CN113855646A

CN113855646A - Response type nano platform for loading phosphorus-containing dendrimer copper complex/toyocamycin and bionic cell membrane as well as preparation and application of response type nano platform

Info

Publication number: CN113855646A
Application number: CN202110949821.XA
Authority: CN
Inventors: 沈明武; 郭云琦; 范钰; 王志强; 詹梦偲; 史向阳
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2021-12-31
Anticipated expiration: 2041-08-18
Also published as: CN113855646B

Abstract

The invention relates to a response type nano platform for loading a phosphorus-containing dendrimer copper complex/toyocamycin and simulating cell membranes, and a preparation method and application thereof. The method comprises the following steps: 1G₃Preparation of Cu NPs solution, 1G₃Preparation of Cu/Toy NPs, preparation of B16 cell membrane suspension CCM, 1G₃-Cu/Toy NPs @ CCM. The method has simple reaction conditions, is easy to operate and separate, and has good development prospect; the prepared reduction response type double-drug-loaded nano platform effectively improves 1G₃The water solubility of Cu is reduced, the toxic and side effect of Toy is reduced, and 1G can be released in response to dissociation of a tumor microenvironment₃Cu and Toy, which provides a new idea for constructing safe, intelligent and efficient drug carriers.

Description

Response type nano platform for loading phosphorus-containing dendrimer copper complex/toyocamycin and bionic cell membrane as well as preparation and application of response type nano platform

Technical Field

The invention belongs to the field of responsive drug-loaded nano-platforms and preparation and application thereof, and particularly relates to a responsive nano-platform loaded with a phosphorus-containing dendrimer copper complex/toyocamycin and a preparation method and application thereof.

Background

Malignant tumor has become the main killer threatening human life and health at present due to its characteristics of fast growth speed, strong metastatic ability, high recurrence rate and the like. Chemotherapy is the most prominent and effective clinical treatment for cancers at intermediate and advanced stages and with high risk of metastasis and recurrence. However, the therapeutic effect of chemotherapy is limited by the problems of poor water solubility, low bioavailability, fast drug metabolism, great toxic and side effects and the like. In order to improve the treatment efficiency of the chemotherapeutic drugs, a tumor microenvironment responsive nano platform can be constructed for loading the chemotherapeutic drugs, delivering the drugs to tumor parts in a targeted manner and releasing the drugs in a responsive manner, so that the toxic and side effects on normal tissues are reduced, and the chemotherapeutic treatment effect is improved. The nano micelle formed by the amphiphilic polymer containing the sensitive bond has a unique core-shell structure, ideal drug loading capacity, enhanced EPR effect and enhanced tumor microenvironment stimulus responsiveness, can integrate hydrophobic drugs and hydrophilic drugs, and realizes the controllable release of the drugs, so that the nano micelle is widely used for constructing tumor diagnosis and treatment integrated nano platforms (Li et al. biomaterials,2020,232,119749).

The phosphorus-containing dendrimer has the same precise molecular structure as the protein and better clinical transformation potential, and has been researched to be used as a gene vector, an antiviral agent and a metal ion vector. The third generation of phosphorus-containing dendrimer has unique skeleton structure and surface chemical properties, and copper ions can be chelated at the tail end of the phosphorus-containing dendrimer through ligand modification to prepare a third generation of phosphorus-containing dendrimer copper complex (1G)₃-Cu). Literature reports before this group (Fan et al Nano Today,2020,33,100899), 1G₃the-Cu has good stability and better T₁Relaxation property, can effectively inhibit pancreatic cancer cell proliferation and induce cell apoptosis, and can be used for in vivo anti-tumor and MR imaging. However, since 1G₃Cu has the defects of poor water solubility, insufficient tumor targeting and the like, so that the application of Cu in tumor diagnosis and treatment is limited.

Toyocamycin (Toy) is a chemotherapeutic drug that acts on the endoplasmic reticulum and inhibits the adaptive regulation of Endoplasmic Reticulum Stress (ERS). In cancer cells, persistent ERS occurs intracellularly due to abnormalities in transcription and metabolism, and excessive cell proliferation (Chen et al Nat. Rev. cancer,2020,21(2), 71-88). During ERS development in cancer cells, chaperone GRP78 is shed from IRE1 α, one of the ERS receptors, dimerizes and autophosphorylates IRE1 α, activating the RNase domain of IRE1 α, which allows splicing of the downstream XBP1 mRNA to produce XBP1-splicing (XBP1s) mRNA. XBP1s mRNA encodes the activated protein XBP1s that restores and maintains endoplasmic reticulum homeostasis to some extent, allowing cancer cells to adapt to persistent ERS and survive (Yoshida et al cell,2001,107(7), 881-. Toy can act on IRE1 alpha-XBP 1 signal channel, and inhibit splicing of IRE1 alpha endoribonuclease to XBP1 mRNA, thereby inhibiting adaptive regulation of ERS and finally inducing apoptosis (Ri et al. Toy has no selectivity to tumor cells, and has great toxic and side effects when used for in vivo treatment.

To exert 1G to the maximum extent₃The efficacy of Cu and Toy, 1G, can be prepared by using amphiphilic polymers containing sensitive bonds₃Integration of-Cu and Toy to construct tumor microenvironment responsive nano platform and improve 1G₃The water solubility of Cu and the selectivity of Toy realize the enhanced integration of tumor diagnosis and treatment. In order to enhance the active targeting property of the nano platform, the nano platform can be further modified. In recent years, the nano platform with biomimetic camouflage of cancer cell membranes has been widely applied to the integration of diagnosis and treatment of tumors due to its excellent biocompatibility, prolonged circulation time in vivo, and ability to actively target to tumor sites and avoid capture and removal of the monocyte macrophage system and reticuloendothelial system. For example, Jia et al prepared B16 cell membrane-wrapped ultra-small ferroferric oxide nanoclusters for targeting delivery of anticancer drug adriamycin (DOX) to tumor sites, thereby realizing enhanced antitumor effect and bimodal T₂/T₁MR imaging (Jia et al nano Today,2021,36, 101022).

In addition, chemotherapy can induce Immunogenic death (ICD) of Tumor cells, release Tumor-associated antigens (Tumor-associated antigens) and damage-associated molecular patterns (DAMPs), stimulate Dendritic Cell (DCs) maturation and presentation of associated antigens to T cells, which can migrate to Tumor tissue and ultimately elicit an antigen-specific immune response. However, tumor cells may highly express immune checkpoint molecules (e.g., PD-L1), inhibiting activation of T cells and thereby evading monitoring and clearance by the immune system. The recognition ability of T cells to tumor cells can be improved by Immune Checkpoint Blockade (ICB) therapy by using a specific immune preparation (such as PD-L1 antibody), and the killing effect of an immune system to the tumor cells can be restored. Thus, combining chemotherapy with ICB therapy results in a sustained immune response that inhibits tumor growth, recurrence or metastasis.

The retrieval of relevant documents and patent results at home and abroad shows that: no simultaneous loading of 1G with PEG-SS-PCL as a carrier has been found₃-Cu and Toy, and coating B16 cell membrane, and combined with PD-L1 antibodyThe related report of the application of the composition in tumor chemotherapy/immunotherapy.

Disclosure of Invention

The invention aims to solve the technical problem of providing a response type nano platform for loading a phosphorus-containing dendrimer copper complex/toyocamycin and a preparation method and application thereof, which are bionic by cell membranes, so as to fill the blank in the prior art.

The invention provides a response type nano platform for loading a phosphorus-containing dendrimer copper complex/toyocamycin with bionic cell membranes, which utilizes amphiphilic polymer PEG-SS-PCL to react with a third-generation phosphorus-containing dendrimer copper complex 1G₃Cu is encapsulated at a hydrophobic end, and toyocamycin Toy is loaded at a hydrophilic end through hydrogen bonding, and then the membrane is coated on a melanoma B16 cell membrane.

The invention also provides a preparation method of the response type nano platform for loading the phosphorus-containing dendrimer copper complex/toyocamycin and simulating the cell membranes, which comprises the following steps:

(1) the third generation of phosphorus-containing dendrimer copper complex 1G₃dissolving-Cu and PEG-SS-PCL in solvent, adding into ultrapure water dropwise under ultrasonic condition, stirring for reaction, dialyzing, filtering, centrifuging to obtain 1G₃-Cu NPs solution;

(2) dissolving toyocamycin Toy in ultrapure water, and adding to 1G in step (1)₃Stirring the solution in-Cu NPs for reaction, and centrifuging to remove unencapsulated Toy to obtain 1G₃-Cu/Toy NPs；

(3) Adding the cell lysis mixed solution into a melanoma B16 cell precipitate, carrying out ice bath, repeatedly freezing and thawing to break cells, centrifuging to obtain a precipitate as a B16 cell membrane, and then re-suspending in a PBS solution to obtain a B16 cell membrane suspension CCM;

(4) subjecting 1G in step (2)₃mixing-Cu/Toy NPs with the B16 cell membrane suspension CCM in the step (3), extruding and centrifuging to obtain 1G₃-Cu/Toy NPs @ CCM, namely a response type nano platform for loading a phosphorus-containing dendrimer copper complex/toyocamycin and simulating cell membranes.

