CN113855646B - 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 - Google Patents

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 Download PDF

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CN113855646B
CN113855646B CN202110949821.XA CN202110949821A CN113855646B CN 113855646 B CN113855646 B CN 113855646B CN 202110949821 A CN202110949821 A CN 202110949821A CN 113855646 B CN113855646 B CN 113855646B
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沈明武
郭云琦
范钰
王志强
詹梦偲
史向阳
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Donghua University
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Abstract

The invention relates to a response type nano platform for loading a phosphorus-containing dendrimer copper complex/toyocamycin and a preparation method and application thereof. The method comprises the following steps: 1G 3 Preparation of Cu NPs solution, 1G 3 Preparation of-Cu/Toy NPs, preparation of B16 cell membrane suspension CCM, 1G 3 -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 1G 3 The water solubility of the-Cu reduces the toxic and side effect of Toy, and the-Cu can release 1G in the response dissociation of a tumor microenvironment 3 Cu and Toy, and 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 a tumor diagnosis and treatment integrated nano platform (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), 1G 3 -Cu has good stability and better T 1 Relaxation 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 1G 3 Cu 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 (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 transcriptional and metabolic abnormalities, too rapid cellular proliferation, and the like (Chen et al. Nat. Rev. Cancer,2020,21 (2), 71-88). During ERS development in cancer cells, chaperone GRP78 is cleaved from IRE1 α in one of the ERS receptors, dimerizes and autophosphorylates IRE1 α, thereby activating the RNase domain of IRE1 α and allowing it to splice downstream XBP1 mRNA to produce XBP1-splicing (XBP 1 s) mRNA. XBP1s mRNA encodes the activated protein XBP1s, which restores and maintains the homeostasis of the endoplasmic reticulum to some extent, allowing cancer cells to adapt to and survive the sustained ERS (Yoshida et al. Cell,2001,107 (7), 881-891). Toy acts on IRE1 α -XBP1 signaling pathway, inhibiting splicing of XBP1 mRNA by IRE1 α endoribonuclease, thereby inhibiting adaptive regulation of ERS, and ultimately inducing apoptosis (Ri et al. Toy is not selective for tumor cells, but has a significant toxic side effect when used in vivo.
To exert 1G to the maximum extent 3 The drug effect of the-Cu and Toy is that 1G can be prepared by using amphiphilic polymer containing sensitive bonds 3 integrating-Cu and Toy to construct a tumor microenvironment responsive nano platform and improve 1G simultaneously 3 The 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 T 2 /T 1 MR 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 can overexpress immune checkpoint molecules (e.g., PD-L1), inhibiting T cell activation and thereby evading immune system surveillance and clearance. The recognition ability of T cells to tumor cells can be improved by Immune Checkpoint Blockade (ICB) therapy and the killing effect of an immune system to the tumor cells can be restored by using a specific immune preparation (such as a PD-L1 antibody). 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 found 3 Cu and Toy, and coat B16 cell membrane, and related reports combined with PD-L1 antibody for 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 1G 3 Cu 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 1G 3 -Cu and PEG-SS-PCL dissolved in solventDropwise adding ultrapure water under the ultrasonic condition, stirring for reaction, dialyzing, filtering and centrifuging to obtain 1G 3 -Cu NPs solution;
(2) Dissolving toyocamycin Toy in ultrapure water, and adding to 1G in step (1) 3 Stirring and reacting in-Cu NPs solution, centrifuging to remove unencapsulated Toy to obtain 1G 3 -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 suspending in a PBS solution to obtain a B16 cell membrane suspension CCM;
(4) Subjecting 1G in step (2) 3 mixing-Cu/Toy NPs with the B16 cell membrane suspension CCM in the step (3), extruding and centrifuging to obtain 1G 3 -Cu/Toy NPs @ CCM, namely a response type nano platform for loading phosphorus-containing dendrimer copper complex/toyocamycin and simulating cell membranes.
Preferably, in the above method, 1G in the step (1) 3 The molar ratio of-Cu to PEG-SS-PCL is 1:3-1:8.
Preferably, in the above method, the volume ratio of the solvent to the ultrapure water in the step (1) is 1:8-1; the solvent is dimethyl sulfoxide DMSO.
Preferably, in the above method, the stirring reaction temperature in the step (1) is room temperature, and the stirring reaction time is 20 to 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 cut-off 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 30min.
Preferably, in the above method, toyocamycin Toy and 1G in step (2) 3 The mass ratio of the-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 to 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 the step (3) is 1 × 10 7 The method comprises the following steps: 2 to 4mL.
Preferably, in the above 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) 3 The ratio of Cu/Toy NPs to B16 cell membrane suspension is 190-210 μ g: 0.4-0.6 mL.
Preferably, in the above method, 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.
Preferably, in the above method, the centrifugation parameters in step (4) are: the centrifugation temperature is 4 ℃, and the centrifugation is carried out for 6min at 10000 rpm.
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 performance 3 the-Cu is encapsulated at the hydrophobic end, toy is loaded at the hydrophilic end through the hydrogen bond effect, the 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 for the accurate diagnosis and treatment integration of the tumor, and the PD-L1 antibody is combined in vivo to realize the accurate diagnosis and treatment integration of the tumorCombination therapy of tumor chemotherapy and immunotherapy.
