR780 modified lipid graft (IR780-PEG 2000-DSPE).
Example 6
The thermosensitive lipid delivery system provided by the embodiment 6 of the invention is obtained by the following method:
taking an inhibitor BMS202(BMS) as a model drug, respectively weighing the inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE) obtained in example 1, thermosensitive lipid (DPPC) and cholesterol, and the balance being ethanol, dissolving the weighed inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE), the thermosensitive lipid DPPC and cholesterol in ethanol, and heating in a water bath at 60 ℃ to obtain an ethanol phase solution (in the solution, the mass percent of the inhibitor BMS202 is 10%, the mass percent of the IR-780 iodide modified lipid graft is 15%, the mass percent of the thermosensitive lipid DPPC is 70%, and the mass percent of the cholesterol is 5%); weighing F68 aqueous solution (the concentration of the aqueous solution is 1mg/mL) according to the volume ratio of the F68 aqueous solution to the ethanol phase solution of 10: 1, rapidly dispersing the ethanol phase solution into the F68 aqueous solution at the temperature of 60 ℃, stirring at the rotating speed of 400rpm for 8min, and cooling to room temperature to prepare the thermosensitive lipid delivery system, namely an IR-780 iodide modified thermosensitive lipid delivery system (IR780/DPPC/BMS, IR NPs).
Example 7
The thermosensitive lipid delivery system provided by the embodiment 7 of the invention is obtained by the following method:
taking an inhibitor BMS202(BMS) as a model drug, respectively weighing the BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE) obtained in example 1, thermosensitive lipid (DPPC) and cholesterol, and the balance of ethanol, dissolving the weighed inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE), the thermosensitive lipid DPPC and cholesterol in ethanol, and heating in a water bath at 55 ℃ to obtain an ethanol phase solution (in the solution, the mass percent of the inhibitor BMS202 is 5%, the mass percent of the IR-780 iodide modified lipid graft is 10%, the mass percent of the thermosensitive lipid DPPC is 65% and the mass percent of the cholesterol is 3%); weighing an F68 aqueous solution (the concentration of the aqueous solution is 0.5mg/mL) according to the volume ratio of the F68 aqueous solution to the ethanol phase solution of 8: 1, rapidly dispersing the ethanol phase solution into the F68 aqueous solution at the temperature of 55 ℃, stirring at the rotating speed of 300rpm for 6min, and cooling to room temperature to prepare a thermosensitive lipid delivery system, namely an IR-780 iodide modified thermosensitive lipid delivery system (IR780/DPPC/BMS, IR NPs).
Example 8
The thermosensitive lipid delivery system provided by the embodiment 8 of the invention is obtained by the following method:
taking an inhibitor BMS202(BMS) as a model drug, respectively weighing the inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE) obtained in example 1, thermosensitive lipid (DPPC) and cholesterol, and the balance being ethanol, dissolving the weighed inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE), the thermosensitive lipid DPPC and cholesterol in ethanol, and heating in a water bath at 65 ℃ to obtain an ethanol phase solution (in the solution, the mass percent of the inhibitor BMS202 is 20%, the mass percent of the IR-780 iodide modified lipid graft is 20%, the mass percent of the thermosensitive lipid DPPC is 75%, and the mass percent of the cholesterol is 6%); weighing an F68 aqueous solution (the concentration of the aqueous solution is 1.5mg/mL) according to the volume ratio of the F68 aqueous solution to the ethanol phase solution of 12: 1, rapidly dispersing the ethanol phase solution into an F68 aqueous solution at 65 ℃, stirring at 500rpm for 10min, and cooling to room temperature to prepare a thermosensitive lipid delivery system, namely an IR-780 iodide modified thermosensitive lipid delivery system (IR780/DPPC/BMS, IR NPs).
