CN116270430A - Diselenide bond crosslinked nanogel loaded with nano gold particles and methotrexate, and preparation and application thereof - Google Patents
Diselenide bond crosslinked nanogel loaded with nano gold particles and methotrexate, and preparation and application thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to a diselenide bond crosslinked nanogel loaded with nano gold particles and methotrexate, and a preparation method and application thereof. The invention has wide sources of raw materials and simple operation process; the prepared nano gel has good colloid stability, X-ray attenuation characteristic, cell compatibility, anti-tumor characteristic and immune regulation tumor microenvironment effect, and has good application potential and industrialization implementation prospect.
Description
Technical Field
The invention belongs to the field of functional high molecular polymers, and particularly relates to a diselenide bond crosslinked nanogel loaded with nano gold particles and methotrexate, and preparation and application thereof.
Background
Malignant tumor has the characteristics of rapid growth, strong transfer capability, high recurrence rate and the like, and becomes a main killer threatening human health, and high-efficiency early diagnosis and accurate treatment are key problems to be overcome in modern medicine. Currently, early diagnostic means for tumors include ultrasound imaging (US), magnetic resonance imaging (MR), computed tomography imaging (CT), and the like. Among many imaging diagnosis techniques, CT is a common clinical diagnosis method due to its characteristics of non-invasiveness, high density resolution, no overlapping of tissue structure images, etc. Iohexol injection is commonly used clinically as a contrast agent. However, the development of clinical medicine is limited due to the short half-life of blood, relatively high concentrations of nephrotoxicity, and the like. In recent years, gold nanoparticles have been widely focused on CT imaging applications, have better stability and biocompatibility in living bodies, and have been very mature in synthetic methods and surface modification methods. Wang et al (Wang et al, biomaterials, 2013,2,34,470-480) surface-functionalized dendrimer-encapsulated Au NPs (Au DENPs) were used as imaging probes for targeted CT imaging of human lung adenocarcinoma.
Meanwhile, the main methods of clinical tumor treatment include chemotherapy, radiotherapy and surgical excision, wherein chemotherapy is the most commonly used cancer treatment in clinic. However, chemotherapy drugs have a short blood circulation in vivo and, because of lack of tumor specificity, damage normal cells while killing tumor cells. These drawbacks limit their therapeutic efficiency and long-term use in biomedical applications. With the development of nanotechnology, the nano-carriers such as micelle, carbon dot, nano-gel, dendritic macromolecule and the like are applied to traditional chemotherapy, so that the blood circulation time of the medicine can be effectively prolonged, the absorption of the medicine at the tumor position is promoted, and the treatment effect is enhanced. Wherein the nano hydrogel is a three-dimensional reticular hydrogel particle formed by physical or chemical crosslinking of hydrophilic or amphiphilic polymer chains. They have good colloid stability, biocompatibility, higher loading capacity and are easy to contact tumor tissues, so that they can be used as excellent drug carriers. Meanwhile, by selecting and designing different polymer networks, a drug-loaded nano hydrogel system with specific intelligent responsiveness can be constructed, so that the release of chemotherapeutic drugs in tumors or tumor microenvironments is promoted, and the drug-loaded nano hydrogel system has great potential in tumor chemotherapy application. For example, the drug delivery system was constructed by Xiang et al (Xiang et al, adv. Sci.,2022,2,2200173) using a poly (tertiary amine oxide) -polycaprolactone amphiphilic block polymer (PTAO-PCL). The nano drug carrying system has the anti-fouling property and is not adsorbed by plasma proteins, so that long blood circulation is realized; after reaching the tumor site, the active extravasation can be adhered to the vascular endothelial cells of the tumor to induce the cells to rapidly transcytose, so as to realize tumor enrichment and show high-efficiency anti-tumor activity. Although the polymer exhibits better antitumor activity, how the polymer functions in cells is still unknown. For example, xv et al (Xv et al, thernostics, 2020,10, 10, 4349-4358.) utilize poly (N-vinylcaprolactam) and disulfide-containing cross-linking agents to construct nanogel drug delivery systems that can undergo redox reactions with Glutathione (GSH) that is overexpressed in the tumor microenvironment, achieving enhanced tumor chemotherapeutic effects by targeted release of the chemotherapeutic doxorubicin through cleavage of the disulfide bond, but when GSH concentrations are low, the disulfide bond cannot be completely broken, impeding drug release. Recently, studies have found that disulfide bonds have a bond energy of 240kJ/mol, diselenide bonds have a bond energy of 172kJ/mol, and diselenide cross-linked network is more reactive than disulfide bonds, and can break under milder conditions, with redox sensitivity superior to disulfide bonds.
Immunosuppressive tumor microenvironment is also an important factor limiting the effectiveness of tumor chemotherapy. In the process of tumorigenesis and development, immune cells in the tumor microenvironment are gradually domesticated into immunosuppressive cells, so that the killing effect of the anticancer drugs on tumor tissues is greatly limited.
There is no report on the preparation of diselenide-bond crosslinked nanogels loaded with gold nanoparticles and methotrexate and the application of the diselenide-bond crosslinked nanogels in tumor diagnosis and treatment.
Disclosure of Invention
The invention aims to solve the technical problem of providing a diselenide bond crosslinked nanogel loaded with nano gold particles and methotrexate, and preparation and application thereof, so as to overcome the defects of difficult discovery in early tumor stage, poor single chemotherapy curative effect and the like.
The invention relates to a diselenide bond cross-linked composite nanogel which is a diselenide bond cross-linked poly-N-vinyl caprolactam nano hydrogel loaded with nano gold particles and medicines.
The diselenide bond crosslinked poly-N-vinyl caprolactam nano hydrogel is a three-dimensional network polymer formed by precipitation polymerization of raw materials containing monomer N-vinyl caprolactam VCL, diselenide bond crosslinked cross-linking agent and acetoacetic acid ethylene glycol methacrylate AAEM; the drug is methotrexate.
The invention relates to a preparation method of diselenide bond crosslinking composite nano gel, which comprises the following steps:
(1) Mixing monomer N-vinyl caprolactam VCL, surfactant, crosslinking agent containing diselenide bond and water, N 2 Stirring in water bath under the environment, adding an initiator and acetoacetic acid ethylene glycol methacrylate (AAEM), continuing stirring reaction, cooling, dialyzing, and freeze-drying to obtain the diselenide-crosslinked poly-N-vinyl caprolactam nano hydrogel PVCL NGs;
(2) Mixing PVCL NGs with water, adding chloroauric acid solution, stirring, adding sodium borohydride ice water solution, continuing stirring reaction, dialyzing, and freeze-drying to obtain the diselenide bond crosslinked nanogel Au@PVCL NGs loaded with the nanogold particles;
(3) And mixing the Au@PVCL NGs, water and the medicine, and stirring for reaction to obtain the diselenide bond crosslinked composite nano gel MTX/Au@PVCL NGs.