Preferably, in the above method, 1G in the step (1)₃The molar ratio of-Cu to PEG-SS-PCL is 1: 3-1: 8.

Preferably, in the method, the volume ratio of the solvent to the ultrapure water in the step (1) is 1: 8-1: 12; the solvent is dimethyl sulfoxide DMSO.

Preferably, in the method, the stirring reaction temperature in the step (1) is room temperature, and the stirring reaction time is 20-30 hours.

Preferably, in the above method, the dialysis in step (1) is: dialyzing in ultrapure water for three days by using a dialysis bag with the molecular weight cutoff of 8000-14000 Da; the filtration was carried out using a microfiltration membrane having a pore size of 1 μm.

Preferably, in the above method, the centrifugation in steps (1) and (2) is ultrafiltration centrifugation, and an ultrafiltration centrifugal tube with a molecular weight cut-off of 3500Da is used for centrifugation at 4500rpm for 30 min.

Preferably, in the above method, toyocamycin Toy and 1G in step (2)₃The mass ratio of-Cu NPs is 1: 1-1: 5.

Preferably, in the above method, the stirring reaction temperature in the step (2) is room temperature, and the stirring reaction time is 20-30 hours.

Preferably, in the above method, the mixed solution of cell lysis in step (3) is a mixed solution of PMSF and hypotonic cell lysis solution, and the volume ratio of PMSF to hypotonic cell lysis solution is 1: 90 to 110.

Preferably, in the above method, the ratio of the melanoma B16 cell sediment to the cell lysis mixture in step (3) is 1 × 10⁷The method comprises the following steps: 2-4 mL.

Preferably, in the method, the ice bath time in the step (3) is 10-20 min; the technological parameters of repeated freeze thawing are as follows: freezing at-20 deg.C, thawing at 37 deg.C, and repeating for 3 times.

Preferably, in the above method, the centrifugation in step (3) is gradient centrifugation, and the gradient centrifugation parameters are: the centrifugation temperature is 4 ℃, the centrifugation is firstly carried out for 10min at 700g of centrifugal force, the sedimentation is removed, then, the centrifugation is carried out for 30min at 14000g of centrifugal force, the supernatant is removed, and the sedimentation is resuspended in PBS solution.

Preferably, in the above method, 1G in the step (4)₃The proportion of the-Cu/Toy NPs to the B16 cell membrane suspension is 190-210 mu g: 0.4-0.6 mL.

Preferably, in the above method, the extruding in the step (4) is repeated 10-15 times by using an Avanti micro extruder with a filter membrane pore size of 400 nm.

Preferably, in the above method, the centrifugation parameters in step (4) are: the centrifugation temperature was 4 ℃ and centrifugation was carried out at 10000rpm for 6 min.

The invention also provides application of the response type nano platform loaded with the phosphorus-containing dendrimer copper complex/toyocamycin in cell membrane biomimetic preparation of tumor diagnosis and treatment agents for combined treatment of MR imaging, chemotherapy and immunotherapy.

The invention utilizes amphiphilic polymer PEG-SS-PCL with reduction responsiveness to prepare the third generation phosphorus-containing dendrimer copper complex 1G with anti-tumor activity and MR imaging performance₃The Cu is encapsulated at the hydrophobic end, Toy is loaded at the hydrophilic end through hydrogen bond action, a melanoma B16 cell membrane is further coated on the surface of the nano platform in a physical extrusion mode to obtain the tumor microenvironment response type double-drug-loaded nano platform which is used for accurate diagnosis and treatment integration of tumors, and the PD-L1 antibody is combined in vivo to realize combined treatment of tumor chemotherapy and immunotherapy.

The invention integrates 1G with PEG-SS-PCL₃-Cu and Toy, improvement 1G₃The water solubility of Cu reduces Toy toxic side effects, induces cancer cell apoptosis, and realizes enhanced chemotherapy and MR imaging. And simultaneously, the PD-L1 antibody is combined in vivo to realize the combined treatment of tumor chemotherapy/immunotherapy.

The physical and chemical properties of the prepared cell membrane bionic reduction response type double-drug-loaded nano platform are represented by Zeta potential and dynamic light scattering analysis (DLS), ultraviolet visible absorption spectrum (UV-vis), Transmission Electron Microscope (TEM), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), nuclear magnetic resonance imaging analyzer and other means. Then, 1G was analyzed and evaluated by the CCK-8 method₃-cytotoxicity of Cu/Toy NPs @ CCM and related control materials; the phagocytosis of the material by different cells is detected by ICP-OES, and 1G is determined₃-immune evasion and homology targeting ability of Cu/Toy NPs @ CCM; detecting the influence of the material on the intracellular GSH level by using a GSH and GSSG detection kit; evaluation by flow cytometryEffect of valence material on intracellular ROS levels; evaluating the influence of the material on the expression of endoplasmic reticulum stress related factors GRP78, p-IRE1 alpha, XBP1, XBP1s and CHOP at protein level by Western blot; evaluating the influence of the material on the mitochondrial membrane potential in the cell by using a laser confocal microscope; evaluating the influence of the material on the expression conditions of apoptosis-related proteins Bax, Bcl-2, P53 and PTEN by Western blot Western blotting; evaluating the influence of the material on the expression condition of the CRT in the cell by using a laser confocal microscope; and finally, establishing a black mouse subcutaneous tumor model for an anti-tumor experiment.

Advantageous effects

(1) The method has simple reaction conditions, is easy to operate and separate, and has good development prospect.

(2) The reduction response type double-drug-loaded nano platform prepared by the invention effectively improves 1G₃The water solubility of Cu is reduced, the toxic and side effect of Toy is reduced, and 1G can be released in response to dissociation of a tumor microenvironment₃Cu and Toy, which provides a new idea for constructing safe, intelligent and efficient drug carriers.

(3) The nano platform prepared by the invention can act on mitochondria to cause the mitochondria to have abnormal functions and induce cancer cells to die through the mitochondria. On the other hand, the nano platform can inhibit the cancer cell endoplasmic reticulum stress from recovering to the steady state, and can enable the cancer cell to generate oxidative stress and aggravate the endoplasmic reticulum stress, so that the nano platform and Toy form a synergistic effect to promote the cancer cell to die through the endoplasmic reticulum. Meanwhile, the related antigen released by the apoptotic cancer cells can also cause immunogenic death, enhancing the therapeutic effect.

(4) After the nano platform prepared by the invention enters a mouse body through tail vein injection, the nano platform can be used for anti-tumor treatment through chemotherapy and immunogenic death caused by the chemotherapy, and can also be used for carrying 1G₃-Cu realizes T₁And (4) MR imaging. Can realize chemotherapy/immunotherapy combined treatment after being combined with the PD-L1 antibody, has enhanced anti-tumor effect, can generate immunological memory effect, and has potential clinical application value.