Example 9
The thermosensitive lipid delivery system provided by the embodiment 9 of the invention is obtained by the following method:
taking an inhibitor BMS202(BMS) as a model drug, respectively weighing the inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE) obtained in example 1, thermosensitive lipid (DPPC) and cholesterol, and the balance of ethanol, dissolving the weighed inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG2000-DSPE), the thermosensitive lipid (DPPC) and the cholesterol in ethanol, and heating in a water bath at 60 ℃ to obtain an ethanol phase solution (in the solution, the mass percent of the inhibitor BMS202 is 5%, the mass percent of the IR-780 iodide modified lipid graft is 20%, the mass percent of the thermosensitive lipid is 65% and the mass percent of the cholesterol is 6%); weighing an F68 aqueous solution (the concentration of the aqueous solution is 1mg/mL) according to the volume ratio of the F68 aqueous solution to the ethanol phase solution of 10: 1, rapidly dispersing the ethanol phase solution into the F68 aqueous solution at the temperature of 60 ℃, stirring at the rotating speed of 400rpm for 8min, and cooling to room temperature to prepare the thermosensitive lipid delivery system, namely an IR-780 iodide modified thermosensitive lipid delivery system (IR780/DPPC/BMS, IR NPs).
Example 10
The thermosensitive lipid delivery system provided by the embodiment 10 of the invention is obtained by the following method:
taking an inhibitor BMS202(BMS) as a model drug, respectively weighing the inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG-DSPE) obtained in example 1, thermosensitive lipid (DPPC) and cholesterol, and the balance of ethanol, dissolving the weighed inhibitor BMS202(BMS), the IR-780 iodide modified lipid graft (IR780-PEG-DSPE), the thermosensitive lipid DPPC and cholesterol in ethanol, and heating in a water bath at 60 ℃ to obtain an ethanol phase solution (in the solution, the mass percent of the inhibitor BMS202 is 20%, the mass percent of the IR-780 iodide modified lipid graft is 15%, the mass percent of the thermosensitive lipid DPPC is 70% and the mass percent of the cholesterol is 5%); weighing an F68 aqueous solution (the concentration of the aqueous solution is 1mg/mL) according to the volume ratio of the F68 aqueous solution to the ethanol phase solution of 10: 1, rapidly dispersing the ethanol phase solution into the F68 aqueous solution at the temperature of 60 ℃, stirring at the rotating speed of 400rpm for 8min, and cooling to room temperature to prepare the thermosensitive lipid delivery system, namely an IR780 modified thermosensitive lipid delivery system (IR780/DPPC/BMS, IR NPs).
Example 11
The embodiment 11 of the invention provides a method for constructing an NIR-responsive biomimetic membrane nanovesicle, which comprises the following steps (tumor cells in the method are exemplified and prepared by 4T1 cells):
collecting4T1 cells (about 1X 10)8Individually), the 4T1 cells were washed three times with PBS buffer and resuspended in 3mL of hypotonic buffer (Tris-HCl 20mM at pH 7.5, KCl 10mM, MgCl 2 mM)2And PMSF mixed solution with the concentration of 1 mM), and repeatedly freezing and thawing for 3 times at-80 ℃ and 37 ℃ respectively to obtain tumor cell fluid after freezing and thawing; carrying out primary centrifugal separation on the frozen and thawed tumor cells for 5min at the temperature of 4 ℃ and the weight of 3200g, and collecting supernatant to obtain primary separated supernatant; performing secondary centrifugal separation on the primary separation supernatant for 20min at the temperature of 4 ℃ and under the condition of 10000g, and collecting the supernatant to obtain secondary separation supernatant; and (3) carrying out centrifugal separation on the secondary separation supernatant for 1h for three times under the conditions of 4 ℃ and 100000g, collecting the separated precipitate, washing the precipitate by using a PBS (phosphate buffer solution) to obtain a cell membrane precipitate, and then carrying out heavy suspension dissolution on the cell membrane precipitate by using the PBS buffer solution to obtain the 4T1 tumor cell membrane. Protein content determination was performed on the collected 4T1 tumor cell membranes using BCA method. The cell membrane solution is stored at-80 ℃ for later use.