The preferred mode of the preparation method is as follows:
the surfactant in the step (1) is Sodium Dodecyl Sulfate (SDS); the initiator is azodicarboxylic ethyl-2-isobutyl amidine hydrate ACMA; the cross-linking agent containing diselenide bond is diacryloyloxy diethyl diselenide;
the mass ratio of VCL, AAEM, the crosslinking agent containing diselenide bond and the surfactant in the step (1) is 20-30:2-3:1-1.5:0.1-0.2.
N in the step (1) 2 The temperature of water bath stirring is 60-70 ℃ under the environment, and the stirring time is 30-40 min; the reaction time of continuous stirring is 4-6 h.
In the step (2), the mass ratio of chloroauric acid to sodium borohydride to PVCL NGs is 1-1.5:1-1.5:5-7.5.
Adding chloroauric acid solution into the step (2), stirring at 0 ℃ for 0.5-2 hours; adding sodium borohydride ice water solution, continuously stirring and reacting at 3-10 deg.c for 8-10 hr.
The mass ratio of the drug to the Au@PVCL NGs in the step (3) is 1-1.5:5-7; the medicine is methotrexate MTX;
the stirring reaction temperature is room temperature, and the stirring reaction time is 8-10 hours.
The dialysis in the steps (1) and (2) adopts a dialysis bag with the molecular weight cut-off of 8000-14000 and the dialysis time of 3-5 days.
The invention relates to application of diselenide bond crosslinking composite nano gel in preparing a chemotherapeutics for CT diagnosis of melanoma and/or enhanced immune regulation of tumor microenvironment.
The invention also provides application of the diselenide bond crosslinked nanogel loaded with the nanogold particles and the methotrexate in tumor CT diagnosis and tumor microenvironment regulation enhancement chemotherapy.
Aiming at the defects of difficult discovery of early tumor, poor single chemotherapy curative effect and the like, the invention designs and synthesizes the multifunctional hybridized nano-hydrogel which can passively target the tumor microenvironment and takes the poly-N-vinyl caprolactam nano-gel as a carrier to load nano-gold particles, thereby realizing CT diagnosis of the nano-hydrogel at the tumor position and enhancing the chemotherapy.
Firstly, preparing and synthesizing a diselenide bond crosslinked poly-N-vinyl caprolactam (PVCL) nanogel rich in acetoacetic acid ethylene glycol methacrylate (AAEM) by a precipitation polymerization method, and then chelating trivalent gold ions with the AAEM and reducing the trivalent gold ions by sodium borohydride to form a poly-N-vinyl caprolactam nanogel (Au@PVCL NGs) loaded with gold nanoparticles; finally, the methotrexate is loaded by utilizing the electrostatic adsorption of the nanogel to form the diselenide bond crosslinking nanogel (MTX/Au@PVCL NGs) loaded with the nanogel particles and the methotrexate.
The invention uses Zeta potential and dynamic light scattering analysis (DLS), ultraviolet visible absorption spectrum (UV-Vis), field emission Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) to characterize the prepared MTX/Au@PVCL NGs. Then, the cytotoxicity of the nanogel was evaluated by CCK-8 method, and the phagocytosis of the nanogel by the cells was evaluated by inductively coupled plasma-emission spectrometry (ICP-OES). TAM typing before and after chemotherapy was examined by flow cytometry. Finally, an anti-tumor experiment of a tumor model in the mouse body is carried out, and the anti-tumor effect of the prepared multifunctional hybrid nanogel in the mouse body is examined. The synthesis of MTX/Au@PVCL NGs and the effect of enhanced tumor chemotherapy treatment are schematically shown in FIG. 1. The specific test results are as follows:
(1) Au@PVCL NGs and MTX optimal feeding ratio selection result
The Au@PVCL NGs are dissolved in ultrapure water, the materials are respectively fed according to the mass ratio of Au@PVCL NGs to MTX=1:0.10, 1:0.25 and 1:0.50, and the uploading rate and the encapsulation rate are calculated through an ultraviolet-visible spectrophotometer. The uploading rate and the encapsulation rate are respectively as follows: 8.94%/95.88%, 15.18%/68.84%, 23.94%/59.67%. Taking the uploading rate and the encapsulation rate into consideration, finally selecting the mass ratio Au@PVCL NGs to prepare MTX/Au@PVCL NGs by MTX=1:0.25.
(2) Zeta potential and hydrodynamic diameter test results
Referring to FIG. 2 of the specification, PVCL NGs have a surface potential of-5.7 mV and hydrodynamic diameter of 171.5nm. After loading the gold nanoparticles, the surface potential became-16.1 mV and the hydrodynamic diameter became 138.3nm, indicating that the gold nanoparticles had been successfully loaded. After further loading of MTX in situ, the surface potential became-9.0 mV and the hydrodynamic diameter became 158.6nm, indicating that MTX was successfully loaded.
Figure 3 of the accompanying drawings shows the particle size change chart of MTX/Au@PVCL NGs stored in PBS and DMEM medium for different time periods, and the sizes of the three nanogels are not changed obviously after the storage for 7 days, so that the nanogels have good colloid stability.
(3) SEM test results
Referring to the attached drawing 4a of the specification, an SEM image shows that MTX/Au@PVCL NGs prepared by loading nano gold particles and MTX are uniform in form and are in a regular sphere shape. The particle size distribution histogram obtained by SEM image statistics shows that the MTX/Au@PVCL NGs have uniform size distribution. Referring to the figure 4b of the specification, the particle size of MTX/Au@PVCL NG is 116.2nm by particle size distribution statistics.
(4) TEM and Mapping element analysis test result
Referring to the attached drawing 5a of the specification, a TEM image shows that the prepared MTX/Au@PVCL NGs are uniform in shape and are in a regular sphere shape. Referring to the specification and the figure 5b which is a Mapping element energy spectrum, N, O, au and Se elements in the hydrogel are observed to be uniformly distributed in each hydrogel particle, and further the MTX/Au@PVCL NGs are successfully prepared.