Drawings

FIG. 1 is 1G prepared in the present invention₃-Cu/Toy NPs @ CCM synthesis and application schematic diagram;

FIG. 2 shows 1G in example 1₃-Cu、1G₃-Cu NPs、1G₃-Cu/Toy NPs、1G₃-Cu/Toy NPs @ CCM and 1G in comparative example 2₃-hydrated particle size (a) and surface potential (b) of Cu/Toy NPs @ RBCM as measured by a nano-particle sizer;

FIG. 3 is 1G prepared in example 1₃-graph of hydrodynamic diameter over time of Cu/Toy NPs @ CCM in water, PBS and 1640 medium;

FIG. 4 is 1G prepared in example 1₃-Cu/Toy NPs (a) and 1G₃-TEM images of Cu/Toy NPs @ ccm (b);

FIG. 5 is 1G prepared in example 1₃-Cu/Toy NPs、CCM、1G₃-Cu/Toy NPs @ CCM (a) and RBCM, 1G prepared in comparative example 2₃-SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of Cu/Toy NPs @ RBCM (b);

FIG. 6 is 1G prepared in example 1₃-Cu/Toy NPs and 1G₃-the drug release kinetics profile of Cu/Toy NPs @ CCM under different conditions;

FIG. 7 shows 1G at different copper concentrations₃-Cu/Toy NPs@CCM、1G₃-Cu/Toy NPs @ CCM + GSH and CuCl₂T of₁MR imaging plots (a) and T₁A linear graph (b) of the inverse of the relaxation time as a function of Cu concentration;

FIG. 8 is 1G prepared in example 1₃-Cu/Toy NPs、1G₃-cell motility profile after 24h incubation of Cu/Toy NPs @ CCM with B16 cells;

FIG. 9 is 1G prepared in example 1₃-Cu/Toy NPs、1G₃-Cu/Toy NPs @ CCM and 1G prepared in comparative example 2₃-intracellular copper content profile after incubation of Cu/Toy NPs @ RBCM with B16 cells and RAW264.7 cells, respectively, for 6 h;

FIG. 10 shows Toy, PEG-PCL, PEG-SS-PCL, and 1G₃-Cu、Insensitive 1G₃-Cu NPs、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃Intracellular GSH levels after 6h of co-incubation of Cu/Toy NPs @ CCM with B16 cellsAnalyzing the graph;

FIG. 11 shows 1G₃-Cu、PEG-PCL、Insensitive 1G₃-Cu NPs (a-b) and Toy, PEG-SS-PCL, 1G₃-Cu NPs、1G₃-Cu/Toy NPs、1G₃-flow cytometric and quantitative cytometric profiles of intracellular ROS levels after 6h of co-incubation of Cu/Toy NPs @ CCM (c-d) with B16 cells;

FIG. 12 shows Toy and 1G₃-Cu NPs、1G₃-Cu/Toy NPs、1G₃-a graph of the results of Western blot assay on GRP78, p-IRE1 α, XBP1, XBP1s and CHOP protein in cells after 24h of co-incubation of Cu/Toy NPs @ CCM with B16 cells, wherein (a) is a protein band diagram of Western blot and (B-f) is a grayscale quantification graph of each protein band;

FIG. 13 shows PEG-SS-PCL, Toy, 1G₃-Cu、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-confocal laser microscopy analysis of JC-1 probe fluorescence change after 6h incubation of Cu/Toy NPs @ CCM with B16 cells;

FIG. 14 shows PEG-SS-PCL, Toy, 1G₃-Cu、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-graph of intracellular JC-1 red/green fluorescence ratio quantitation after 6h incubation of Cu/Toy NPs @ CCM with B16 cells;

FIG. 15 shows PEG-SS-PCL, Toy, 1G₃-Cu、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-a result graph of Western blot test on Bax, Bcl-2, P53 and PTEN proteins in cells after the Cu/Toy NPs @ CCM and the B16 cells are co-incubated for 24h, wherein (a) is a protein band diagram of the Western blot, and (B-e) is a gray scale quantitative graph of each protein band;

FIG. 16 shows PEG-SS-PCL, Toy, 1G₃-Cu、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-confocal laser microscopy analysis of CRT expression in cells after 24h incubation of Cu/Toy NPs @ CCM with B16 cells;

FIG. 17 shows PEG-SS-PCL, Toy, 1G₃-Cu、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-quantitative analysis of HMGB-1 content in cell culture broth after 24h co-incubation of Cu/Toy NPs @ CCM with B16 cells;

FIG. 18 shows an example 15Through the tail vein injection of PBS, PEG-SS-PCL, Toy, 1G₃-Cu NPs、1G₃-Cu/Toy NPs、1G₃-Cu/Toy NPs @ RBCM and 1G₃-recording a graph of relative tumor volume change (a) and mouse relative body weight change (b) over 14 days after Cu/Toy NPs @ CCM;

FIG. 19 shows the results of the analysis of example 16 in PBS, Anti-PD-L1, 1G₃-Cu/Toy NPs @ CCM and 1G₃-recording a graph of relative tumor volume change (a) and mouse relative body weight change (b) over 14 days after Cu/Toy NPs @ CCM + Anti-PD-L1 treatment;

FIG. 20 is a graph of flow cytometric analysis of the typing of CD4+/CD8+ T cells in tumor tissue after 14 days of treatment in each experimental group in example 16;

FIG. 21 is a graph of flow cytometric analysis of the typing of CD44+/CD62L + T cells in the spleen after 9 days of treatment in each experimental group in example 17.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Unless otherwise specified, all chemical reagents were used without further purification. Third generation phosphorus-containing dendrimer copper Complex 1G₃Cu from the group of professor topics of national center for science research, france, j.p. PEG-SS-PCL and PEG-PCL were purchased from Siemens Rexi Biotech, Inc. (SiEn, China). Toy was purchased from Shanghai Wei atlas Biotech, Inc. (Shanghai, China). B16 cells (mouse melanoma cell line) and RAW264.7 cells (mouse macrophage line) were from the institute of biochemistry and cell biology, chinese academy of sciences. RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin double antibody and trypsin were purchased from Hangzhou Gino biomedical technologies, Inc. (Hangzhou, China). BCA assay kit, PMSF, cell lysate, GSH and GSSH detection kit, ROS detection probe (DCFH-DA), JC-1 detection kit and Anti-CRT purchased from BzunyanTech Ltd (Shanghai, China). Cell Counting Kit-8(CCK-8) was purchased from Shanghai seven sea Biotechnology, Inc. (Shanghai, China). The ELISA kit for HMGB-1 was purchased from Shanghai Ming color Biotech Co., Ltd (Shanghai, China). C57BL/6 black mice were purchased from Shanghai Slek laboratory animal center (Shanghai, China). Anti-CD8-PE, Anti-CD4-FITC, Anti-CD44-FITC, Anti-CD62L-APC were purchased from Thermo Fisher Scientific (Waltham, MA).

Example 1

(1) 1.3mg of the third generation phosphorus-containing dendritic copper complex 1G₃Cu and 1.0mg PEG-SS-PCL were dissolved in 500. mu.L DMSO, and then the solution was added dropwise to 5mL of ultrapure water under ultrasonic conditions, and the reaction was stirred for 24 hours. Dialyzing with dialysis bag with molecular weight cut-off of 8000-14000Da in ultrapure water for 3 days to remove DMSO and excessive PEG-SS-PCL, and filtering the product with microporous membrane with pore diameter of 1 μm to remove unsupported 1G₃-Cu, and finally centrifuging at 4500rpm for 30min in an ultrafiltration centrifuge tube with molecular weight cutoff of 3500Da to obtain 1G₃-Cu NPs. Product 1G by ICP₃-Cu content in Cu NPs, calculating 1G₃The upper loading rate of Cu was 99.2 wt%, and the encapsulation rate was 57.6 wt%.

(2) 0.5mg of toyocamycin Toy was dissolved in 1mL of ultrapure water, and then added to 1mL of 1G₃adding-Cu NPs solution (1.5mg/mL), stirring and mixing for 24h, centrifuging at 4500rpm for 30min in an ultrafiltration centrifuge tube with molecular weight cutoff of 3500Da to remove unloaded Toy to obtain 1G₃-Cu/Toy NPs. The amount of unloaded Toy was calculated by measuring the absorbance at 279nm of the liquid outside the ultrafiltration tube by UV, and thus Toy was calculated as 39.4 wt% and the encapsulation efficiency was 11.6 wt%.

(3) Mixing 30 μ L PMSF with 3mL hypotonic cell lysate, collecting 1 × 10 cells of B16 in logarithmic growth phase⁷And centrifuging at 1000rpm for 5min to obtain cell precipitate, adding mixed hypotonic cell lysate into the cell precipitate, performing ice bath for 15min, and repeatedly freezing and thawing at (-20 deg.C, 37 deg.C, and repeating for three times). Setting the centrifugal temperature at 4 ℃, centrifuging for 10min at 700g of centrifugal force, and removing precipitates; centrifuging at 14000g centrifugal force for 30min, and removingSupernatant, and resuspension of the pellet in 1mL PBS solution, B16 cell membrane suspension (CCM) can be obtained.