The NIR response type bionic membrane nano vesicles (M/IR780/DPPC/BMS, M/IR NPs) are prepared by a filter membrane extrusion method, and the specific method is as follows:
the extracted 4T1 tumor cell membrane (M) was mixed with the thermosensitive lipid delivery system (IR780/DPPC/BMS, IR NPs) obtained in example 6 at a set mass ratio (mass of IR NPs: mass of M protein: 2: 1), extruded by a liposome extruder, and the extruded mixed solution was filtered sequentially through 400nm, 200nm, and 100nm polycarbonate filters to obtain NIR-responsive biomimetic membrane nanovesicles (M/IR780/DPPC/BMS, M/IR NPs).
In order to verify the performance of the IR-780 iodide modified lipid grafts obtained in the present invention, the following tests were performed by taking the NIR responsive biomimetic cell membrane nanovesicles obtained in example 11 as an example:
1) and (3) observing the distribution of the NIR response type bionic membrane nano vesicles with different mass ratios in the body of the 4T1 tumor-bearing mouse by using a small animal living body imager. Fig. 3 shows that after the NIR-responsive biomimetic membrane nanovesicle is injected into tail vein, when the mass ratio of the IR780 heat-sensitive lipid delivery system to the 4T1 tumor cell membrane is 2: 1, the distribution of the NIR-responsive biomimetic membrane nanovesicle at the tumor site is significantly stronger than that of other groups, and when the mass ratio of the IR780 heat-sensitive lipid delivery system to the 4T1 tumor cell membrane is 2.0, the NIR-responsive biomimetic membrane nanovesicle has stronger tumor targeting ability, and the ratio is selected as the preferable coating ratio of the tumor cell membrane to the IR780 heat-sensitive lipid delivery system.
Preparing the same concentration of IR780 modified thermosensitive lipid drug delivery system (IR NPs) and NIR response type bionic membrane nano vesicle (M/IR NPs) (1.0mg/mL) drug-loaded nanoparticle solution, and respectively measuring the particle size and surface potential of the IR NPs and the M/IR NPs by adopting a particle size and surface potential meter. As shown in FIGS. 4 and 5, it can be seen from FIGS. 4 and 5 that the particle diameters of the IR NPs and M/IR NPs were 95.5. + -. 0.6nm and 252.4. + -. 18.3nm, respectively, and the surface potentials were 12.7. + -. 0.46mV and-12.8. + -. 2.08mV, respectively. The result shows that the IR NPs nanoparticles have increased particle size after being wrapped by cell membranes, and the charges are inverted from positive charges to negative charges, which are similar to the charges of the cell membranes.
In addition, taking the drug-loaded nanoparticle solution of IR NPs and M/IR NPs (1.0mg/mL) with the same concentration, diluting the solution to 0.1mg/mL by using deionized water, dripping the solution to a copper net coated with a carbon film, dyeing by using 2% (w/v) uranyl acetate, absorbing the redundant liquid by using filter paper, and respectively observing the form and the particle size of the M/IR NPs by using a transmission electron microscope. The structure of the measurement is shown in FIG. 6, and it can be seen from FIG. 6 that M/IR NPs are spherical, have obvious core-shell structure and have a size of about 200nm, further verifying that cell membranes are successfully wrapped on the surface of the IR NPs.
The tumor cell membrane protein of the M/IR NPs is characterized by adopting gel electrophoresis and a western blot method. The 4T1 cell lysate, 4T1 cell membrane solution and M/IR NPs solution containing equal amount of protein were heated at 100 deg.C for 10 min. The separation was carried out in a Mini-Protean Tetra system using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie blue staining was performed and after overnight destaining imaging was performed. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes for western blot analysis. The membranes were incubated overnight at 4 ℃ with EpCAM antibody (Abcam, A1177), N-cadherin antibody (Immunoway, YM0465) and galectin-3 antibody (Abcam, ab 76245). Horseradish peroxidase (HRP) conjugated anti-mouse/rabbit secondary antibodies were incubated for 1h at room temperature. Finally, protein signals were detected using a ChemiDoc imaging system, and the detection results are shown in fig. 7. As can be seen from FIG. 7A, the protein pattern of M/IR NPs is consistent with that of purified cell membrane, indicating that the membrane protein after the cell membrane is fused with the IR NPs nanoparticle remains good.