(5) In vitro drug responsive release results
Referring to fig. 6 of the specification, in the condition that the pH=7.4 (the pH simulating the physiological environment) and hydrogen peroxide is not present, only about 18% of MTX is released within 3 days, which proves that the material can avoid leakage of medicines in the in-vivo circulation process and maintain better stability. In addition, the drug release rate was below 40% in 7 days in the presence of ph=7.4 and hydrogen peroxide and in the absence of ph=6.5, indicating that the tumor microenvironment of the overexpressed hydrogen peroxide caused partial hydrogel rupture, accelerating MTX release. Notably, in the presence of hydrogen peroxide at ph=6.5, the structural drug of the hydrogel is further increased due to the enhanced oxidizing property of hydrogen peroxide, so that more drug is released, the release rate reaches 50% in 10 hours, and the release rate reaches 68% in 72 hours, and the results show that the diselenide bond crosslinking agent in MTX/au@pvcl NGs has remarkable hydrogen peroxide responsiveness.
(6) Evaluation of in vitro imaging Performance
Referring to the attached figure 7 of the specification, the X-ray attenuation effect of MTX/Au@PVCL NGs is detected, aqueous solutions of MTX/Au@PVCL NGs with Au concentrations of 5, 10, 30, 40 and 50mM are prepared, then a CT imager is used for measuring the X-ray attenuation characteristics of MTX/Au@PVCL NGs with different concentrations, and the MTX/Au@PVCL NGs show excellent X-ray attenuation coefficients, so that the CT imaging performance is good.
(7) Results of CCK-8 cytotoxicity experiments
Referring to FIG. 8 of the specification, toxicity of PVCL NGs and Au@PVCL NGs on cells of different concentrations was studied by CCK-8 method using mouse fibroblast L929 and mouse melanoma cell B16-F10 as models, and PBS-treated cells were used as control groups. Compared with a control group, the PVCL NGs and the Au@PVCL NGs have no obvious cytotoxicity to L929 and B16-F10 cells respectively in a test concentration range, and the cell survival rate is above 85%, which indicates that the PVCL NGs and the Au@PVCL NGs have good cell compatibility.
Referring to FIG. 9 of the specification, toxicity of MTX and MTX/Au@PVCL NGs at different concentrations on L929 and B16-F10 cells respectively was studied by CCK-8 method using mouse fibroblast L929 and mouse melanoma cell B16-F10 as models, and PBS-treated cells were used as control groups. Compared with a control group, the addition of MTX and MTX/Au@PVCL NGs improves the toxicity to L929 cells and the killing effect to B16-F10 cells, and the MTX/Au@PVCL NGs has stronger toxicity to tumor cells than MTX alone. In addition, MTX and MTX/Au@PVCL NGs are less cytotoxic to normal L929 at higher MTX concentrations than B16-F10 cells.
(8) Results of cell phagocytosis experiments
Referring to FIG. 10 of the specification, B16-F10 cells in logarithmic growth phase were collected according to 1X 10 5 Density of individual cells per well was seeded on 12-well plates at 37℃with 5% CO 2 After cells are attached, the fresh culture medium containing MTX/Au@PVCL NGs is replaced, incubated with the cells for 6 hours, the original culture medium is discarded, the cells in the holes are digested and collected by PBS three times, the supernatant is removed by centrifugation, and the cells are digested by 1mL aqua regia for 24 hours. Finally, 3mL of ultrapure water was added to each sample. Quantitative analysis of Au in the sample by ICP-OES shows that MTX/Au@PVCL NGs can be effectively ingested by cancer cells, and the cell ingestion rate increases with increasing Au concentration.
(9) Apoptosis experimental results
Referring to fig. 11 of the specification, the apoptosis was further examined using a flow cytometer. The results show that the cells in the PBS control group, the PVCL NGs and the Au@PVCL NGs are basically in a normal survival state, and in the MTX group and the MTX/Au@PVCL NGs, the B16-F10 cells undergo obvious apoptosis, and the apoptosis rate is 35% and 39% respectively, which indicates that the chemotherapeutic drug MTX can cause apoptosis so as to effectively kill tumor cells.
(10) Evaluation of cell cycle regulatory Effect
Referring to FIG. 12 of the specification, B16-F10 cells in logarithmic growth phase were collected according to 1X 10 5 The density of each cell per well is inoculated on a 12-well plate, after the cells are attached, fresh culture media containing PVCL NGs, au@PVCL NGs, MTX and MTX/Au@PVCL NGs are replaced, and B16-F10 cells are incubated for 12 hours. The results show that MTX and MTX/Au@PVCL NGs treated cells showed a larger change in cell cycle distribution than the PBS control group, which significantly increased the time in the G1 phase, indicating that MTX, and MTX/Au@PVCL NGs can inhibit the growth and proliferation of tumor cells by modulating the cell cycle, preventing the mitotic DNA of the cells from self-replicating.
(11) Evaluation of in vitro macrophage repolarization Effect
Referring to FIG. 13 of the specification, murine macrophage RAW264.7 in log phase was collected according to 2X 10 5 Density of individual cells per well was seeded on 6-well plates at 37℃with 5% CO 2 After overnight incubation under conditions, the medium was discarded, washed three times with PBS, replaced with fresh medium containing 40ng/mL interleukin-4 (IL-4) and incubated for 24 hours, and RAW264.7 was induced to M2 type. Incubation was performed for 24 hours using PBS, lipopolysaccharide (LPS), PVCL NGs, au@PVCL NGs, MTX, and MTX/Au@PVCL NGs, respectively, LPS as a positive control. Macrophages were then digested, centrifuged, collected, stained with fluorescent-labeled FITC-CD206 antibody and PE-CD86 antibody for 30 minutes, and the fluorescence intensity of the samples was measured with a flow cytometer. Experimental results show that the fluorescence intensity of CD206/CD86 in PVCL NGs group has no obvious change, while the fluorescence intensity of CD206 in Au@PVCL NGs group, MTX and MTX/Au@PVCL NGs group is gradually reduced, while the fluorescence intensity of CD86 is gradually reduced The strength is gradually increased. The M1/M2 ratios of LPS positive control, PVCL NGs, au@PVCL NGs, MTX and MTX/Au@PVCL NGs were 7.6, 0.1, 1.3, 3.4 and 4.2, respectively. These results indicate that the experimental group containing the gold nanoparticles and MTX was able to transform TAM from M2 type, which promotes tumor growth, to M1 type, which inhibits tumor growth.
(12) Evaluation of blood compatibility
Referring to FIG. 14 of the specification, the haemocompatibility of MTX/Au@PVCL NGs was determined by a haemolysis experiment. The prepared MTX/Au@PVCL NGs with different concentrations (10, 50, 100 and 100 mug/mL) are incubated with erythrocytes for 2 hours, and the hemolysis rate is less than 5% by measurement, which shows that the MTX/Au@PVCL NGs have good blood compatibility.