The invention integrates 1G with PEG-SS-PCL 3 -Cu and Toy, improvement 1G 3 The water solubility of Cu reduces the toxic and side effect of Toy, 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 method 3 -cytotoxicity of Cu/Toy nps @ ccm and related control materials; detecting phagocytosis of material by different cells by ICP-OES, and determining 1G 3 -immune evasion and homologous 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; evaluating the effect of the material on intracellular ROS levels using flow cytometry; evaluating the influence of the material on the protein level expression of endoplasmic reticulum stress related factors GRP78, p-IRE1 alpha, XBP1s and CHOP by Western blot; evaluating the influence of the material on the mitochondrial membrane potential in the cell by using a laser confocal microscope; the influence of the material on the expression conditions of apoptosis-related proteins Bax, bcl-2, P53 and PTEN is evaluated by Western blot protein blotting; evaluating the influence of the material on the CRT expression condition in the cell by using a laser confocal microscope; and finally establishing a black mouse subcutaneous tumor model for carrying out 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 1G 3 The water solubility of-Cu reduces the toxic and side effect of Toy, and 1G can be released in response dissociation of tumor microenvironment 3 Cu and Toy, and 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 recovery of the stress of the endoplasmic reticulum of the cancer cells to a steady state, and can enable the cancer cells to generate oxidative stress and aggravate the stress of the endoplasmic reticulum, so that the nano platform and Toy form a synergistic effect and promote the apoptosis of the cancer cells 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 1G 3 -Cu realizes T 1 And (4) MR imaging. Can realize chemotherapy/immunotherapy combined treatment after combining 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 invention 3 -Cu/Toy NPs @ CCM synthesis and application schematic;
FIG. 2 shows 1G in example 1 3 -Cu、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs、1G 3 -Cu/Toy NPs @ CCM and 1G in comparative example 2 3 -hydrated particle size (a) and surface potential (b) as measured by a nano-particle sizer of Cu/Toy nps @ rbcm;
FIG. 3 is 1G prepared in example 1 3 -graph of hydrodynamic diameter over time of Cu/Toy nps @ ccm in water, PBS and 1640 medium;
FIG. 4 is 1G prepared in example 1 3 -Cu/Toy NPs (a) and 1G 3 -TEM images of Cu/Toy nps @ ccm (b);
FIG. 5 is 1G prepared in example 1 3 -Cu/Toy NPs、CCM、1G 3 -Cu/Toy NPs @ CCM (a) and RBCM, 1G prepared in comparative example 2 3 A SDS-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of-Cu/Toy NPs @ RBCM (b);
FIG. 6 is 1G prepared in example 1 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs @ CCM kinetics profiles of drug release under different conditions;
FIG. 7 shows 1G for different copper concentrations 3 -Cu/Toy NPs@CCM、1G 3 -Cu/Toy NPs @ CCM + GSH and CuCl 2 T of 1 MR imaging plots (a) and T 1 A linear graph (b) of the inverse of the relaxation time as a function of Cu concentration;
FIG. 8 is 1G prepared in example 1 3 -Cu/Toy NPs、1G 3 -cell motility diagram after 24h of co-incubation of Cu/Toy NPs @ CCM with B16 cells;
FIG. 9 is 1G prepared in example 1 3 -Cu/Toy NPs、1G 3 -Cu/Toy NPs @ CCM and 1G prepared in comparative example 2 3 -Cu/Toy NPs @ RBCM and B16 cell and RAW264.7 cell respectively after incubation for 6 h;
FIG. 10 shows Toy, PEG-PCL, PEG-SS-PCL, and 1G 3 -Cu、Insensitive 1G 3 -Cu NPs、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -graph of intracellular GSH levels after 6h co-incubation of Cu/Toy nps @ ccm with B16 cells;
FIG. 11 shows 1G 3 -Cu、PEG-PCL、Insensitive 1G 3 -Cu NPs (a-b) and Toy, PEG-SS-PCL, 1G 3 -Cu NPs、1G 3 -Cu/Toy NPs、1G 3 -intracellular ROS horizontal flow cytometry and quantitation plots after 6h of co-incubation of Cu/Toy NPs @ CCM (c-d) with B16 cells;
FIG. 12 shows Toy, 1G 3 -Cu NPs、1G 3 -Cu/Toy NPs、1G 3 -Cu/Toy NPs @ CCM and B16 cells were incubated for 24h, and then Western blot assay results of GRP78, p-IRE1 alpha, XBP1s and CHOP proteins in the cells are shown, wherein (a) is a protein band diagram of Western blot, and (B-f) is a gray scale quantification graph of each protein band;
FIG. 13 shows PEG-SS-PCL, toy, 1G 3 -Cu、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs @ CCM and B16 cells incubated for 6h, then the fluorescence change of JC-1 probe is analyzed by confocal laser microscopy;
FIG. 14 shows PEG-SS-PCL, toy, 1G 3 -Cu、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and1G 3 -Cu/Toy NPs @ CCM and B16 cells after incubation for 6h, and then quantitatively analyzing the ratio of JC-1 red/green fluorescence in the cells;
FIG. 15 shows PEG-SS-PCL, toy, 1G 3 -Cu、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 A Western blot test result graph of Bax, bcl-2, P53 and PTEN proteins in cells after co-incubation of Cu/Toy NPs @ CCM and B16 cells 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, 1G 3 -Cu、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -laser confocal 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, 1G 3 -Cu、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -quantitative analysis of HMGB-1 content in cell culture broth after 24h of co-incubation of Cu/Toy NPs @ CCM with B16 cells;
FIG. 18 shows the injection of PBS, PEG-SS-PCL, toy, 1G via tail vein in example 15 3 -Cu NPs、1G 3 -Cu/Toy NPs、1G 3 -Cu/Toy NPs @ RBCM and 1G 3 -after Cu/Toy nps @ ccm, a plot of relative tumor volume change (a) and a plot of relative mouse body weight change (b) over 14 days was recorded;
FIG. 19 shows the results of the analysis of example 16 in PBS, anti-PD-L1, 1G 3 -Cu/Toy NPs @ CCM and 1G 3 -Cu/Toy NPs @ CCM + Anti-PD-L1 treatment, relative tumor volume change profile (a) and mouse relative body weight change profile (b) over 14 days were recorded;
FIG. 20 is a graph of flow cytometric analysis of the typing of CD4+/CD8+ T cells in tumor tissues 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 chemicals were used without further purification. Third generation phosphorus-containing dendrimer copper Complex 1G 3 Cu 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 Virginia atlantoaxial 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 are purchased from Biyunnan biotechnology limited (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 1G 3 Cu 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 cutoff 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 unloaded 1G 3 -Cu, and finally centrifuging at 4500rpm for 30min in an ultrafiltration centrifuge tube with molecular weight cutoff of 3500Da to obtain 1G 3 -Cu NPs. Product 1G by ICP 3 in-Cu NPsThe Cu content was calculated to be 1G 3 The upper loading of Cu was 99.2wt% and the encapsulation efficiency was 57.6wt%.