(4) 200. mu.g of 1G₃-Cu/Toy NPs were mixed with 0.5mL CCM, the solution was extruded 11 times using an Avanti micro-extruder, centrifuged at 10000rpm for 6min to remove excess cell membranes and 1G was obtained₃-Cu/Toy NPs@CCM。

Example 2

Taking appropriate amount of 1G₃Cu, dissolved completely in DMSO and then added dropwise to ultrapure water to make 1G of 4mM copper₃Cu solution (1% DMSO, ultrapure water as solvent); and the appropriate amount of 1G prepared in example 1 was taken₃-Cu NPs、1G₃-Cu/Toy NPs、1G₃-Cu/Toy NPs @ CCM and 1G prepared in comparative example 2₃-Cu/Toy NPs @ RBCM, diluted with ultrapure water, formulated into a 1mg/mL solution for determination of hydrated particle size and surface potential. The results are shown in FIG. 2, 1G₃The hydrated particle size of-Cu is 185.8nm, the hydrated particle size is increased to 230.4nm after micelle formation under the action of PEG-SS-PCL, the potential is changed to-3.02 mV, and the change of the hydrated particle size and the potential proves that 1G is generated₃Successful synthesis of Cu NPs. When 1G₃After loading of the Cu NPs with the chemotherapeutic drug Toy, the hydrated particle size is increased to 238.4nm, and the potential is further reduced to-6.81 mV, which proves the successful loading of Toy. 1G after coating B16 cell membrane or erythrocyte membrane by physical compression₃-Cu/Toy NPs @ CCM and 1G₃The hydrated particle size of the-Cu/Toy NPs @ RBCM was increased to 280.6nm and 275.5nm, respectively, and the potential was decreased to-16.1 mV and-17.2 mV, respectively, demonstrating that 1G₃Successful coating of cell membranes on the surface of Cu/Toy NPs. 1G₃The hydrated particle size of-Cu/Toy NPs @ CCM in various solutions (water, PBS, 1640 medium) was almost unchanged (see FIG. 3), demonstrating that 1G₃the-Cu/Toy NPs @ CCM has good colloidal stability.

Example 3

1G prepared in example 1 was taken₃-Cu/Toy NPs and 1G₃Characterization of dimensions and morphology was performed by Cu/Toy NPs @ CCM. 1G₃TEM image of-Cu/Toy NPs is shown in FIG. 4(a), 1G₃the-Cu/Toy NPs are in the form of uniform spheres with a size of about 185 nm. 1G prepared as shown in FIG. 4(b)₃the-Cu/Toy NPs @ CCM size was about 220nm and the thickness of the coated cell membrane was about 25 nm.

Example 4

1G prepared in example 1 was taken₃-Cu/Toy NPs、CCM、1G₃-Cu/Toy NPs @ CCM and RBCM, 1G prepared in comparative example 2₃Cu/Toy NPs @ RBCM was characterized by SDS-PAGE to confirm cell membrane coating and retention of cell membrane proteins. Determination of CCM, 1G by BCA protein quantification kit₃-Cu/Toy NPs @ CCM, RBCM and 1G₃-protein content in Cu/Toy NPs @ RBCM, and adjusting the protein content in each sample to 0.8mg/mL with PBS solution. Add 5. mu.L of protein Marker to the first protein lane, and add 15. mu.L of 1G₃Cu/Toy NPs (concentration 1G)₃1G corresponding to a protein concentration of 0.8mg/mL in Cu/Toy NPs @ CCM₃Concentration of-Cu/Toy NPs), CCM and 1G₃-Cu/Toy NPs @ CCM was added to the protein lanes at a current of 100A for a time of 30 min. As shown in FIG. 5(a), 1G₃the-Cu/Toy NPs group did not run out of the protein band, whereas 1G₃the-Cu/Toy NPs @ CCM group and the CCM group run out similar protein bands, and the B16 cell membrane is proved to be in 1G₃Successful coating of the Cu/Toy NPs surface and retention of B16 cell membrane proteins. Similarly, protein Marker, RBCM and 1G₃SDS-PAGE experiments in lanes of-Cu/Toy NPs @ RBCM-added protein, as shown in FIG. 5(b), 1G₃Similar protein bands were run out from the-Cu/Toy NPs @ RBCM group and the RBCM group, demonstrating that the erythrocyte membrane is at 1G₃Successful coating of the Cu/Toy NPs surface and preservation of erythrocyte membrane proteins.

Example 5

Buffer solutions with pH 7.4 and pH 7.4(GSH concentration 10mM) were prepared for analysis of 1G, respectively₃-Cu/Toy NPs and 1G₃-Toy responsive release behavior in Cu/Toy NPs @ CCM. 1G to be prepared₃the-Cu/Toy NPs were dissolved in 1mg/mL of the above different buffer solutions with 1mL and placed in dialysis bags (molecular cut-off of 3500Da), and the prepared 1G was used₃-Cu/Toy NPs @ CCM was dissolved to a solution of 1mg/mL with 1mL of a buffer solution having pH 7.4(GSH concentration 10mM) and placed in a dialysis bag (molecular cut-off 3500Da), which was then placed in a dialysis bag containing 9mL of different buffer solutionsIn a container, the mixture was shaken in a constant temperature shaker at 37 ℃. At different time points, 1mL of the dialysate bag external solution was aspirated, 1mL of the corresponding buffer solution was replenished into the container, and the absorbance at 279nm of the withdrawn solution was measured. After the sustained release is finished, 1G is drawn₃-Cu/Toy NPs and 1G₃-Cu/Toy NPs @ CCM drug release profiles under different conditions. As shown in FIG. 6, 1G₃-Cu/Toy NPs release slowly with a 3-day drug release rate of 32.8% in pH 7.4 (no GSH) buffer, whereas Toy release rate of 65.2% in pH 7.4(GSH concentration 10mM) buffer, since GSH can make 1G available₃The Cu/Toy NPs dissociate to accelerate the release of Toy. In addition, 1G was added to a buffer solution at pH 7.4(GSH concentration 10mM)₃The release rate of the-Cu/Toy NPs @ CCM group Toy is slightly lower than that of 1G under the same conditions in the initial stage of drug release₃The Cu/Toy NPs group, which is probably due to the coated cell membrane blocking the burst release of Toy. But 1G₃The total 3-day release rate of Toy in 72h for-Cu/Toy NPs @ CCM was 63.8%, which was close to 1G₃Total release rate of Toy from Cu/Toy NPs under these conditions, demonstrating that wrapping the cell membrane does not affect the total release of Toy. These results show that 1G₃The release of Toy in-Cu/Toy NPs @ CCM is GSH-responsive and can achieve enhanced chemotherapy at Toy tumor microenvironment-responsive release.

Example 6

1G prepared in example 1 was taken₃Characterization of its T by-Cu/Toy NPs @ CCM₁Relaxation behavior. CuCl with different concentrations is determined by a magnetic resonance imaging analyzer₂Or 1G₃-T of Cu/Toy NPs @ CCM solution₁Relaxation time and collecting T at different concentrations₁MR imaging. At the same time, 1G was measured at different concentrations₃-T after reaction of Cu/Toy NPs @ CCM with 10mM GSH solution₁Relaxation time and acquisition of T₁MR imaging. As shown in FIG. 7(a), 1G₃The magnetic resonance imaging signal intensity of the-Cu/Toy NPs @ CCM is weak, and after the reaction with GSH, 1G₃The magnetic resonance imaging signal intensity of the-Cu/Toy NPs @ CCM reaches a stronger level, and the magnetic resonance imaging signal intensity gradually increases (the circular magnetic resonance contrast area gradually becomes brighter) along with the increase of the concentration of the copper ions, which indicates that T₁MR imaging is increasingly effective. As shown in FIG. 7(b), 1G was calculated by further fitting₃Relaxation rate r of-Cu/Toy NPs @ CCM₁The value was 0.1875mM^-1s^-1And 1G₃R of-Cu/Toy NPs @ CCM + GSH₁The value rises to 0.6682mM^-1s^-1With CuCl₂(0.6958mM^-1s^-1) And (4) approaching. As the above results demonstrate, 1G was prepared₃the-Cu/Toy NPs @ CCM can release 1G in response to dissociation under the action of GSH₃Cu, selective enhancement of relaxivity at tumor sites, achieving precise T₁And (4) MR imaging.