Furthermore, western blot analysis results showed the presence of tumor cell membrane biomarkers EpCAM, N-cadherin and galectin-3 on both purified cell membranes and M/IR NPs, as shown in FIG. 7B, indicating that this method preserves specific membrane proteins.
Constructing a 4T1 breast cancer tumor-bearing animal model, and detecting the in vivo diagnostic imaging capability of the multifunctional bionic membrane nano vesicle. To verify whether M/IR NPs could target tumor tissues, we injected M/IR NPs tail vein into 4T1 tumor-bearing mice. A4T 1 tumor-bearing animal model inoculated with 2w tumor cells is taken and randomly divided into two groups, 0.2mL of IR NPs and M/IR NPs solution (BMS dosage is 5mg/kg) are respectively injected into tail vein, 2h, 8h, 24h, 48h and 72h after injection, pentobarbital sodium (10mg/kg) anesthetizes mice, a mouse living body imager observes the time-lapse distribution of the IR NPs or the M/IR NPs in the tumor-bearing animal model and the imaging condition of tumor tissues, takes a fluorescence photo (an optical filter is ICG), and measures the average signal intensity of tumor part carriers. As shown in FIG. 8A, it can be seen from FIG. 8A that the fluorescence signals of M/IR NPs began to be distributed in the tumor region 2h after the injection, while the fluorescence signals of the IR NPs group were not observed at the tumor site. The fluorescence at the tumor sites was significantly enhanced in the M/IR NPs group as compared to the IR NPs group with time (FIGS. 8A and 8B). The M/IR NPs with homologous tumor cell membrane coatings accumulate in tumor tissues more quickly and strongly, and the results show that the M/IR NPs can realize homologous active targeting and are used for diagnostic imaging of tumors.
Measuring the absorption spectrum of the IR NPs or M/IR NPs nano-carrier in a 400-900 nm wave band by adopting an ultraviolet-visible spectrophotometry; the detection result is shown in fig. 9, and it can be known from analysis of fig. 9 that a Near Infrared (NIR) fluorescent probe IR780 has a strong characteristic absorption at a wavelength of 795nm, the IR NPs or M/IR NPs nanocarriers still maintain a strong absorption in the near infrared region after being modified by the IR780, and the maximum absorption peak at 795nm is substantially consistent with the absorption characteristic of the IR780, indicating that the IR NPs or M/IR NPs nanocarriers have a good near infrared absorption characteristic.
Equal volumes of PBS, free IR780/DMSO solution containing IR780 at 5.0. mu.g/mL, IR NPs and aqueous M/IR NPs were placed in an incubator. Irradiating with near infrared laser (1W/cm) with wavelength of 808nm2)3min for each sample, recording the real-time temperature change trend of the M/IR NPs nano-carrier irradiated by the laser by using an infrared thermal imager, and evaluating the photothermal conversion capability of the M/IR NPs; the results are shown in FIG. 10, and it can be seen from an analysis of FIG. 10 that the measured value is 1W/cm2The laser irradiation was continued for 3min, and the temperatures of the free IR780, IR NPs and M/IR NPs solutions were raised to 53.8 deg.C, 54.4 deg.C and 58.8 deg.C, respectively, while the blank PBS group had almost no change in temperature and was set at 35.4 deg.C. The multifunctional bionic membrane nano vesicle M/IR NPs have good photo-thermal conversion capability.
Drug responsive release study
To determine whether near-infrared laser irradiation-induced photothermal conversion could trigger effective drug release, the drug release behavior of M/IR NPs at different time points was further determined.