(2) 0.5mg of toyocamycin Toy was dissolved in 1mL of ultrapure water and then added to 1mL of 1G 3 adding-Cu NPs solution (1.5 mg/mL), stirring and mixing for 24h, centrifuging at 4500rpm for 30min in an ultrafiltration centrifuge tube with molecular weight cutoff of 3500Da to remove unsupported Toy to obtain 1G 3 -Cu/Toy NPs. The absorbance of the liquid outside the ultrafiltration centrifugal tube at 279nm is measured by ultraviolet, and the amount of the non-loaded Toy is calculated, so that the loading rate of Toy is 39.4wt% and the encapsulation rate is 11.6wt%.
(3) Mixing 30 μ L PMSF with 3mL hypotonic cell lysate, collecting 1 × 10B 16 cells in logarithmic growth phase 7 And centrifuging at 1000rpm for 5min to obtain cell precipitate, adding mixed hypotonic cell lysis solution into the cell precipitate, ice-bathing for 15min, and repeatedly freezing and thawing at (-20 deg.C, 37 deg.C for three times). Setting the centrifugal temperature at 4 ℃, centrifuging for 10min at 700g of centrifugal force, and removing precipitates; centrifuging at 14000g for 30min, removing supernatant, and suspending the precipitate in 1mL PBS solution to obtain B16 cell membrane suspension (CCM).
(4) 200. Mu.g of 1G 3 -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 obtained 3 -Cu/Toy NPs@CCM。
Example 2
Taking appropriate amount of 1G 3 Cu, completely dissolved in DMSO and then added dropwise to ultrapure water to prepare 1G of 4mM copper 3 -Cu solution (1% dmso, ultrapure water as solvent); and the appropriate amount of 1G prepared in example 1 was taken 3 -Cu NPs、1G 3 -Cu/Toy NPs、1G 3 -Cu/Toy NPs @ CCM and 1G prepared in comparative example 2 3 -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, 1G 3 The 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 that1G 3 Successful synthesis of Cu NPs. When 1G 3 After the-Cu NPs are loaded 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 means of physical compression 3 -Cu/Toy NPs @ CCM and 1G 3 The hydrated particle size of-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 1G 3 Successful coating of cell membranes on the surface of Cu/Toy NPs. 1G 3 The hydrated particle size of-Cu/Toy NPs @ CCM in various solutions (water, PBS, 1640 medium) was almost unchanged (FIG. 3), demonstrating that 1G 3 the-Cu/Toy NPs @ CCM has good colloidal stability.
Example 3
1G prepared in example 1 was taken 3 -Cu/Toy NPs and 1G 3 Characterization of dimensions and morphology was carried out by-Cu/Toy NPs @ CCM. 1G 3 TEM image of-Cu/Toy NPs is shown in FIG. 4 (a), 1G 3 the-Cu/Toy NPs are in the form of uniform spheres with a size of about 185nm. 1G prepared as shown in FIG. 4 (b) 3 the-Cu/Toy NPs @ CCM size is about 220nm and the thickness of the coated cell membrane is about 25nm.
Example 4
1G prepared in example 1 was taken 3 -Cu/Toy NPs、CCM、1G 3 -Cu/Toy NPs @ CCM and RBCM, 1G prepared in comparative example 2 3 the-Cu/Toy NPs @ RBCM was characterized by SDS-PAGE to verify the coating of the cell membrane and the retention of the cell membrane proteins. CCM, 1G determination by BCA protein quantification kit 3 -Cu/Toy NPs @ CCM, RBCM and 1G 3 -Cu/Toy NPs @ RBCM, and PBS solution was used to adjust the protein content in each sample to 0.8mg/mL. Add 5. Mu.L of protein Marker to the first protein lane, and add 15. Mu.L of 1G 3 Cu/Toy NPs (concentration 1G) 3 1G corresponding to the protein concentration of 0.8mg/mL in-Cu/Toy NPs @ CCM 3 Concentration of-Cu/Toy NPs), CCM and 1G 3 The protein lane was fed with-Cu/Toy NPs @ CCM at a current of 100A for 30min. As shown in FIG. 5 (a), 1G 3 The group of-Cu/Toy NPs did not run out of the protein band, whereas 1G 3 Similar protein bands were run out from the-Cu/Toy NPs @ CCM group and the CCM group, demonstrating B16 cell membrane at 1G 3 Successful coating of the Cu/Toy NPs surface and retention of B16 cell membrane proteins. Similarly, protein Marker, RBCM and 1G 3 SDS-PAGE experiment using the protein lane-Cu/Toy NPs @ RBCM as shown in FIG. 5 (b) 3 Similar protein bands are run out from the-Cu/Toy NPs @ RBCM group and the RBCM group, and the fact that the erythrocyte membrane is 1G is proved 3 Successful coating of the Cu/Toy NPs surface and preservation of erythrocyte membrane proteins.