Example 7

Detection of 1G prepared in example 1 Using B16 cells as a cell model₃-Cu/Toy NPs and 1G₃-cytotoxicity of Cu/Toy NPs @ CCM. B16 cells were collected at logarithmic growth phase at 1X 10 per well⁴Individual cells were seeded in 2 96-well plates in 5% CO₂Incubation was carried out at 37 ℃ for 12 h. Discarding the original culture medium, adding 1G with different concentrations into each well plate₃-Cu/Toy NPs or 1G₃-Cu/Toy NPs@CCM([Cu]1, 2, 10, 25, 50, 100, 200, 400 μ M) with cells in 5% CO₂And co-culturing at 37 ℃ for 24 h. The plates were then removed, the original medium discarded, washed three times with PBS, fresh medium containing 10% (v/v) CCK-8 was added and incubation continued in the incubator for 3 h. And finally, testing the light absorption value of each hole at the position with the wavelength of 450nm by using a multifunctional microplate reader, taking the cells treated by the PBS as a blank control, and recording the cell activity as 100%. The results are shown in fig. 8, where the cytotoxicity of both materials increased gradually with increasing Cu concentration in the experimental concentration range. While at the same Cu concentration, 1G₃Cytotoxicity ratio of-Cu/Toy NPs @ CCM group 1G₃High group of-Cu/Toy NPs, which may be due to 1G₃The cell membrane of B16 coated on the-Cu/Toy NPs @ CCM has homologous targeting, and increases phagocytosis of B16 cells on materials, so that the effect of inhibiting cancer cell proliferation is enhanced. 1G₃-Cu/Toy NPs with 1G₃Half maximal Inhibitory Concentration (IC) after 24h of co-incubation of Cu/Toy NPs @ CCM with B16 cells₅₀) 29.3. mu.M and 12.7. mu.M, respectively, demonstrate that under the same experimental operating conditions, 1G₃The cytotoxicity of-Cu/Toy NPs @ CCM on B16 cells is stronger than that of 1G₃-Cu/Toy NPs。

Example 8

Evaluation of cell pairs for 1G Using B16 cells and RAW264.7 cells as cell models₃-Cu/Toy NPs、1G₃-Cu/Toy NPs @ CCM and 1G₃-phagocytic Capacity of Cu/Toy NPs @ RBCM, and 1G was evaluated therefrom₃-homologous targeting ability and immune evasion ability of Cu/Toy NPs @ CCM. B16 cells or RAW264.7 cells were treated according to 1X 10⁵The density of each cell per well was individually inoculated in 12-well plates using RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin, and 10% FBS in 5% CO₂Incubation was carried out at 37 ℃ for 12 h. Discarding the original culture medium, adding the culture medium containing 1G₃-Cu/Toy NPs、1G₃-Cu/Toy NPs @ CCM and 1G₃-Cu/Toy NPs@RBCM([Cu]80 μ M) with cells in 5% CO₂And co-culturing at 37 ℃ for 6 h. Then, the cells in the 12-well plate are digested, centrifuged and counted, 1mL of aqua regia is added for digestion for 3h, and the content of the Cu element in the cells is detected by ICP-OES after digestion is stopped. The results are shown in FIG. 9, B16 cell pairs 1G₃The phagocytosis amount of-Cu/Toy NPs @ CCM is obviously higher than 1G₃-Cu/Toy NPs and 1G₃Cu/Toy NPs @ RBCM, indicating that coating B16 cell membrane can increase 1G pair of B16 cells₃Phagocytosis of Cu/Toy NPs @ CCM, conferring its cognate targeting ability. And RAW264.7 cell pair 1G₃-Cu/Toy NPs @ CCM and 1G₃Phagocytosis of-Cu/Toy NPs @ RBCM was similar, but significantly lower than for 1G₃Phagocytosis amount of-Cu/Toy NPs, which indicates that the prepared nano platform can escape phagocytosis of macrophage RAW264.7 by coating B16 cell membrane or erythrocyte membrane, and immune evasion capability is endowed. In summary, the coating of B16 cell membrane resulted in 1G₃the-Cu/Toy NPs @ CCM has the homologous targeting capability and the immune evasion capability at the same time, can prolong the blood circulation time in vivo, improve the targeting property and enhance the 1G₃Enrichment of-Cu/Toy NPs @ CCM at the tumor site.

Example 9

The effect of different materials on intracellular Glutathione (GSH) levels was evaluated using B16 cells as a cell model.Cells were plated at 2X 10 per well⁵The cells were seeded at a density in 6-well plates in RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. The original medium was discarded, and 10% PBS-containing medium or Toy-containing medium (8.6. mu.M, 1G concentration) was added thereto₃in-Cu/Toy NPs [ Cu]Toy concentration at 20 μ M); PEG-PCL and PEG-SS-PCL (concentration of 2. mu.M, concentration of Insensitive 1G)₃-Cu NPs or 1G₃-Cu NPs [ Cu]PEG-PCL or PEG-SS-PCL concentration at 20 μ M); 1G₃-Cu(1％DMSO)、Insensitive 1G₃-Cu NPs、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-Cu/Toy NPs@CCM([Cu]20 μ M) with cells in 5% CO₂Incubate at 37 ℃ for 6 h. Then digesting and centrifuging the cells in the 6-well plate, repeatedly freezing and thawing and crushing the cells at the temperature of 37 ℃ in liquid nitrogen, and then detecting the content of the GSH in the cells by using the kit according to the instruction of the GSH and GSSG detection kit. The results are shown in FIG. 10, which is relative to PEG-PCL and Insensitive 1G prepared from PEG-PCL₃-Cu NPs, PEG-SS-PCL containing disulfide bond and 1G prepared by using PEG-SS-PCL₃the-Cu NPs can significantly reduce intracellular GSH levels, since disulfide bonds can be cleaved by reducing GSH, thereby consuming intracellular GSH. Toy also reduces intracellular GSH levels, since Toy inhibits the adaptive regulation of cancer cell endoplasmic reticulum stress, thereby exacerbating endoplasmic reticulum stress. Increased endoplasmic reticulum stress in cancer cells can induce increased intracellular levels of ROS, and when ROS levels are increased, peroxidases catalyze reactions of GSH and ROS to convert GSH to oxidized glutathione (GSSG) in order to maintain intracellular redox balance (Ding et al adv. sci.,2021,8, 2002404). Thus, 1G loaded with Toy₃-Cu/Toy NPs compared to 1G₃-Cu NPs have an enhanced ability to deplete intracellular GSH. While at the same Cu concentration, 1G₃Intracellular GSH levels in the-Cu/Toy NPs @ CCM group to 1G₃The lower group of-Cu/Toy NPs, because the homologous targeting of B16 cell membranes coated on the surface of the material enhances phagocytosis of the material by cells, thereby consumingMore intracellular GSH.

Example 10

B16 cells were used as a cell model to evaluate the effect of different materials on intracellular Reactive Oxygen Species (ROS) levels. Cells were plated at 2X 10 per well⁵The cells were seeded at a density in 6-well plates in RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. The original medium was discarded, and a medium containing 10% PBS or a medium containing Toy (concentration 8.6. mu.M) was added, respectively; PEG-PCL, PEG-SS-PCL (concentration 2. mu.M); 1G₃-Cu(1％DMSO)、Insensitive 1G₃-Cu NPs、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-Cu/Toy NPs@CCM([Cu]20 μ M) with cells in 5% CO₂Incubate at 37 ℃ for 6 h. Discarding the original culture medium, and incubating the cells with the DCFH-DA probe diluted with the serum-free culture medium for 30min, wherein the colorless DCFH-DA probe can be oxidized into DCF with green fluorescence by ROS in the cells. After the incubation is finished, the original culture medium is discarded, the cells in all the well plates are digested, centrifuged, collected and resuspended in PBS, and the intensity of green fluorescence in the cells is detected by a flow cytometer. Based on the experimental results of the effects of different materials on intracellular GSH levels, it was first verified that 1G, which does not consume intracellular GSH, is not consumed₃-Cu, PEG-PCL and Insensitive 1G₃-effect of Cu NPs on intracellular ROS levels. As shown in FIGS. 11(a-b), 1G₃-Cu, PEG-PCL and Insensitive 1G₃There was no significant change in the mean ROS levels in the Cu NPs group of cells. As shown in FIG. 11(c-d), Toy and PEG-SS-PCL all enhanced the intracellular green fluorescence intensity to form 1G₃-Cu NPs and 1G₃Further enhancement of intracellular green fluorescence intensity after Cu/Toy NPs indicates increased intracellular ROS levels. 1G₃The cells of the-Cu/Toy NPs @ CCM group showed the strongest green fluorescence, indicating that more ROS were produced in the cells. GSH consumption and ROS production both indicate 1G produced₃the-Cu/Toy NPs @ CCM can break the redox balance in cancer cells to cause oxidative stress in the cancer cells, thereby further aggravating the endoplasmic reticulum stress state and inducing the cancer cells to wither through the endoplasmic reticulum pathwayAnd (7) death.