(13) Evaluation of in vivo antitumor Effect
Referring to FIG. 15 of the description, 4-6 week female C57BL/6 mice for experiments were purchased from Shanghai Laike laboratory animal center (China, shanghai). Will be 1X 10 6 The B16-F10 cells were inoculated into the right leg of the mice until the tumor volume reached about 100mm 3 On the left and right, mice were randomly divided into 5 groups (5 per group) and then each mouse was injected with 100 μl of PBS solution containing different materials by intratumoral injection: first group PBS (control group), second group PVCL NGs (carrier group), third group Au@PVCL NGs (gold-coated carrier group), fourth group MTX (drug group), fifth group MTX/Au@PVCL NGs (Jin Zaiti + drug group). Tumor volumes and mouse body weights were recorded in mice over 11 days. The results show that the tumor volumes of mice in the control and vehicle groups increased rapidly with time. In addition, the tumor growth of the drug group is inhibited to a certain extent, and the tumor growth of the mice treated by the Jin Zaiti + drug is obviously and effectively inhibited. Experimental results prove that the MTX/Au@PVCL NGs nano-hydrogel synthesized by the method can be applied to tumor treatment in mice, and can effectively inhibit tumor growth.
Referring to FIG. 16 of the specification, on day 11 after treatment, mice tumors were shaved and H & E, TUNEL and Ki-67 staining was performed to observe necrosis, apoptosis and proliferation of tumor tissue. H & E, TUNEL and Ki-67 staining results indicate that the MTX/Au@PVCL NGs group is capable of producing the greatest degree of tumor cell necrosis, apoptosis and proliferation inhibition compared to the other experimental groups.
Referring to figure 17 of the specification, mice on day 11 are taken out for H & E staining test, and the cell morphology of different treatment groups and the cell morphology of the control group are not obviously abnormal, namely no obvious pathology appears, which indicates that the injection of MTX/Au@PVCL NGs does not generate obvious toxic or side effect on normal tissues and organs, and has good biocompatibility.
(14) Evaluation of in vivo tumor microenvironment immunoregulatory Effect
Referring to figure 18 of the specification, to further verify the immune therapy of the organism, each group of mice on day 11 of the treatment process is sacrificed, spleen tissues are taken out under the aseptic condition, sheared and ground and filtered by a 400-mesh filter screen to obtain a cell suspension, T lymphocyte suspensions are obtained by nylon wool columns, and after the obtained T cells are respectively marked by antibodies with different fluorescence FITC-CD4 and PE-CD8, quantitative analysis is carried out on CD4+ T cells and CD8+ T cells in the spleen tissues by a flow cytometer. The results show that the spleen infiltration CD4+T and CD8+T cell content effects of MTX (drug group) and MTX/Au@PVCL NGs (Jin Zaiti + drug package) are superior to those of the vector group. In addition, spleen-infiltrating cd4+ T and cd8+ T cells content of MTX/au@pvcl NGs (package Jin Zaiti + drug) were highest.
Referring to FIG. 19 of the specification, to assess the phenotypic changes of tumor-associated macrophages. Groups of mice on day 11 of the treatment were sacrificed, their tumor tissues were removed, tissue fixed, and the fixed samples were paraffin-embedded and sectioned for immunofluorescent staining of M1 macrophages (iNOS, red) and M2 macrophages (Arg-1, green) markers. As a result, it was found that MTX (drug group) and MTX/Au@PVCL NGs (package Jin Zaiti + drug group) showed an increase in iNOS (M1 marker) fluorescence signal, whereas Arg-1 (M2 marker) fluorescence signal was decreased, MTX/Au@PVCL NGs (package Jin Zaiti + drug group) showed the strongest red fluorescence signal and the weakest green fluorescence signal. Experiments show that MTX/Au@PVCL NGs can repolarize macrophages from M2 to M1. MTX/Au@PVCL NGs regulate the tumor microenvironment into an anti-tumor state by regulating macrophage polarization.
(15) CT imaging results of tumors in vivo
Referring to description attached 20, a B16-F10 subcutaneous tumor model was constructed in a black mouse, and the CT imaging effect of tumor sites was evaluated by intratumoral injection of PBS solution of MTX/au@pvcl NGs (100 μl, [ Au ] =50 mM). The tumor site signal peaks after 30 minutes of injection, and the tumor signal value is: 74HU. The result proves that the material has good tumor CT imaging effect in tumor-bearing mice after the gold nanoparticles are coated, and can be used as a contrast agent for in vivo tumor imaging.
Advantageous effects
(1) The invention has simple process, easy operation and separation, wide raw material sources and good development and application prospect;
(2) The diselenide bond crosslinking nanogel loaded with the nano gold particles and the methotrexate has good colloid stability, X-ray attenuation characteristic, hydrogen peroxide responsive drug release performance and cancer cell killing effect;
(3) The invention utilizes the diselenide bond crosslinking nanogel loaded with the nano gold particles and the methotrexate to realize CT diagnosis of tumor parts, and simultaneously chemotherapeutic drugs MTX in the nanogel can effectively kill tumor cells, and the drug MTX and the nano gold particles jointly regulate TAM to be converted into an anti-tumor M1 type, thereby regulating and controlling tumor microenvironment to enhance the chemotherapeutic effect.