Example 5
Buffer solutions of pH =7.4 and pH =7.4 (GSH concentration =10 mM) were prepared for analysis of 1G, respectively 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs @ CCM Toy responsive release performance. 1G to be prepared 3 the-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 3500 Da), and the prepared 1G was used 3 -Cu/Toy nps @ ccm dissolved as a 1mg/mL solution with 1mL of a buffer solution of ph =7.4 (GSH concentration =10 mM) and placed in a dialysis bag (molecular cut-off 3500 Da), which was then placed in a container containing 9mL of different buffer solutions and shaken in a 37 ℃ constant temperature shaker. 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 drawn 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs @ CCM drug release profiles under different conditions. As shown in FIG. 6, 1G 3 The Cu/Toy NPs release slowly with a 3-day drug release rate of 32.8% in pH =7.4 (no GSH) buffer, while the 3-day release rate of Toy is 65.2% in pH =7.4 (GSH concentration =10 mM) buffer, since GSH can make 1G 3 The Cu/Toy NPs dissociate leading to a faster release of Toy. Further, in a buffer of pH =7.4 (GSH concentration =10 mM), 1G 3 The release rate of-Cu/Toy NPs @ CCM group Toy is slightly lower than 1G under the same condition in the initial stage of drug release 3 The Cu/Toy NPs group, which is probably due to the coated cell membrane block the burst of Toy. But 1G 3 The 3-day total release rate of Toy in 72h of-Cu/Toy NPs @ CCM is 63.8%, and is close to 1G 3 Total release rate of Toy in Cu/Toy NPs under this condition, demonstrating that coating cell membranes does not affect the overall ToyAnd (4) releasing. These results show that 1G 3 The release of Toy in-Cu/Toy NPs @ CCM has GSH responsiveness, and can release Toy in tumor microenvironment responsiveness, so that enhanced chemotherapy is realized.
Example 6
1G prepared in example 1 was taken 3 Characterization of its T by-Cu/Toy NPs @ CCM 1 Relaxation behavior. CuCl with different concentrations is determined by a magnetic resonance imaging analyzer 2 Or 1G 3 T of-Cu/Toy NPs @ CCM solution 1 Relaxation time, and collecting T at different concentrations 1 MR imaging. At the same time, 1G was measured at different concentrations 3 -T after reaction of Cu/Toy NPs @ CCM with 10mM GSH solution 1 Relaxation time and acquisition of T 1 MR imaging. As shown in FIG. 7 (a), 1G 3 The magnetic resonance imaging signal intensity of-Cu/Toy NPs @ CCM is weak, and after reaction with GSH, 1G 3 The magnetic resonance imaging signal intensity of-Cu/Toy NPs @ CCM reaches a stronger level, and the magnetic resonance imaging signal intensity also gradually increases (the circular magnetic resonance contrast area gradually becomes brighter) along with the increase of the concentration of copper ions, which indicates that T 1 MR imaging is increasingly effective. As shown in FIG. 7 (b), 1G was calculated by further fitting 3 Relaxation rate r of-Cu/Toy NPs @ CCM 1 The value was 0.1875mM -1 s -1 And 1G is 3 R of-Cu/Toy NPs @ CCM + GSH 1 The value rises to 0.6682mM -1 s -1 With CuCl 2 (0.6958mM -1 s -1 ) And (4) approaching. As the above results demonstrate, 1G was prepared 3 the-Cu/Toy NPs @ CCM can release 1G in response to dissociation under the action of GSH 3 Cu, selective enhancement of relaxivity at tumor sites, achieving precise T 1 And (4) MR imaging.
Example 7
Detection of 1G prepared in example 1 Using B16 cells as a cell model 3 -Cu/Toy NPs and 1G 3 Cytotoxicity of Cu/Toy NPs @ CCM. B16 cells were collected at logarithmic growth phase at 1X 10 per well 4 Density of individual cells in 2 96-well plates, put at 5% CO 2 Incubation was carried out at 37 ℃ for 12h. Discarding the original culture medium, adding 1G with different concentrations into each well plate 3 -Cu/Toy NPs or 1G 3 -Cu/Toy NPs@CCM([Cu]=1, 2, 10, 25, 50, 100, 200, 400 μ M) with cells at 5% co 2 And co-culturing at 37 ℃ for 24h. 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 3h. 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, 1G 3 Cytotoxicity ratio of-Cu/Toy NPs @ CCM group 1G 3 High group of-Cu/Toy NPs, which may be due to 1G 3 The B16 cell membrane coated on the-Cu/Toy NPs @ CCM has homologous targeting property, increases phagocytosis of the B16 cell to the material, and further enhances the effect of inhibiting cancer cell proliferation. 1G 3 -Cu/Toy NPs with 1G 3 Half maximal Inhibitory Concentration (IC) after 24h of co-incubation of-Cu/Toy NPs @ CCM with B16 cells 50 ) Respectively 29.3. Mu.M and 12.7. Mu.M, and it was confirmed that under the same experimental operating conditions, 1G was present 3 The cytotoxicity of-Cu/Toy NPs @ CCM on B16 cells is stronger than that of 1G 3 -Cu/Toy NPs。
Example 8