Transferring a certain volume of the IR NPs and M/IR NPs drug-loaded nanoparticle solution, diluting the solution to 1.0mL by deionized water, and placing the solution in a dialysis bag (MWCO 3.5 kDa). 20mL of PBS with pH 6.8 was used as release medium, and samples were periodically taken at 0.5h, 1h, 2h, and 4h with constant temperature shaking (37 ℃, 75rpm), and fresh release medium was replaced. At 4h, the mixture was irradiated with laser (808nm, 1W/cm)2) After 3min, the sampling was continued at regular time and the fresh release medium was replaced. The concentration of each group of drug BMS is measured by HPLC, and the BMS cumulative release amount and the cumulative release percentage of the drug-loaded nanoparticles before and after laser irradiation are calculated according to a standard curve, and the specific result is shown in FIG. 11.
As can be seen in FIG. 11, the drug release from both M/IR NPs and IR NPs was slower at the beginning of 4 h. However, when the M/IR NPs or IR NPs solution is subjected to a 808nm laser (1W/cm)23min), the release rate of BMS was faster, and at 72h, the cumulative release rates of M/IR NPs and IR NPs reached 77.1% and 80.8%, respectively. The cumulative release efficiency of M/IR NPs and IR NPs is not significantThe difference indicates that the wrapping of the cell membrane has no influence on the drug release. In the absence of laser irradiation, the M/IR NPs and the IR NPs showed slow release behavior throughout the release process, with 72h cumulative release rates of 45.9% and 49.1%, respectively.
The morphological changes of the M/IR NPs and the IR NPs at different temperatures (37 ℃ and 56 ℃) are observed by adopting a transmission electron microscope, the specific result is shown in FIG. 12, and as can be seen from FIG. 12, when the temperature is 37 ℃, the M/IR NPs and the IR NPs are in regular spherical shapes and have the sizes of about 200nm and 100nm respectively; when the temperature is raised to 56 ℃, the membrane structure of the surface of the M/IR NPs starts to expand and crack, and the size of the M/IR NPs is obviously increased to about 400 nm; while the IR NPs structure starts to disintegrate and melt into small particles with a particle size of less than 10 nm.
Evaluation of in vivo antitumor efficacy of multifunctional biomimetic membrane nanovesicle
The invention takes a 4T1 tumor-bearing mouse as an animal model to evaluate the in vivo anti-tumor curative effect of the multifunctional bionic membrane nano vesicle M/IR NPs. Taking 4T1 breast cancer with volume of 200mm3The left and right tumor-bearing mice were 25, randomly divided into 5 groups (n ═ 5), and the group 1 without laser irradiation: saline, laser irradiation group 4 group: physiological saline, BMS, IR NPs, M/IR NPs, (BMS dose was 5 mg/kg). The first administration is 1d, the last day tail vein administration is 200 μ L/d, and the laser irradiation group performs laser irradiation on tumor region for 3min (808nm, 0.5W/cm) after each administration for 1d2) The group without laser irradiation was not treated, and the treatment was performed 5 times and 5 times with laser irradiation. Within the treatment period, the tumor size of each group of nude mice was measured with a digital vernier caliper every 1d, and the tumor volume was calculated and the body weight was recorded, referring to formula (1).
Tumor volume V (mm)3)＝a×b2/2 (1)
In the formula: a is the major diameter of the tumor and b is the minor diameter of the tumor
As shown in FIG. 13, the tumor volume continued to increase in the saline group, while the tumor volume was significantly reduced after laser irradiation with M/IR NPs; compared with BMS Laser (+) and IR NPs Laser (+) treatment groups, the M/IR NPs treatment group with the same dosage is most effective in inhibiting tumor growth under Laser irradiation, tumor tissues of tumor-bearing mice are dissected and weighed after the treatment period is finished, and as shown in figure 14, the tumor weight of the M/IR NPs Laser irradiation group is the smallest, and the anti-tumor effect is most obvious.