Drawings
FIG. 1 is a schematic diagram of synthesis and application of MTX/Au@PVCL NGs nanogel prepared by the method;
FIG. 2 shows Zeta potential diagrams (a) and particle size distribution diagrams (b) of PVCL NGs, au@PVCL NGs and MTX/Au@PVCL NGs prepared by the method of the invention;
FIG. 3 is a graph showing the variation of the hydration particle size of MTX/Au@PVCL NGs prepared by the method of the invention in PBS or DMEM solution at different time periods;
FIG. 4 is a SEM image (a) and particle size distribution histogram (b) of MTX/Au@PVCL NGs prepared according to the present invention;
FIG. 5 shows TEM images (a) of MTX/Au@PVCL NGs and TEM and Mapping element analysis images (b) of MTX/Au@PVCL NGs prepared by the method;
FIG. 6 is an in vitro drug response release curve of MTX/Au@PVCL NGs prepared by the invention under different conditions;
FIG. 7 is a graph showing the linear relationship between CT values of MTX/Au@PVCL NGs and Au concentrations at different Au concentrations according to the present invention;
FIG. 8 shows the cell viability of L929 cells (a) and B16-F10 cells (B) prepared according to the present invention after treatment with different concentrations ([ PVCL NGs ] = 0-300 μg/mL) for 24 hours, as measured by CCK8 method, of PVCL NGs and Au@PVCL NGs;
FIG. 9 shows the cell viability of L929 cells (a) and B16-F10 cells (B) prepared according to the present invention after treatment for 24 hours at different concentrations ([ MTX ] =0-40. Mu.g/mL) as measured by CCK-8 method for MTX and MTX/Au@PVCL NGs;
FIG. 10 shows the phagocytosis of Au element after co-incubation of MTX/Au@PVCL NGs prepared according to the invention with B16-F10 cells for 6 hours;
FIG. 11 is a graph of apoptosis flow assay and histogram of apoptosis quantification after 4 hours of co-incubation of Au@PVCL NGs, MTX and MTX/Au@PVCL NGs prepared by the invention with B16-F10 cells;
FIG. 12 is a graph showing cell cycle profiles of PVCL NGs, au@PVCL NGs, MTX and MTX/Au@PVCL NGs prepared according to the present invention after 12 hours of co-incubation with B16-F10 cells;
FIG. 13 is a graph (a) and a histogram (b) of quantitative analysis of the flow assay of murine macrophage RAW 264.7 by PVCL NGs, au@PVCL NGs, MTX and MTX/Au@PVCL NGs and lipopolysaccharide prepared according to the present invention;
FIG. 14 is a photograph of a quantitative analysis chart (a) of the blood compatibility of MTX/Au@PVCL NGs prepared by the method of the present invention at different concentrations and a photograph of a real photographed solution of the blood compatibility experiment;
FIG. 15 is a graph of change in body weight (a) and relative volume (B) of each group of melanoma-bearing mice in B16-F10 groups treated with PBS, PVCL NGs, au@PVCL NGs, MTX or MTX/Au@PVCL NGs for 11 days;
FIG. 16 is a graph showing the results of staining with H & E, TUNEL and Ki-67 of tumor tissue of tumor-bearing mice after B16-F10 melanoma-bearing mice received PBS, PVCL NGs, au@PVCL NGs, MTX or MTX/Au@PVCL NGs for 11 days;
FIG. 17 is a graph showing H & E staining results of heart, liver, spleen, lung, kidney sections of tumor-bearing mice after 11 days of treatment with PBS, PVCL NGs, au@PVCL NGs, MTX or MTX/Au@PVCL NGs in B16-F10 melanoma-bearing mice;
FIG. 18 is a graph (a) showing the flow assay of CD4+, CD8+ T cell expression in spleen tissue, a graph (B) showing the quantitative analysis of CD4+ T lymphocytes, and a graph (c) showing the quantitative analysis of CD8+ T lymphocytes after 11 days of treatment with PBS, PVCL NGs, au@PVCL NGs, MTX, or MTX/Au@PVCL NGs in B16-F10 melanoma-bearing mice;
FIG. 19M1 macrophage (iNOS, red) and M2 macrophage (Arg-1, green) markers immunofluorescence sections;
FIG. 20 is a graph of CT images and quantification of CT values of tumors of mice prepared according to the present invention in PBS (100. Mu.L, [ Au ] = 0.1M) before and after intratumoral injection for different time periods.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
Example 1
(1) 469.5mg of N-Vinylcaprolactam (VCL), 5mg of 2-bromoethyl acrylate diselenether (diselener) and 8.8mg of sodium dodecyl sulfate were dissolved in 44mL of ultrapure water, and 5mL of an aqueous solution of ethylene glycol acetoacetate methacrylate (AAEM, 14 mg/mL) was added thereto, followed by addition of a solution of sodium dodecyl sulfate in N 2 Stirring at 70deg.C for 30min. Then, 1mL of an aqueous solution of azodicarbonethyl-2-isobutyl amidine (ACMA, 11.7 mg/mL) was added to the above solution. The reaction was stirred for 4 hours. Then, the reacted solution is put into a dialysis bag with the molecular weight cut-off of 8000-14000 for dialysis for 3 days, and freeze-dried to obtain the poly-N-vinyl caprolactam nano hydrogel (PVCL NGs).
(2) PVCL NGs (180 mg) prepared in the step (1) were taken and dispersed in 18mL of ultrapure water, then 1mL of aqueous chloroauric acid solution (30 mg/mL) was added dropwise, stirred in an ice bath for 30min, then 1mL of an ice water solution of sodium borohydride (22.5 mg/mL) was added rapidly thereto as a reducing agent, and stirred overnight. And then, placing the reacted solution into a dialysis bag with the molecular weight cutoff of 8000-14000, dialyzing the solution against water for 3 days, and freeze-drying to obtain the diselenide bond crosslinked nanogel (Au@PVCL NGs) loaded with the nanogold particles.
(3) Dispersing Au@PVCL NGs (15 mg) prepared in the step (2) in 15mL of ultrapure water, slowly dropwise adding 500 mu L of MTX methanol solution according to the mass ratio of Au@PVCL NGs to MTX=1:0.10, 1:0.25 and 1:0.50, and stirring overnight. Centrifuging the reacted solution (1000 rpm,10 minutes), separating supernatant (MTX/Au@PVCL NGs), collecting MTX precipitate, repeating the steps three times, dissolving the MTX precipitate in a methanol solution, calculating the mass of the MTX precipitate by an ultraviolet-visible spectrophotometer, determining the mass of MTX in NG solution by subtracting the mass of MTX in the precipitate from the initial total mass of MTX, and calculating the loading rate and encapsulation efficiency. The uploading rate and the encapsulation rate are respectively as follows: 8.94%/95.88%, 15.18%/68.84%, 23.94%/59.67%. And finally, selecting the mass ratio Au@PVCL NGs to MTX=1:0.25 to prepare the poly-N-vinyl caprolactam nanogel (MTX/Au@PVCL NGs) loaded with the nanogold particles and the MTX by comprehensively considering the uploading rate and the encapsulation rate. Upload rate (%) = (loaded MTX weight)/(loaded MTX weight+au@pvcl NGs carrier weight) ×100%; encapsulation efficiency (%) = (loaded MTX weight)/(total MTX weight dosed) ×100%.