Evaluation of cell pairs for 1G Using B16 cells and RAW264.7 cells as cell models 3 -Cu/Toy NPs、1G 3 -Cu/Toy NPs @ CCM and 1G 3 Phagocytic ability of-Cu/Toy NPs @ RBCM, and 1G was evaluated based on the phagocytic ability 3 The homologous targeting ability and the immune evasion ability of-Cu/Toy NPs @ CCM. B16 cells or RAW264.7 cells were treated at 1X 10 5 The density of each cell per well was inoculated in 12-well plates using RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS, and 5% CO 2 And incubated at 37 ℃ for 12h. Discarding the original culture medium, adding the culture medium containing 1G 3 -Cu/Toy NPs、1G 3 -Cu/Toy NPs @ CCM and 1G 3 -Cu/Toy NPs@RBCM([Cu]= 80. Mu.M) medium and cells at 5% CO 2 And co-culturing at 37 ℃ for 6h. Then digesting, centrifuging and counting cells in a 12-well plate, adding 1mL of aqua regia for digestion for 3h, detecting the Cu element in the cells by ICP-OES after digestion is stoppedThe content of the element. The results are shown in FIG. 9, for B16 cell pair 1G 3 The phagocytosis amount of-Cu/Toy NPs @ CCM is obviously higher than 1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs @ RBCM, demonstrating that coating B16 cell membrane can increase B16 cell to 1G 3 Phagocytosis of-Cu/Toy NPs @ CCM, conferring its cognate targeting ability. And RAW264.7 cell pair 1G 3 -Cu/Toy NPs @ CCM and 1G 3 Phagocytosis of-Cu/Toy NPs @ RBCM was similar, but significantly lower than for 1G 3 Phagocytosis amount of-Cu/Toy NPs, which shows that both the coated B16 cell membrane and the red cell membrane can enable the prepared nano platform to escape phagocytosis of macrophage RAW264.7, and endow the prepared nano platform with immune evasion capability. In summary, the coating of B16 cell membranes resulted in 1G 3 the-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 1G 3 Enrichment 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 cellular model. Cells were plated at 2X 10 per well 5 The density of individual cells was inoculated into 6-well plates using RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS, and placed at 5% CO 2 Incubation was carried out at 37 ℃ for 24h. Discarding the original medium, adding 10% PBS-containing medium or Toy (8.6. Mu.M, 1G concentration) containing 3 in-Cu/Toy NPs [ Cu]Toy concentration corresponding to 20 μ M); PEG-PCL and PEG-SS-PCL (concentration of 2. Mu.M, concentration of Insensitive 1G) 3 -Cu NPs or 1G 3 -Cu NPs [ Cu](ii) = the corresponding PEG-PCL or PEG-SS-PCL concentration at 20 μ M); 1G 3 -Cu(1％DMSO)、Insensitive 1G 3 -Cu NPs、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs@CCM([Cu]= 20. Mu.M) medium and cells at 5% CO 2 Incubate at 37 ℃ for 6h. 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, relative to PEG-PCL and the PEG-PCL preparedInsensitive 1G 3 -Cu NPs, PEG-SS-PCL containing disulfide bond and 1G prepared by using PEG-SS-PCL 3 the-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 ROS levels, and when ROS levels are increased, peroxidases catalyze the reaction 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 Toy 3 -Cu/Toy NPs compared to 1G 3 -Cu NPs have an enhanced ability to deplete intracellular GSH. While at the same Cu concentration, 1G 3 Intracellular GSH levels in the-Cu/Toy NPs @ CCM group to 1G 3 the-Cu/Toy NPs group was lower, since the homologous targeting of the B16 cell membrane coated on the material surface enhanced phagocytosis of the material by cells, consuming more 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 well 5 The density of individual cells was inoculated into 6-well plates using a medium RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS, charged with 5% CO 2 Incubation was carried out at 37 ℃ for 24h. Discard the original medium, add 10% PBS-containing medium or Toy (8.6. Mu.M concentration), respectively; PEG-PCL, PEG-SS-PCL (concentration 2. Mu.M); 1G 3 -Cu(1％DMSO)、Insensitive 1G 3 -Cu NPs、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs@CCM([Cu]= 20. Mu.M) medium and cells at 5% CO 2 Incubate at 37 ℃ for 6h. 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 incubation, the original medium was discarded, washed three times with PBS, and all the wells were platedThe cells were digested, centrifuged, collected, resuspended in PBS, and the intensity of green fluorescence in the cells was detected by flow cytometry. 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 consumed 3 -Cu, PEG-PCL and Insensitive 1G 3 The effect of Cu NPs on intracellular ROS levels. As shown in FIGS. 11 (a-b), 1G 3 -Cu, PEG-PCL and Insensitive 1G 3 There 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 1G 3 -Cu NPs and 1G 3 Further enhancement of intracellular green fluorescence intensity after Cu/Toy NPs, indicating increased intracellular ROS levels. 1G 3 The cell group-Cu/Toy NPs @ CCM showed the strongest green fluorescence, indicating that more ROS are produced intracellularly. Both GSH consumption and ROS production indicate 1G produced 3 the-Cu/Toy NPs @ CCM can destroy the redox balance in the cancer cell to cause the oxidative stress in the cancer cell, thereby further aggravating the endoplasmic reticulum stress state and inducing the cancer cell to die through the endoplasmic reticulum pathway.