Example 11

In order to verify the effect of the prepared dual-drug-loaded nano platform on the endoplasmic reticulum stress state of cancer cells, B16 cells are used as a model to evaluate the influence of different materials on the expression level of endoplasmic reticulum stress-related proteins (GRP78, pIRE1 alpha, XBP1u, XBP1s and CHOP). Cells were plated at 2X 10 per well⁵The cells were seeded at a density in 6-well plates in RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. The original medium was discarded, and a medium containing 10% PBS or a medium containing Toy (concentration 8.6. mu.M) was added, respectively; 1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-Cu/Toy NPs@CCM([Cu]20 μ M) with cells in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. After the incubation was completed, the original medium was discarded, washed three times with PBS, then all the cells of the well plate were digested, centrifuged, collected, lysed on ice and centrifuged at 12000rpm for 5min at 4 ℃, the supernatant protein solution was collected, the protein concentration was determined, and then SDS-PAGE electrophoresis, membrane transfer, immunoreaction, ECL chemical developer fixation experiments were sequentially performed, and the contents of GRP78, pIRE1 α, XBP1u, XBP1s and CHOP in the cells were investigated, and β -actin was used as an internal reference, and the results are shown in fig. 12. The expression of marker proteins GRP78 and pIRE1 α representing the degree of endoplasmic reticulum stress were up-regulated to different degrees by treatment with each group of materials, indicating that the endoplasmic reticulum stress was increased (FIG. 12 (b-c)). This is due to 1G on the one hand₃Cu NPs can exacerbate endoplasmic reticulum stress by causing oxidative stress in cancer cells; on the other hand, Toy acts on IRE1 alpha-XBP 1 signal channel in endoplasmic reticulum stress, so that the shearing of the pIRE1 alpha to XBP1u is inhibited, the content of XBP1u in cells is increased, the expression of XBP1s capable of restoring the homeostasis of the endoplasmic reticulum is reduced, and the mechanism of restoring the homeostasis of the endoplasmic reticulum is cut off, so that the stress state of the endoplasmic reticulum is aggravated. As shown in fig. 12(d-e), increased endoplasmic reticulum stress upregulated the expression of XBP1u in cells treated with each group of materials; in the cells treated by the Toy-containing material, the expression of XBP1s is obviously reduced, and the action path of Toy in the cells is verified. Finally, to verify the weighted innerThe effect of reticulum stress on apoptosis was analyzed for the expression of the marker protein CHOP, which represents apoptosis of cells via the endoplasmic reticulum pathway. As shown in FIG. 12(f), CHOP expression was up-regulated to various degrees in the cells treated with each group of materials, indicating that 1G was produced₃the-Cu/Toy NPs @ CCM can cause cancer cells to undergo apoptosis via the endoplasmic reticulum pathway by aggravating endoplasmic reticulum stress and inhibiting the recovery of homeostasis from endoplasmic reticulum stress.

Example 12

In order to verify the effect of the prepared double-drug-loaded nano platform on the functions of the mitochondria of cancer cells, B16 cells are taken as a cell model to evaluate the influence of different materials on the mitochondrial membrane potential in the cells. Cells were plated at 2X 10 per dish⁵The density of each cell was inoculated into a confocal cell culture dish in RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. The original medium was discarded, and a medium containing 10% PBS or a medium containing Toy (concentration 8.6. mu.M) was added, respectively; PEG-SS-PCL (concentration 2. mu.M); 1G₃-Cu(1％DMSO)、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-Cu/Toy NPs@CCM([Cu]20 μ M) with cells in 5% CO₂Incubate at 37 ℃ for 6 h. The original culture medium is discarded, washed three times by PBS, and incubated for 20min with a staining working solution of a mitochondrial membrane potential detection probe (JC-1). After the incubation was completed, the supernatant was aspirated, washed three times with JC-1 staining buffer, 1mL of the medium was added, and then the change in red/green fluorescence in the cells was observed by confocal laser microscopy. When the mitochondrial membrane potential is higher, the fluorescent probe JC-1 is gathered in a matrix of mitochondria and shows red fluorescence; at low mitochondrial membrane potential, JC-1 cannot aggregate in the matrix of mitochondria, where JC-1 is a monomer and exhibits green fluorescence. As shown in fig. 13-14, the cells treated with PEG-SS-PCL and Toy showed weaker green fluorescence and lower red/green fluorescence ratio compared to PBS group, probably because PEG-SS-PCL and Toy could up-regulate intracellular ROS levels, thus having an effect on mitochondrial membrane potential (Guo et al. chem. mater.,2019,31(24), 10071-. To pass through 1G₃Significant increase in green fluorescence in Cu-treated cellsStrong and significant decrease in red/green fluorescence ratio, indicating 1G₃Cu may act on mitochondria, causing a significant decrease in mitochondrial membrane potential. To form 1G₃Cu NPs, Toy-loaded and cell membrane-coated, cell pairs 1G₃Increased phagocytosis of-Cu/Toy NPs @ CCM further decreased mitochondrial membrane potential, inducing mitochondrial dysfunction.

Example 13

To further explore 1G₃The molecular mechanism of inhibition of cancer cell growth by Cu/Toy NPs @ CCM was modeled by B16 cells to evaluate the effect of different materials on the expression levels of apoptosis-related proteins (Bax, Bcl-2, P53 and PTEN) in cells. Cells were plated at 2X 10 per well⁵The cells were seeded at a density in 6-well plates in RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. The original medium was discarded, and 10% PBS-containing medium or Toy (concentration 8.6. mu.M) was added, respectively; PEG-SS-PCL (concentration 2. mu.M); 1G₃-Cu(1％DMSO)、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-Cu/Toy NPs@CCM([Cu]20 μ M) with cells in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. After the incubation was completed, the original medium was discarded, washed three times with PBS, then the cells of all the well plates were digested, centrifuged, collected, lysed on ice and centrifuged at 12000rpm for 5min at 4 ℃, the supernatant protein solution was collected, the protein concentration was determined, and then SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were sequentially performed, and the contents of Bax, Bcl-2, P53, and PTEN in the cells were investigated, and β -actin was used as an internal reference, and the results are shown in fig. 15. In the mitochondrial pathway of apoptosis, the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 control the permeability of the mitochondrial outer membrane by regulating the mitochondrial membrane potential, and maintain the normal function of mitochondria. As shown in FIG. 15(b-c), through 1G₃The intracellular Bax protein level after Cu treatment was significantly increased and the Bcl-2 protein level was significantly decreased, while the changes in Bax and Bcl-2 affected the intracellular mitochondrial membrane potential, consistent with the results of the experiments on mitochondrial membrane potential in example 12 (FIGS. 13-14). The results of this experiment show that 1G₃Cu can act on Bax and Bcl-2, thereby changing the mitochondrial membrane potential and leading cells to undergo apoptosis through the mitochondrial pathway. To form 1G₃Cell pairs 1G after Cu NPs, Toy-loaded and cell membrane-coated₃Increased phagocytosis of-Cu/Toy NPs @ CCM, further increased Bax protein expression level, and further decreased Bcl-2 protein expression level. To further verify 1G₃The pro-apoptotic effect of Cu/Toy NPs @ CCM, and the expression levels of the intracellular pro-apoptotic proteins P53 and PTEN were determined by Western blotting experiments. See FIGS. 15(d-e), Toy and 1G₃Both P53 and PTEN expression can be increased by-Cu to form 1G₃After the-Cu/Toy NPs coat the cell membrane, the expression of P53 and PTEN is remarkably increased, indicating that 1G₃the-Cu/Toy NPs @ CCM can induce apoptosis.

The above experimental results show that 1G is prepared₃-Cu/Toy NPs @ CCM can break the redox balance in cancer cells, aggravate endoplasmic reticulum stress in cells, and inhibit adaptive regulation of endoplasmic reticulum stress; meanwhile, the protein related to mitochondria can be regulated and controlled, and mitochondrial dysfunction is induced, so that cancer cells are induced to die through endoplasmic reticulum and mitochondrial pathways.

Example 14

To verify the prepared 1G₃Whether Cu/Toy NPs @ CCM can cause cancer cell immunogenic death (ICD) or not is evaluated by taking B16 cells as a model, and the expression condition of an ICD marker Calreticulin (CRT) and the release condition of a high mobility group protein B1(HMGB-1) are evaluated after the cells are treated by different materials.