Example 2
PVCL NGs, au@PVCL NGs and MTX/Au@PVCL NGs solutions (500. Mu.g/mL) prepared in example 1 were used to measure surface potential and hydrodynamic diameter. As a result of the measurement, as shown in FIG. 2, PVCL NGs have a surface potential of-5.75 mV and a hydrodynamic diameter of 171.5nm. After loading the gold nanoparticles, the surface potential became-16.1 mV and the hydrodynamic diameter became 138.3nm, indicating successful loading of the gold nanoparticles. After further loading of MTX in situ, the surface potential became-9.0 mV and the hydrodynamic diameter became 158.6nm, indicating successful loading of MTX. Meanwhile, as shown in the figure 3, after MTX/Au@PVCL NGs are stored in PBS and DMEM solutions for 7 days, the size is not changed greatly, which indicates that the prepared multifunctional nano hydrogel has good colloid stability. The MTX/Au@PVCL NGs are observed through SEM, and the results are shown in FIG. 4a, and the results show that the prepared MTX/Au@PVCL NGs are uniform in morphology and are in regular round sphere shape. The particle size distribution histogram obtained by the SEM image statistics shows that the MTX/Au@PVCL NGs are uniformly distributed in size, and the particle size is 116.1+/-21.19 nm. In addition, as shown in fig. 5a, after the gold nanoparticles and the MTX are loaded, the obtained MTX/Au@PVCL NGs are still in a spherical shape, the size is not changed significantly, and the gold nanoparticles can be clearly observed to be uniformly distributed in the nanogel from an enlarged view. As shown in fig. 5b, the Mapping element energy spectrum can observe that N, O, au and Se elements in the hydrogel are uniformly distributed in the hydrogel particles.
Example 3
The MTX/Au@PVCL NGs solution (500. Mu.g/mL) prepared in example 1 was taken and its in vitro drug-responsive release properties were studied. MTX/Au@PVCL NGs were dissolved in 5.0mL of buffer and added to a dialysis bag. Then, the mixture was shaken at 37℃and 90rpm and immersed in 45mL of the buffer. Buffers were in the following three groups: first group ph=7.4 (H 2 O 2 (-), second group ph=6.5 (H) 2 O 2 (-), third group ph=7.4+h 2 O 2 ([H 2 O 2 ]=0.1 mM), ph=6.5+h for the fourth group 2 O 2 ([H 2 O 2 ]=0.1 mM). At each time point, 5.0mL of release solution was removed and 5.0mL of the same fresh buffer was replenished. The release test was repeated three times under the same conditions. The absorbance at 320nm of the released MTX was determined by UV-vis spectroscopy. As shown in fig. 6, only about 22.4% of MTX was released within the first 3 days, demonstrating that the material remained stable under normal physiological conditions, avoiding excessive drug leakage. Drug release rates within 7 days of the second and third groups were 28.9% and 45.2%, respectively. It is notable that the fourth group of drug release rate was approximately 50% at 24 hours and reached 70.3% at 72 hours, because hydrogen peroxide was more oxidizing in an acidic environment, which can break more diselenide bonds in MTX/Au@PVCL NGs and release more drug.
Example 4
The MTX/Au@PVCL NGs solution prepared in example 1 is taken to examine the in-vitro X-ray attenuation characteristics, and MTX/Au@PVCL NGs aqueous solutions with Au concentrations of 5, 10, 20, 30 and 60mM are respectively prepared, and then the X-ray attenuation characteristics of the MTX/Au@PVCL NGs with different concentrations are measured by a CT imager. As shown in FIG. 7, MTX/Au@PVCL NGs exhibit excellent X-ray attenuation coefficients, indicating good CT imaging performance.
Example 5
Collecting B16-F10 cells and L929 cells in logarithmic growth phase according to 1X 10 4 Cell density per well was seeded on 96 well cell culture plates and placed in 5% CO 2 Incubate overnight at 37 ℃. After discarding the medium, 90. Mu.L of serum-free medium was changed per well, and 10. Mu.L of PVCL NGs and Au@PVCL NGs ([ PVCL NGs) containing different concentrations were added]=10, 50, 100, 200, 300 μg/mL) of PBS solution, 10 μl of PBS was added as a control group. The cell culture plates were kept on 5% CO 2 Incubation was continued for 24 hours at 37 ℃. The original medium was then discarded, 100. Mu.L of a fresh medium solution containing 10% CCK-8 was added, and after further incubation for 3 hours, the medium was placed in a multifunctional microplate reader and absorbance was measured at a test wavelength of 450nm, and the results are shown in FIG. 8. Compared with a PBS control group, the PVCL NGs and the Au@PVCL NGs have no obvious cytotoxicity to B16-F10 cells and L929 cells within the test concentration range, and the cell survival rate is above 90%, which indicates that the PVCL NGs and the Au@PVCL NGs have good cell compatibility.
Collecting B16-F10 cells and L929 cells in logarithmic growth phase according to 1X 10 4 Cell density per well was seeded on 96 well cell culture plates and placed in 5% CO 2 Incubate overnight at 37 ℃. After discarding the medium, 90. Mu.L of serum-free medium was replaced per well and 10. Mu.L of medium containing different concentrations of MTX and MTX/Au@PVCL NGs ([ MTX)]=1, 5, 10, 40 μg/mL) of PBS solution, 10 μl of PBS was added as a control group. The cell culture plates were then further placed at 5% CO 2 Incubation was continued for 24 hours at 37 ℃. The original medium was then discarded, 100. Mu.L of a fresh medium solution containing 10% CCK-8 was added, and after further incubation for 3 hours, the medium was placed in a multifunctional microplate reader and the absorbance was measured at a test wavelength of 450nm, as followsFig. 9 shows the same. MTX and MTX/Au@PVCL NGs showed significant cytotoxicity against B16-F10 cells and L929 cells over the range of concentrations tested, compared to the PBS control. Compared with a control group, the MTX and MTX/Au@PVCL NGs have obvious toxicity to L929 cells and effective killing effect to B16-F10 cells, and the MTX/Au@PVCL NGs have stronger toxicity to tumor cells than MTX alone. In addition, MTX and MTX/Au@PVCL NGs are less cytotoxic to normal L929 cells than B16-F10 cells.
Example 6
Collecting B16-F10 cells in logarithmic growth phase according to 1X 10 5 Cell density per well was seeded on 12 well cell culture plates and placed in 5% CO 2 Incubate overnight at 37 ℃. After discarding the medium, 900. Mu.L of serum-free medium was replaced per well and 100. Mu.L of Au@PVCL NGs ([ Au ] containing different concentrations was added]=2, 10, 20, 40, 80 μg/mL) in PBS. The cell culture plates were kept on 5% CO 2 Incubation was continued for 4 hours at 37 ℃, medium was discarded and washed three times with PBS. The cells were digested with pancreatin and centrifuged, and the supernatant was discarded, and the resulting precipitate was digested with 1mL of aqua regia for 24 hours, and after complete digestion, 3mL of ultrapure water was added, and the Au concentration in the sample was measured with ICP-OES. As shown in figure 10, the results of quantitative analysis on Au show that the materials can be effectively absorbed by cells, and the absorption rate of the materials is increased along with the increasing of the concentration of the materials, so that the prepared Au@PVCL NGs can be well phagocytized by cancer cells.