In vivo anti-tumor metastasis research of multifunctional bionic membrane nano vesicle
Treatment of both primary tumors and tumor metastases is important for cancer patients, but inhibition of tumor metastasis is more challenging. The invention further evaluates the potential of the multifunctional bionic membrane nano vesicle (M/IR NPs) induced immune-photothermal combination therapy for metastatic tumors. Injecting 100 mu L of 4T1 cell suspension (containing 1X 10 cells) into BALB/c female mice at 4-6 weeks and right mammary fat pad6Individual cells), a 4T1 in situ breast cancer metastasis model was established. And (3) continuously culturing tumor-bearing mice, taking the tumor-bearing mice with different volumes of primary tumors, dissecting lung tissues, and observing the lung metastasis condition by HE (high-intensity eosin) staining. When tumor volume reached the requirement, the mouse model was treated with the above dosing method (n-5).
After 21 days of the treatment cycle, different groups of lung tissue were collected and analyzed for the number of lung metastases from each treatment group (fig. 15A and 15B). As can be seen from FIGS. 15A and 15B, the mean number of lung metastasis nodules was reduced by 52.3%, 67.1% and 91.9% in the BMS-, IR NPs-and M/IR NPs-treated groups, respectively, when irradiated with laser light, as compared to the saline-treated group. The transfer inhibition rate of the M/IR NPs Laser irradiation treatment group is significantly higher than that of the BMS Laser (+) and IR NPs Laser (+) treatment groups. These results indicate that M/IR NPs with combined immune-photothermal therapy and homologous targeting ability are more effective in inhibiting the formation of micrometastases than either immunotherapy or photothermal therapy alone.
T cell infiltration of tumor microenvironment
The anti-tumor immune response is mainly dependent on activated T cells. Among them, CD8+After T cells are activated, the T cells are usually differentiated into cytotoxic T Cells (CTLs) and can specifically kill tumor cells. After the administration is finished, taking tumor tissues of each group of model animals, grinding by using a cell homogenizer, preparing single cell suspension, and detecting CD8 in the tumor tissues+Proportion of T cells. Adopting fluorescence labeled antibodies APC anti-mouse CD3 epsilon and PerCP/cyanine5.5 anti-mouse CD8a to label cells, incubating for 20min at 4 ℃, washing for 3 times by PBS, analyzing a tumor group by a flow cytometerCD8 in weaving+Percentage of T cells.
As a result, as shown in FIG. 16, it can be seen from the data in FIG. 16 that the M/IR NPs laser irradiation group CD3+CD8+The percentage of T cells was 71.2%, which was 2.70 times that of the saline laser group, suggesting that CTLs efficiently penetrated the tumor tissue. Notably, the M/IR NPs laser irradiated group tumor tissue infiltrated CD8+The proportion of T cells is 1.30 times of that of the IR NPs laser irradiation group, which shows that the photothermal immune combined preparation M/IR NPs with the homologous targeting capability can enhance the immune response of T lymphocytes and make 'cold' tumors more sensitive to immunotherapy.
Cytokine detection
CD8+T cells produce the cytokine interferon-gamma (IFN- γ), which is a key indicator of a helper T cell type 1(Th1) immune response that effectively suppresses tumor growth and progression. Further, the level of secreted IFN-. gamma.in the primary tumor was measured by flow cytometry and immunofluorescence staining, and the maximum amount of secreted IFN-. gamma.was found in the M/IR NPs Laser (+) treated group, as shown in FIG. 17.
Therefore, M/IR NPs-induced immune photothermal therapy is combined with the advantages of homologous targeting and NIR response release, so that the sensitivity of cold tumors to anti-PD immunotherapy is increased, the systemic immune response is enhanced, and the tumor growth and metastasis are effectively inhibited.
In conclusion, the IR-780 iodide modified lipid graft provided by the invention not only lays a foundation for preparing a thermosensitive lipid drug delivery system and an NIR response type bionic membrane nano vesicle, but also realizes long-term reversion of tumor immunosuppression state by blocking a PD-1/PD-L1 pathway, exerts an antitumor immune response synergistically, inhibits the growth of primary tumors and metastatic foci, and further effectively realizes the purposes of improving the tumor treatment effect and reducing the tumor recurrence and metastasis rate.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.