First, cells were plated at 2X 10 cells per dish⁵The density of each cell was inoculated into a confocal cell culture dish in RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. The original medium was discarded, and a medium containing 10% PBS or a medium containing Toy (concentration 8.6. mu.M) was added, respectively; PEG-SS-PCL (concentration 2. mu.M); 1G₃-Cu(1％DMSO)、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-Cu/Toy NPs@CCM([Cu]20 μ M) with cells in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. Discard original medium, wash three times with PBSAfter fixation with 2.5% glutaraldehyde solution for 15min and washing with PBS three times, the immunostaining blocking solution was added, and after blocking for 60min, diluted primary Antibody (CRT Rabbit Monoclonal Antibody) was added and incubated at room temperature for 1 h. After the incubation was completed, the primary antibody was aspirated, washed three times with PBS, and incubated at room temperature for 1 hour with the addition of a fluorescently labeled secondary antibody staining solution (Cy3-labeled Goat Anti-Rabbit IgG). After the incubation was completed, the secondary antibody staining solution was aspirated, washed three times with PBS, stained for 10min with DAPI, washed three times with PBS after the completion, added 0.5mL of PBS, and then the fluorescence in the cells was observed by confocal laser microscopy. When CRT in cancer cells everts to the cell surface, Dendritic Cells (DCs) are promoted to enter the tumor area and stimulate DCs maturation, enhancing their recognition and phagocytosis of antigens from apoptotic tumor cells, and presenting the relevant antigens to T cells. The eversion condition of the CRT after the cancer cells are processed by different materials can be observed through a laser confocal microscope. As shown in fig. 16, the fluorescence of CRT was hardly detected in cancer cells treated with PBS, because CRT is expressed in the endoplasmic reticulum of cells under normal conditions. PEG-SS-PCL had no significant effect on CRT expression in cells, which was observed with Toy and 1G₃The apparent fluorescence signal was detected on the surface of Cu-treated B16 cells, indicating Toy and 1G₃Cu can turn the CRT out. 1G₃The cells of the-Cu/Toy NPs @ CCM group showed the strongest fluorescence signals, indicating that the coated cell membrane can increase the phagocytosis of the material by the cells, and Toy and 1G₃The synergistic effect of-Cu everts more CRT to promote maturation of DCs.

Cells were plated at 2X 10 per well⁵The cells were seeded at a density in 6-well plates in RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. The original medium was discarded, and a medium containing 10% PBS or a medium containing Toy (concentration 8.6. mu.M) was added, respectively; PEG-SS-PCL (concentration 2. mu.M); 1G₃-Cu(1％DMSO)、1G₃-Cu NPs、1G₃-Cu/Toy NPs and 1G₃-Cu/Toy NPs@CCM([Cu]20 μ M) with cells in 5% CO₂Incubation was carried out at 37 ℃ for 24 h. After incubation, cell culture fluid is collected and passes through ELISA kit of HMGB-1And measuring the absorbance of the cell culture solution at 450nm by using a microplate reader, and calculating the content of HMGB-1 in the cell culture solution. HMGB-1 is localized in the nucleus, and when ICD occurs in cancer cells, HMGB-1 is triggered to be released from the nucleus to the outside of the cells, and the released HMGB-1 can stimulate DCs to mature and present tumor antigens to T cells. As shown in FIG. 17, cells were incubated with Toy or 1G₃after-Cu co-culture, the released HMGB-1 was significantly higher than that of the PBS group. To pass through 1G₃Cu/Toy NPs @ CCM treated cells, the amount of HMGB-1 released was 2.5 times that of PBS group.

Overall, eversion of CRT and release of HMGB-1 were demonstrated at Toy and 1G₃Cu-based chemotherapy induces ICD in B16 cells, releases the associated antigen, stimulates DCs to mature and present the antigen to T cells, and ultimately induces activation of antigen-specific CD8+ cytotoxic T lymphocytes, enhancing anti-tumor immunity.

Example 15

A mouse subcutaneous tumor model is constructed to verify the in vivo anti-tumor activity of different materials, and all animal experiments are carried out strictly according to the standards of the animal protection Association. 4-week female C57BL/6 mice for the experiment were purchased from Shanghai Slek laboratory animal center (China, Shanghai). 2 x 10 to⁶A single B16 cell was inoculated into the right hind leg of the mouse until the tumor volume reached about 100mm³On the left and right, the mice were randomly divided into 7 groups (6 mice per group). The specific grouping is as follows: control (PBS, 0.1 mL); PEG-SS-PCL (concentration 0.4mM, 0.1 mL); toy (concentration 1.72mM, 0.1 mL); 1G₃-Cu NPs([Cu]＝4mM，0.1mL)；1G₃-Cu/Toy NPs([Cu]＝4mM，0.1mL)；1G₃-Cu/Toy NPs@RBCM([Cu]＝4mM，0.1mL)；1G₃-Cu/Toy NPs@CCM([Cu]4mM, 0.1mL) by tail vein injection. The day of treatment initiation was day 1, the treatment was performed by tail vein injection on

days

1,3, 5, and 7, and the weight and tumor size of the mice were recorded every 2 days during the treatment period, and the tumor volume was (L × W) when the tumor length of the mice was L and the tumor width was W²)/2. The relative tumor volumes of the groups are shown in FIG. 18(a), and the tumor volumes of the PBS group mice rapidly increased with time, and the PEG-SS-PCL group, Toy group and 1G group₃The tumor growth of the-Cu NPs group mice is inhibited to different degrees. Compared with Toy groups and 1G₃Group of-Cu NPs, 1G₃The in vivo antitumor effect of the-Cu/Toy NPs group is better, because the prepared nanoparticles can be Toy and 1G₃Cu delivered to the tumor site by EPR effect, and Toy with 1G₃The synergistic effect of-Cu NPs enhances the therapeutic effect. Cell membrane-coated 1G₃-Cu/Toy NPs @ RBCM and 1G₃-Cu/Toy NPs @ CCM shows enhanced antitumor effect due to prolonged blood circulation time. Wherein, 1G₃The best treatment effect of the-Cu/Toy NPs @ CCM group is probably because the homologous targeting protein on the B16 cell membrane improves the targeting property of the material, so that the material is enriched at a tumor part. As can be seen in FIG. 18(b), the weight change of each group of mice was insignificant, indicating 1G₃the-Cu/Toy NPs @ CCM has good biocompatibility in vivo.

Example 16

To verify 1G₃-the ability of Cu/Toy NPs @ CCM to induce ICD in tumor cells in vivo and to achieve a combination chemotherapy/immunotherapy with 1G₃-Cu/Toy NPs @ CCM in combination with the PD-L1 antibody, for a second in vivo anti-tumor experiment. 2 x 10 to⁶A single B16 cell was inoculated into the right hind leg of the mouse until the tumor volume reached about 100mm³On the left and right, the mice were randomly divided into 4 groups (6 mice per group). The specific grouping is as follows: control group (PBS, 0.1mL), Anti-PD-L1 (concentration 1mg/mL, 0.1mL), 1G₃-Cu/Toy NPs@CCM([Cu]＝4mM，0.1mL)、1G₃-Cu/Toy NPs@CCM+Anti-PD-L1([Cu]4mM, 0.1 mL; Anti-PD-L1 concentration was 1mg/mL, 0.1 mL). The injection mode is 1G₃-Cu/Toy NPs @ CCM tail vein injection, Anti-PD-L1 intratumoral injection. Day of treatment initiation was recorded as

day

1, 1G₃-Cu/Toy NPs @ CCM was treated by tail vein injection on

days

1,3, 5 and 7, Anti-PD-L1 was treated by intratumoral injection on

days

2, 4, 6 and 8, mouse body weight and tumor size were recorded every 2 days during treatment, mouse tumor length was recorded as L, and tumor volume was (L x W) when width was recorded as W²)/2. After the treatment, tumor tissues of each group of mice were taken out under aseptic conditions on day 14, and were minced, ground, and filtered through a 400-mesh sieve to obtain a cell suspension. First by tumor infiltration of various animalsSeparating lymphocytes by using the tissue lymphocyte separation kit, separating T lymphocyte suspension by using a nylon hair column, marking the obtained T lymphocytes by using Anti-CD4-FITC and Anti-CD8-PE respectively, and quantitatively analyzing CD4+ T cells and CD8+ T cells in tumor tissues by using a flow cytometer.

As shown in fig. 19(a), compared with the PBS group, the tumor growth of the Anti-PD-L1 group is somewhat inhibited, which indicates that the Anti-PD-L1 can block the binding of PD-1/PD-L1, improve the recognition ability of T cells to tumor cells, and realize the immunotherapy of tumors. 1G₃The relative tumor volumes of the-Cu/Toy NPs @ CCM group were significantly reduced, whereas those of the 1G group₃The tumor volume was minimal in the-Cu/Toy NPs @ CCM + Anti-PD-L1 group treated mice, since the synergy of chemotherapy and immunotherapy was the best therapeutic effect. As can be seen in FIG. 19(b), the weight change of each group of mice was insignificant, indicating 1G₃The therapy of-Cu/Toy NPs @ CCM + Anti-PD-L1 has good biocompatibility in vivo.