Example 7
First, B16-F10 cells were cultured at 1X 10 5 The density of individual cells/wells was inoculated into 12-well plates, after overnight incubation, the medium was decanted, and medium containing Au@PVCL NGs, MTX and MTX/Au@PVCL NGs was added for co-incubation for 24 hours, PBS was used as a control group. And pouring out the culture medium, washing 3 times by PBS, digesting the cells by pancreatin, transferring into a 5mL centrifuge tube, centrifuging for 5 minutes at 1000 revolutions to remove supernatant, adding 1mL of PBS, blowing uniformly, putting into an ice box, dyeing by an Annexin V/PI apoptosis detection kit, and loading into a machine for flow detection. As shown in FIG. 11, cells were apoptotic after MTX and Au@PVCL NGs treatment at 35% and 39% apoptosis rate, respectively, and MTX/Au@PVCL NGs showed more remarkable fineness Apoptosis phenomenon, these results demonstrate that chemotherapeutic drug MTX can cause apoptosis to effectively kill tumor cells.
Example 8
Collecting B16-F10 cells in logarithmic growth phase according to 1X 10 5 Cell density per well was seeded on 12 well cell culture plates and placed in 5% CO 2 Incubate overnight at 37 ℃. After the medium was discarded, 900. Mu.L of medium was changed per well, and MTX and prepared PVCL NGs, au@PVCL NGs and MTX/Au@PVCL NGs (100. Mu.g/mL) were added to co-culture with B16-F10 cells for 12 hours, PBS was used as a control group. The cells were then washed, digested, centrifuged and collected in 1.5mL centrifuge tubes. To the tube, 1mL of 70% ethanol precooled in an ice bath was added to fix the cells for 12 hours. The cells were centrifuged, washed 3 times, the supernatant was aspirated, 0.5mL of pyridine iodide staining solution was added to each well, the cells were resuspended, and the cells were incubated at 37℃for 30min in the dark. The cell cycle was then analyzed by flow cytometry. As a result, as shown in fig. 12, PVCL NGs, au@pvcl NGs treated cells did not show a large change in cell cycle distribution compared to the control group. In contrast, after MTX and MTX/Au@PVCL NGs treatment, the cell rate at the G1 phase increases, suggesting that MTX and MTX/Au@PVCL NGs can inhibit the growth and proliferation of tumor cells by blocking the self-replication of DNA of cell mitosis.
Example 9
RAW 264.7 cells were first cultured at 1X 10 5 The individual cells/well density was seeded into 12-well plates, cultured overnight, and then treated with fresh medium containing IL-4 (40 ng/mL) for 12 hours to convert it to M2-type macrophages. PBS was used as a control group, MTX (5. Mu.g/mL) and prepared PVCL NGs, au@PVCL NGs and MTX/Au@PVCL NGs (50. Mu.g/mL) were CO-cultured with RAW 264.7 cells for 12 hours, respectively, and incubated at 37℃and 5% CO 2 Co-cultivation was carried out for 24 hours under the conditions. After that, the cells were gently washed 2 times with PBS, and the cells were collected and resuspended in 500. Mu.L of PBS. mu.L of anti-CD206-FITC and anti-CD86-PE were added to the above cell suspension, and incubated for 30 minutes under dark conditions for staining. And then centrifuged and washed to remove excess antibody, and resuspended in PBS solution for flow cytometry analysis. Analysis by flow cytometry, resultsAs shown in FIG. 13, there was no significant change in the fluorescence intensity of cells in the PVCL NGs group, while the expression of CD206 was decreased in the Au@PVCL NGs, MTX, and MTX/Au@PVCL NGs groups, while the expression of CD86 was gradually increased. The M1/M2 ratios of LPS positive control, PVCL NGs, au@PVCL NGs, MTX and MTX/Au@PVCL NGs were 7.6, 0.1, 1.3, 3.4 and 4.2, respectively. These results indicate that the experimental group containing the gold nanoparticle and MTX can transform TAM from M2 type for promoting tumor growth to M1 type for inhibiting tumor growth, thereby being capable of better inhibiting tumor growth.
Example 10
1.5mL of blood was taken from the eye vein of the mice and dispersed in 3.5mL of PBS. After repeated centrifugation (2000 rpm,10 min) and redispersion, the obtained purified Red Blood Cells (RBCs) were dispersed with 5mL PBS for use. 900. Mu.L of MTX/Au@PVCL NGs dispersion at various concentrations (10, 20, 40, 80, 160. Mu.g/mL) was prepared, 100. Mu.L of RBCs dispersion was added to each, incubated at 37℃for 2 hours, each sample was centrifuged at 13000rpm for 15min, and the supernatant was assayed for absorbance at 540nm using UV-vis spectroscopy. Likewise, water and PBS-treated RBCs were set as positive and negative controls, respectively. The hemolysis rate of each group of samples was calculated by the following formula:
hemolysis (%) = (OD sample-OD negative)/(OD positive-OD negative) ×100%
Wherein, OD sample, OD positive and OD negative represent absorbance at 540nm of sample group, positive control group and negative control group, respectively. As shown in FIG. 14, MTX/Au@PVCL NGs have good anti-hemolytic properties.
Example 11
The C57BL/6 female mice purchased from Shanghai Laek laboratory animal center for 4-6 weeks were subjected to animal experiments, all strictly according to the animal ethics committee standard of Donghua university. According to 2X 10 6 The individual cell/mouse dose was subcutaneously injected in the right leg of C57BL/6 female mice. To the tumor volume of about 100mm 3 At this time, the tumor-bearing mice were randomly divided into 5 groups (5 per group): intratumoral injection of PBS (100 μL) (control group), intratumoral injection of PVCL NGs (100 μL) (vehicle group), intratumoral injection of Au@PVCL NGs (100 μL) (gold-coated vehicle group), intratumoral injection of MTX (100 μL) (drug group), tumorMTX/Au@PVCL NGs (100. Mu.L) (package Jin Zaiti + drug group) were injected internally. As shown in fig. 15 (a), the results of the weight monitoring of mice were shown, and after the mice were treated in each group, no significant effect was exerted on the weight of the mice. The results of the tumor volume monitoring in mice are shown in fig. 15 (b), the tumor volume of the control group increases most rapidly, and the tumor continues to increase after the tumor growth is inhibited for a period of time by the au@pvcl NGs vehicle group and the MTX alone drug chemotherapy group. Meanwhile, the tumor growth of mice in the MTX/Au@PVCL NGs package Jin Zaiti + drug group is obviously inhibited. The results show that the MTX/Au@PVCL NGs prepared by the method has good treatment effect.