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 (GRP 78, pIRE1 alpha, XBP1u, XBP1s and CHOP). Cells were plated at 2X 10 per well 5 The density of individual cells was inoculated into 6-well plates using RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS, and placed at 5% CO 2 Incubation was carried out at 37 ℃ for 24h. Discard the original medium, add 10% PBS-containing medium or Toy (8.6. Mu.M concentration), respectively; 1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs@CCM([Cu]= 20. Mu.M) medium and cells at 5% CO 2 Incubation was carried out at 37 ℃ for 24h. After the incubation was completed, the original medium was discarded, washed three times with PBS, then all the cells in the well plate were digested, centrifuged, collected, the cells were 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 was sequentially performedPAGE electrophoresis, membrane transfer, immunoreaction, ECL chemical developer fixation experiments, and the intracellular contents of GRP78, pIRE1 alpha, XBP1u, XBP1s and CHOP were investigated, and beta-actin was used as an internal reference, and the results are shown in FIG. 12. The expression of the marker proteins GRP78 and pIRE 1. Alpha. Which represent the degree of endoplasmic reticulum stress was up-regulated to various 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 hand 3 Cu 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 pIRE1 alpha to XBP1u is inhibited, the content of XBP1u in cells is increased, the expression of XBP1s capable of recovering endoplasmic reticulum steady state is reduced, and the mechanism of recovering endoplasmic reticulum steady state is cut off, so that the stress state of endoplasmic reticulum is aggravated. As shown in FIG. 12 (d-e), increased endoplasmic reticulum stress up-regulated the expression of XBP1u in cells treated with each group of materials; in the cells treated by the material containing Toy, the expression of XBP1s is obviously reduced, and the action path of Toy in the cells is verified. Finally, to verify the effect of increased endoplasmic reticulum stress on apoptosis, the expression of the marker protein CHOP, representing apoptosis of cells via the endoplasmic reticulum pathway, was analyzed. 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 produced 3 the-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 dish 5 The density of each cell was inoculated into a confocal cell culture dish using a medium of RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS, charged at 5% CO 2 Incubation was carried out at 37 ℃ for 24h. Discard the original medium, add 10% PBS-containing medium or Toy (8.6. Mu.M concentration), respectively; PEG-SS-PCL (concentration 2. Mu.M); 1G 3 -Cu(1％DMSO)、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs@CCM([Cu]= 20. Mu.M) medium and cells at 5% CO 2 Incubate at 37 ℃ for 6h. 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 can up-regulate intracellular ROS levels, thus having an effect on mitochondrial membrane potential (Guo et al. Chem. Mater.,2019,31 (24), 10071-10084). To pass through 1G 3 Significant increase in green fluorescence and significant decrease in red/green fluorescence ratio in Cu-treated cells, indicating 1G 3 Cu may act on mitochondria, causing a significant decrease in mitochondrial membrane potential. To form 1G 3 -Cu NPs, loaded with Toy and coated with cell membrane, cell pair 1G 3 Phagocytosis of-Cu/Toy NPs @ CCM is increased, and mitochondrial membrane potential is further decreased to induce mitochondrial dysfunction.
Example 13
To further explore 1G 3 The molecular mechanism of inhibiting the growth of cancer cells by Cu/Toy NPs @ CCM is that B16 cells are used as a model to evaluate the influence of different materials on the expression level of apoptosis-related proteins (Bax, bcl-2, P53 and PTEN) in cells. Cells were plated at 2X 10 per well 5 The density of individual cells was inoculated into 6-well plates using RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS, and placed at 5% CO 2 Incubation was carried out at 37 ℃ for 24h. Discard the original medium, add 10% PBS-containing medium or Toy (8.6. Mu.M concentration), respectively; PEG-SS-PCL (concentration 2. Mu.M); 1G 3 -Cu(1％DMSO)、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs@CCM([Cu]= 20. Mu.M) medium and cells at 5% CO 2 Incubation was carried out at 37 ℃ for 24h. 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 1G 3 The 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 1G 3 Cu acts on Bax and Bcl-2, thereby changing mitochondrial membrane potential and leading cells to undergo apoptosis through a mitochondrial pathway. To form 1G 3 Cell pairs 1G after-Cu NPs, toy loaded and cell membranes coated 3 Phagocytosis of-Cu/Toy NPs @ CCM is increased, the Bax protein expression level is further increased, and the Bcl-2 protein expression level is further decreased. To further verify 1G 3 The apoptosis promoting effect of-Cu/Toy NPs @ CCM, and the expression level of the intracellular apoptosis-promoting proteins P53 and PTEN is determined by Western blotting experiments. FIG. 15 (d-e), toy and 1G 3 Both P53 and PTEN expression can be increased by-Cu to form 1G 3 after-Cu/Toy NPs coat cell membranes, the expression of P53 and PTEN is remarkably increased, indicating that 1G 3 the-Cu/Toy NPs @ CCM can induce apoptosis.
The above experimental results show that 1G is prepared 3 the-Cu/Toy NPs @ CCM can break the redox balance in cancer cells, aggravate the endoplasmic reticulum stress of the cells and inhibit the adaptive regulation of the endoplasmic reticulum stress; at the same time, can regulate and control the related protein of mitochondria to induce the dysfunction of mitochondria, thereby inducing the cancer cells to pass through endoplasmic reticulumAnd mitochondrial pathway apoptosis.
Example 14
To verify the prepared 1G 3 Whether the @ CCM of Cu/Toy 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 dish 5 The density of individual cells was inoculated into a confocal cell culture dish using RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS, and placed at 5% CO 2 And incubating at 37 ℃ for 24h. Discard the original medium, add 10% PBS-containing medium or Toy (8.6. Mu.M concentration), respectively; PEG-SS-PCL (concentration 2. Mu.M); 1G 3 -Cu(1％DMSO)、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs@CCM([Cu]= 20. Mu.M) medium and cells at 5% CO 2 Incubation was carried out at 37 ℃ for 24h. Discarding the original culture medium, washing with PBS three times, fixing with 2.5% glutaraldehyde solution for 15min, washing with PBS three times, adding immunostaining blocking solution, blocking for 60min, adding diluted primary Antibody (CRT Rabbit Monoclonal Antibody), and incubating at room temperature for 1h. 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 (Cy 3-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 CRT eversion condition of cancer cells 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 cellsToy and 1G 3 The apparent fluorescence signal was detectable on the surface of Cu-treated B16 cells, indicating Toy and 1G 3 Cu can turn the CRT out. 1G 3 The 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 1G 3 Cu synergy everts more CRTs to promote maturation of DCs.