The quantitative analysis of CD4+ T cells and CD8+ T cells in each group of tumor tissues is shown in FIG. 20, Anti-PD-L1 and 1G₃Both the-Cu/Toy NPs @ CCM caused the increase of tumor-infiltrating CD4+ T cells and CD8+ T cells, indicating that Anti-PD-L1 and 1G₃the-Cu/Toy NPs @ CCM restored the T cell immune response function to varying degrees. And 1G₃The content of CD4+ T cells and CD8+ T cells in the-Cu/Toy NPs @ CCM group is higher than that in the Anti-PD-L1 group because 1G₃the-Cu/Toy NPs @ CCM can induce tumor cells to generate ICD, stimulate the maturation of dendritic cells and enhance the antigen presenting capacity of the dendritic cells, and finally induce the activation of CD4+ T cells and CD8+ T cells. 1G₃The content of tumor-infiltrating CD4+ T cells and CD8+ T cells in the group of-Cu/Toy NPs @ CCM + Anti-PD-L1 is highest, which indicates that the content is based on 1G₃Chemotherapy-induced tumor cell ICD of-Cu/Toy NPs @ CCM combined with Anti-PD-L1-based immune checkpoint blockade therapy elicits the most effective immune response, resulting in enhanced combination therapy of tumors.

Example 17

To verify 1G₃The ability of the combination therapy of-Cu/Toy NPs @ CCM + Anti-PD-L1 to generate an immunological memory in vivo, for the third timeInternal antitumor experiments. 2 x 10 to⁶A single B16 cell was inoculated into the right hind leg of the mouse until the tumor volume reached about 100mm³On the left and right, the mice were randomly divided into 4 groups (6 mice per group). The specific grouping is as follows: control group (PBS, 0.1mL), Anti-PD-L1 (concentration 1mg/mL, 0.1mL), 1G₃-Cu/Toy NPs@CCM([Cu]＝4mM，0.1mL)、1G₃-Cu/Toy NPs@CCM+Anti-PD-L1([Cu]4mM, 0.1 mL; Anti-PD-L1 concentration was 1mg/mL, 0.1 mL). The injection mode is 1G₃-Cu/Toy NPs @ CCM tail vein injection, Anti-PD-L1 intratumoral injection. Day of treatment initiation was recorded as

day

1, 1G₃-Cu/Toy NPs @ CCM was treated by tail vein injection on

days

1,3, 5, 7, and Anti-PD-L1 was treated by intratumoral injection on

days

2, 4, 6, 8. After treatment, three mice in each group are randomly selected on day 9, the spleen of each mouse is taken out under the aseptic condition, the three mice are cut into pieces, ground and filtered by a 400-mesh filter screen to obtain cell suspension, T lymphocyte suspension is separated by a nylon hair column, the obtained T lymphocytes are respectively marked by Anti-CD44-FITC and Anti-CD62L-APC, and central memory T cells (T cells) in the spleen are detected by a flow cytometer_CMCD44+ CD62L +) and effector memory T cells (T)_EMCD44+ CD62L-) for quantitative analysis.

Memory T cells can be classified as T_CMAnd T_EMTwo types, T_CMCan multiply and maintain immunological memory for a long time, is more sensitive to antigen stimulation, and can be differentiated into T after being stimulated_EM(ii) a And T_EMThe immune effect is produced immediately after stimulation by the antigen. As shown in FIG. 21, Anti-PD-L1 and 1G₃both-Cu/Toy NPs @ CCM can cause T_CMAnd T_EMIndicates that Anti-PD-L1 and 1G are increased₃The Cu/Toy NPs @ CCM activated the immune memory T cells to varying degrees. And 1G₃-T of the Cu/Toy NPs @ CCM set_CMAnd T_EMThe content was higher than that of Anti-PD-L1 group, which is probably due to that Anti-PD-L1 injected intratumorally only achieved immune checkpoint blockade in the tumor area, whereas 1G injected tail vein₃the-Cu/Toy NPs @ CCM has an extended blood circulation time and can induce ICD by continuous chemotherapy to enhance the production of immune memory T cells. 1G₃-Cu/Toy NPs@CCM+Anti-PD-L1 group T_CMAnd T_EMHighest content, indicating 1G₃The combined treatment of-Cu/Toy NPs @ CCM and Anti-PD-L1 can activate more immune memory T cells, enhance the defense capability to tumor antigens, and inhibit tumor recurrence or metastasis.

Comparative example 1

1.3mg of third generation phosphorous-containing dendrimer copper complex 1G₃Cu and 1.0mg PEG-PCL were dissolved in 500. mu.L DMSO, and then the solution was added dropwise to 5mL of ultrapure water under ultrasonic conditions, and the reaction was stirred for 24 hours. Dialyzing with dialysis bag with molecular weight cut-off of 8000-14000Da in ultrapure water for 3 days to remove DMSO and unreacted PEG-PCL, and filtering the product with microporous membrane with pore diameter of 1 μm to remove unsupported 1G₃Cu, and finally centrifuging the product for 30min at 4500rpm by an ultrafiltration centrifugal tube with the molecular weight cutoff of 3500Da to obtain the intensive 1G₃-Cu NPs。

Comparative example 2

(1) Collecting C57BL/6 black mouse whole blood, centrifuging at 4 deg.C under 800g centrifugal force for 5min, washing with 1mL PBS solution for three times to remove serum to obtain 1 × 10⁸And (4) precipitating the red blood cells. The obtained erythrocyte pellet is resuspended in 3mL of 0.25 × PBS solution and ice-cooled for 20min to induce rupture of erythrocyte membrane, then centrifuged at 800g centrifugal force for 5min to remove hemoglobin, and the pellet is resuspended in 1mL of PBS solution to obtain erythrocyte membrane suspension (RBCM).

(2) 200. mu.g of 1G₃-Cu/Toy NPs were mixed with 0.5mL RBCM, the solution was extruded 11 times using an Avanti micro-extruder, centrifuged at 10000rpm for 6min to remove excess cell membranes to give 1G₃-Cu/Toy NPs@RBCM。

Claims

1. A responsive nano platform for bionic cell membrane and loaded with phosphorus-containing dendrimer copper complex/toyocamycin is characterized in that amphiphilic polymer PEG-SS-PCL is used to react with the third generation of phosphorus-containing dendrimer copper complex 1G₃Cu is encapsulated at a hydrophobic end, and toyocamycin Toy is loaded at a hydrophilic end through hydrogen bonding, and then the membrane is coated on a melanoma B16 cell membrane.

2. A preparation method of a response type nano platform for bionic cell membrane loaded with phosphorus-containing dendrimer copper complex/toyocamycin comprises the following steps:

(2) dissolving toyocamycin Toy in ultrapure water, and adding to 1G in step (1)₃Stirring and reacting in-Cu NPs solution, and centrifuging to obtain 1G₃-Cu/Toy NPs；

(3) Adding the cell lysis mixed solution into a melanoma B16 cell precipitate, performing ice bath, repeatedly freezing and thawing, centrifuging to obtain a precipitate as a B16 cell membrane, and then suspending in a PBS solution to obtain a B16 cell membrane suspension CCM;

3. The method according to claim 2, wherein 1G in the step (1)₃The molar ratio of-Cu to PEG-SS-PCL is 1: 3-1: 8; the volume ratio of the solvent to the ultrapure water is 1: 8-1: 12; the solvent is dimethyl sulfoxide DMSO.

4. The preparation method according to claim 2, wherein the stirring reaction temperature in the step (1) is room temperature, and the stirring reaction time is 20-30 h.

5. The method according to claim 2, wherein step (2) comprises reacting toyocamycin Toy with step 1G₃The mass ratio of-Cu NPs is 1: 1-1: 5; the stirring reaction temperature is room temperature, and the stirring reaction time is 20-30 h.

6. The preparation method according to claim 2, wherein the cell lysis mixture in step (3) is a mixture of PMSF (phenylmethylsulfonyl chloride) and hypotonic cell lysis solution; the ratio of the melanoma B16 cell sediment to the cell lysis mixture is 1 × 10⁷The method comprises the following steps: 2-4 mL.

7. The preparation method according to claim 2, wherein the ice bath time in the step (3) is 10-20 min; the technological parameters of repeated freeze thawing are as follows: freezing at-20 deg.C, thawing at 37 deg.C, and repeating for 3 times.

8. The method according to claim 2, wherein 1G in the step (4)₃The proportion of the-Cu/Toy NPs to the B16 cell membrane suspension is 190-210 mu g: 0.4-0.6 mL.

9. The method according to claim 2, wherein the extruding in the step (4) is repeated 10 to 15 times by using an Avanti micro extruder with a filter membrane pore size of 400 nm.

10. The use of the cell membrane-biomimetic phosphorus-containing dendrimer-loaded copper complex/toyocamycin responsive nano-platform of claim 1 in the preparation of a tumor diagnostic agent for combined therapy of MR imaging, chemotherapy and immunotherapy.