11 days after treatment, tumor tissues of mice subjected to different treatments were shaved, and H & E, TUNEL and Ki-67 staining was performed to observe necrosis, apoptosis and proliferation of the tumor tissues. Experimental results as shown in fig. 16, H & E, TUNEL and Ki-67 staining results indicate that MTX/au@pvcl NGs are capable of producing the greatest degree of tumor cell necrosis, apoptosis and proliferation inhibition. Meanwhile, the main tissue organ is subjected to H & E staining, and the result is shown in figure 17, the cell morphology in each tissue is not different from that in a control group, and the injection of the material does not generate obvious toxic or side effect on the normal tissue organ, so that the tissue organ has good biocompatibility.
Example 12
To further verify the immunoregulatory effect of nanogel, on day 11 of the treatment, mice of each group were sacrificed and their spleen tissues were removed under sterile conditions, sheared and ground and filtered through a 400 mesh filter to obtain cell suspensions, T lymphocyte suspensions were obtained by nylon wool column, and the obtained T cells were labeled with antibodies with different fluorescence FITC-CD4 and PE-CD8, respectively, and then CD4 in the spleen tissues was purified by flow cytometry + T cells and CD8 + T cells were quantitatively analyzed. The results are shown in FIG. 18, spleen-invasive CD4 of MTX (drug group) and MTX/Au@PVCL NGs (vector+drug) + T and CD8 + The T cell content effect is better than that of the vector group. In addition, spleen-infiltrating CD4 of MTX/Au@PVCL NGs (vector+drug) + T and CD8 + The highest T cell content, these results demonstrate that the gold nanoparticle and MTX can effectively promote T cell immune responses.
Example 13
To evaluate tumor-associated macrophage polarization distribution, groups of mice on day 11 of the treatment were sacrificed, tumor tissues of the mice undergoing different treatments were shaved, tissue fixation was performed, paraffin-embedded samples after fixation, and sections were cut, and immunofluorescent staining was performed on markers of tumor-associated macrophages M1 and tumor-associated macrophages M2. As a result, as shown in FIG. 19, the iNOS (M1 marker) fluorescence signal of MTX (drug group) and MTX/Au@PVCL NGs (package Jin Zaiti + drug group) increased, whereas the Arg-1 (M2 marker) fluorescence signal decreased, the MTX/Au@PVCL NGs (package Jin Zaiti + drug group) had the strongest red fluorescence signal and the weakest green fluorescence signal. Experiments show that MTX/Au@PVCL NGs repolarize macrophages from M2 type to M1 type, and the tumor microenvironment is changed from a pro-tumor state to an anti-tumor state.
Example 14
The animal experiments were performed with C57BL/6 female mice purchased from Shanghai Laike laboratory animal center for 4-6 weeks. According to 2X 10 6 The individual cell/mouse dose was subcutaneously injected in the right leg of C57BL/6 female mice. To the tumor volume of about 100mm 3 At this time, the CT imaging effect of the tumor site was evaluated by intratumoral injection of PBS solution (100. Mu.L) of MTX/Au@PVCL NGs. As shown in FIG. 20, the CT signal of the tumor site of the mice was significantly enhanced (74 HU) after 0.5 hours of injection of MTX/Au@PVCL NGs, which was much higher than the CT signal value (33 HU) of the tumor site before injection. The results show that MTX/Au@PVCL NGs have good tumor CT imaging effect in tumor-bearing mice, and can be used as a contrast agent for in vivo tumor imaging.
Claims (10)
1. The diselenide bond crosslinked composite nanogel is characterized in that the composite nanogel is a diselenide bond crosslinked poly-N-vinyl caprolactam nanogel loaded with nano gold particles and medicines.
2. The diselenide bond crosslinked composite nanogel according to claim 1, wherein the diselenide bond crosslinked poly (N-vinyl caprolactam) nanogel is a three-dimensional network polymer formed by precipitation polymerization of raw materials containing monomer N-vinyl caprolactam VCL, diselenide bond-containing crosslinking agent and acetoacetic acid ethylene glycol methacrylate AAEM; the drug is methotrexate.
3. A preparation method of diselenide bond crosslinking composite nano gel comprises the following steps:
(1) Mixing monomer N-vinyl caprolactam VCL, surfactant, crosslinking agent containing diselenide bond and water, N 2 Stirring in water bath under the environment, adding an initiator and acetoacetic acid ethylene glycol methacrylate (AAEM), continuing stirring reaction, cooling, dialyzing, and freeze-drying to obtain the diselenide-crosslinked poly-N-vinyl caprolactam nano hydrogel PVCL NGs;
(2) Mixing PVCL NGs with water, adding chloroauric acid solution, stirring, adding sodium borohydride ice water solution, continuing stirring reaction, dialyzing, and freeze-drying to obtain the diselenide bond crosslinked nanogel Au@PVCL NGs loaded with the nanogold particles;
(3) And mixing the Au@PVCL NGs, water and the medicine, and stirring for reaction to obtain the diselenide bond crosslinked composite nano gel MTX/Au@PVCL NGs.
4. The method according to claim 3, wherein the surfactant in the step (1) is Sodium Dodecyl Sulfate (SDS); the initiator is azodicarboxylic ethyl-2-isobutyl amidine hydrate ACMA; the cross-linking agent containing diselenide bond is diacryloyloxy diethyl diselenide;
the mass ratio of VCL, AAEM, the crosslinking agent containing diselenide bond and the surfactant in the step (1) is 20-30:2-3:1-1.5:0.1-0.2.
5. The method according to claim 3, wherein N in the step (1) 2 The temperature of water bath stirring in the environment is 60-70 ℃, and the stirring time is 30-40 min; the reaction time of continuous stirring is 4-6 h.
6. The preparation method according to claim 3, wherein the mass ratio of chloroauric acid, sodium borohydride and PVCL NGs in the step (2) is 1-1.5:1-1.5:5-7.5.
7. The preparation method according to claim 3, wherein the stirring temperature of the chloroauric acid solution added in the step (2) is 0 ℃, and the stirring time is 0.5-2 hours; adding sodium borohydride ice water solution, continuously stirring and reacting at 3-10 deg.c for 8-10 hr.
8. The preparation method according to claim 3, wherein the mass ratio of the drug to the Au@PVCL NGs in the step (3) is 1-1.5:5-7; the medicine is methotrexate MTX; the stirring reaction temperature is room temperature, and the stirring reaction time is 8-10 hours.
9. The method according to claim 3, wherein the dialysis in the steps (1) and (2) is carried out by using a dialysis bag having a molecular weight cut-off of 8000 to 14000 and a dialysis time of 3 to 5 days.
10. Use of the diselenide-bonded cross-linked composite nanogel of claim 1 in the preparation of a chemotherapeutic agent for CT diagnosis of melanoma and/or enhancement of tumor microenvironment immunomodulation.
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