Cells were plated at 2X 10 per well 5 The density of individual cells was inoculated into 6-well plates using RPMI-1640 complete medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS, and placed at 5% CO 2 Incubation was carried out at 37 ℃ for 24h. Discard the original medium, add 10% PBS-containing medium or Toy (8.6. Mu.M concentration), respectively; PEG-SS-PCL (concentration 2. Mu.M); 1G 3 -Cu(1％DMSO)、1G 3 -Cu NPs、1G 3 -Cu/Toy NPs and 1G 3 -Cu/Toy NPs@CCM([Cu]=20 μ M) medium and cells in 5% 2 Incubation was carried out at 37 ℃ for 24h. And after the incubation is finished, collecting the cell culture solution, and measuring the absorbance of the cell culture solution at 450nm by using an enzyme-linked immunosorbent assay (ELISA) kit of HMGB-1 to calculate 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 1G 3 after-Cu co-culture, the released HMGB-1 was significantly higher than that of the PBS group. To pass through 1G 3 The amount of HMGB-1 released by the cells treated with-Cu/Toy NPs @ CCM was 2.5 times that of PBS.
Overall, eversion of CRT and release of HMGB-1 was demonstrated at Toy and 1G 3 Cu-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
Mouse subcutaneous tumor models were constructed to verify the in vivo anti-tumor activity of different materials, and all animal experiments were performed strictly in accordance with the animal protection society standards. 4 week female for experimentC57BL/6 mice were purchased from Shanghai Slek laboratory animal center (China, shanghai). 2 x 10 to 6 A single B16 cell was inoculated into the right hind leg of the mouse until the tumor volume reached about 100mm 3 On 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, 0.1mL); toy (concentration 1.72mM, 0.1mL); 1G 3 -Cu NPs([Cu]＝4mM，0.1mL)；1G 3 -Cu/Toy NPs([Cu]＝4mM，0.1mL)；1G 3 -Cu/Toy NPs@RBCM([Cu]＝4mM，0.1mL)；1G 3 -Cu/Toy NPs@CCM([Cu]=4mm, 0.1ml), the injection mode is 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 W 2 )/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, the PEG-SS-PCL group, the Toy group and the 1G group 3 Tumor growth was inhibited to various degrees in the-Cu NPs group of mice. Compared with Toy group and 1G 3 Group of-Cu NPs, 1G 3 The in vivo antitumor effect of the-Cu/Toy NPs group is better, because the prepared nano particles can convert Toy and 1G 3 Cu is delivered to the tumor site by the EPR effect, and Toy with 1G 3 The synergistic effect of-Cu NPs enhances the therapeutic effect. Cell membrane-coated 1G 3 -Cu/Toy NPs @ RBCM and 1G 3 -Cu/Toy NPs @ CCM shows enhanced antitumor effect due to prolonged blood circulation time. Wherein, 1G 3 The 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 1G 3 the-Cu/Toy NPs @ CCM has good biocompatibility in vivo.
Example 16
To verify 1G 3 The ability of-Cu/Toy NPs @ CCM to induce tumor cells to develop ICD in vivo and to achieve a combination therapy of chemotherapy/immunotherapy with 1G 3 -Cu/Toy NPs @ CCM in combination with PD-L1 antibody,a second in vivo anti-tumor experiment was performed. 2 x 10 to 6 A single B16 cell was inoculated into the right hind leg of the mouse until the tumor volume reached about 100mm 3 On 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, 0.1 mL), anti-PD-L1 (concentration 1mg/mL,0.1 mL), 1G 3 -Cu/Toy NPs@CCM([Cu]＝4mM，0.1mL)、1G 3 -Cu/Toy NPs@CCM+Anti-PD-L1([Cu]=4mm,0.1ml; anti-PD-L1 concentration is 1mg/mL,0.1 mL). The injection mode is 1G 3 -Cu/Toy NPs @ CCM tail vein injection, anti-PD-L1 intratumoral injection. Day of treatment initiation was recorded as day 1, 1G 3 Treating by tail vein injection of-Cu/Toy NPs @ CCM on days 1,3, 5 and 7, treating by Anti-PD-L1 intratumoral injection on days 2, 4, 6 and 8, recording the weight and the tumor size of the mice once every 2 days during the treatment period, recording the tumor length of the mice as L and the width as W, and then, the tumor volume is (L multiplied by W) 2 )/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. Firstly, separating lymphocytes through various animal tumor infiltration tissue lymphocyte separation kits, then separating T lymphocyte suspension through nylon columns, marking the obtained T lymphocytes with Anti-CD4-FITC and Anti-CD8-PE respectively, and carrying out quantitative analysis on 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 inhibited to some extent, which indicates that the Anti-PD-L1 can block the binding of PD-1/PD-L1, improve the recognition capability of T cells on tumor cells, and realize the immunotherapy on tumors. 1G 3 The relative tumor volume of the-Cu/Toy NPs @ CCM group was significantly reduced, while that of the 1G group 3 The tumor volume of mice treated with the group-Cu/Toy NPs @ CCM + Anti-PD-L1 was minimal, since chemotherapy and immunotherapy synergized to maximize therapeutic efficacy. As can be seen in FIG. 19 (b), the weight change of each group of mice was insignificant, indicating 1G 3 The therapy of-Cu/Toy NPs @ CCM + Anti-PD-L1 has good biocompatibility in vivo.
Quantitative analysis of CD4+ T cells and CD8+ T cells in each group of tumor tissues is shown in FIG. 20, anti-PD-L1And 1G 3 both-Cu/Toy NPs @ CCM can cause the increase of tumor infiltrating CD4+ T cells and CD8+ T cells, indicating that Anti-PD-L1 and 1G 3 the-Cu/Toy NPs @ CCM restored the T cell immune response function to varying degrees. And 1G 3 The content of CD4+ T cells and CD8+ T cells in the group of-Cu/Toy NPs @ CCM are higher than that in the group of Anti-PD-L1, because 1G 3 the-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. 1G 3 The 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 1G 3 The tumor cell ICD caused by chemotherapy of-Cu/Toy NPs @ CCM can cause the most effective immune response after being combined with Anti-PD-L1-based immune checkpoint blocking therapy, and the enhanced combined treatment of the tumor is realized.
Example 17
To verify 1G 3 The ability of the combination therapy of-Cu/Toy NPs @ CCM + Anti-PD-L1 to generate immunological memory in vivo for the third in vivo Anti-tumor experiment. 2 x 10 to 6 A single B16 cell was inoculated into the right hind leg of the mouse until the tumor volume reached about 100mm 3 On 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, 0.1 mL), anti-PD-L1 (concentration 1mg/mL,0.1 mL), 1G 3 -Cu/Toy NPs@CCM([Cu]＝4mM，0.1mL)、1G 3 -Cu/Toy NPs@CCM+Anti-PD-L1([Cu]=4mm,0.1ml; anti-PD-L1 concentration is 1mg/mL,0.1 mL). The injection mode is 1G 3 -Cu/Toy NPs @ CCM tail vein injection, anti-PD-L1 intratumoral injection. Day of treatment initiation was recorded as day 1, 1G 3 -Cu/Toy NPs @ CCM on days 1,3, 5, 7 for treatment by tail vein injection, and Anti-PD-L1 on days 2, 4, 6, 8 for treatment by intratumoral injection. 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 pillar, 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 CM CD44+ CD62L +) and effector memory T cells (T) EM CD44+ CD 62L-) for quantitative analysis.
Memory T cells can be classified as T CM And T EM Two types, T CM Can 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 is EM The immune effect is produced immediately after stimulation by the antigen. As shown in FIG. 21, anti-PD-L1 and 1G 3 T can be induced by-Cu/Toy NPs @ CCM CM And T EM Indicates that Anti-PD-L1 and 1G are increased 3 the-Cu/Toy NPs @ CCM activated immune memory T cells to varying degrees. And 1G 3 T of the group-Cu/Toy NPs @ CCM CM And T EM The content was higher than that of Anti-PD-L1 group, which is probably due to that Anti-PD-L1 injected intratumorally achieves immune checkpoint blockade only in the tumor region, while 1G injected tail vein 3 -Cu/Toy NPs @ CCM has an extended blood circulation time and can enhance the production of immune memory T cells by triggering ICD by continuous chemotherapy. 1G 3 -Cu/Toy NPs @ CCM + Anti-PD-L1 group T CM And T EM Highest content, indicating 1G 3 The combined treatment of-Cu/Toy NPs @ CCM and Anti-PD-L1 can activate more immune memory T cells, enhance the defense capacity to tumor antigens, and inhibit tumor recurrence or metastasis.
Comparative example 1
1.3mg of third generation phosphorous-containing dendrimer copper complex 1G 3 Cu 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 cutoff 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 unloaded 1G 3 Cu, 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 1G 3 -Cu NPs。
Comparative example 2
(1) Collecting C57BL/6 black mouse whole blood, setting centrifugation temperature at 4 deg.C, centrifuging at 800g centrifugal force for 5min, and centrifuging withSerum was removed by washing three times with 1mL of PBS solution to obtain 1X 10 8 And (4) precipitating the red blood cells. The obtained erythrocyte pellet is resuspended in 3mL of 0.25 XPBS 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 1G 3 -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 1G 3 -Cu/Toy NPs@RBCM。

Claims (10)

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 1G 3 Cu 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:
(1) The third generation of phosphorus-containing dendrimer copper complex 1G 3 dissolving-Cu and PEG-SS-PCL in solvent, adding into ultrapure water dropwise under ultrasonic condition, stirring for reaction, dialyzing, filtering, centrifuging to obtain 1G 3 -Cu NPs solution;
(2) Dissolving toyocamycin Toy in ultrapure water, adding to 1G in step (1) 3 Stirring and reacting in-Cu NPs solution, and centrifuging to obtain 1G 3 -Cu/Toy NPs；
(3) Adding the cell lysis mixed solution into a melanoma B16 cell precipitate, carrying out 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;
(4) Subjecting 1G in step (2) 3 mixing-Cu/Toy NPs with the B16 cell membrane suspension CCM in the step (3), extruding and centrifuging to obtain 1G 3 -Cu/Toy NPs@CCM is a response type nano platform for bionic cell membrane loading phosphorus-containing dendrimer copper complex/toyocamycin.
3. The method according to claim 2, wherein 1G in the step (1) 3 The 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; the solvent is dimethyl sulfoxide DMSO.
4. The method according to claim 2, wherein the reaction temperature in step (1) is room temperature and the reaction time is 20 to 30 hours.
5. The method according to claim 2, wherein step (2) comprises mixing toyocamycin Toy with 1G 3 -the mass ratio of the 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 mixed liquor is 1 x 10 7 The method comprises the following steps: 2 to 4mL.
7. The preparation method according to claim 2, wherein the ice bath time in the step (3) is 10 to 20min; 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) 3 The ratio of Cu/Toy NPs to B16 cell membrane suspension is 190-210 μ g: 0.4-0.6 mL.
9. The method according to claim 2, wherein the extrusion in the step (4) is repeated 10 to 15 times using an Avanti micro-extruder having a filter 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.
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