Nanocomposite as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, and discloses a nano-composite and a preparation method and application thereof, the nano-composite is used for realizing diagnosis and treatment integration of glioblastoma, and the nano-composite comprises nanogel, nano-particles loaded with ultra-small Fe3O4, temozolomide and PD-L1siRNA. The preparation process of the nano material is relatively easy, and the USIO NGs-TMZ / siRNA compound is relatively stable in vivo, so that the loaded gene can be prevented from being degraded by enzyme in vivo in the transportation process, the siRNA is promoted to realize rapid endosome escape, and the tumor treatment effect is enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a kind of nanocomposite and its preparation method and application. BACKGROUND

[0002] Glioblastoma (GBM) is the most common primary central nervous system (CNS) malignancy, accounting for 45.2% of all primary malignant brain tumors and 54% of all intracranial gliomas. GBM has high invasiveness, with a median survival of only 12-15 months. The current multimodal comprehensive treatment for GBM includes surgical treatment, radiotherapy, chemotherapy, and molecular targeted therapy. The treatment principle is to maximize the safe resection, reduce the tumor volume, and accurately diagnose the pathology, so as to provide a reliable basis for the follow-up treatment. The traditional postoperative radiotherapy is combined with Temozolomide (TMZ) at a dose of 60 Gy, 2 Gy per fraction for 6 weeks, aiming to improve the local control rate and survival time of the tumor. So far, it is still an important physical treatment method. The FDA-approved chemotherapy and molecular targeted drugs mainly include Temozolomide for early GBM treatment and Bevacizumab for recurrent GBM. Tumor treating fields (TTF) is a non-invasive physical mode for tumor treatment, which induces DNA double-strand breaks (DSBs) by low-intensity and medium-frequency alternating electric fields, weakens the rejoining of DSBs, and interferes with cell mitosis, selectively killing proliferating tumor cells. Although this multimodal treatment has been continuously improved, it has prolonged the survival of patients to a certain extent, but the recurrence rate and five-year survival rate have not been significantly improved. The reason is that GBM is essentially different from other solid tumors in the system: due to the existence of blood-brain barrier and blood-brain tumor barrier, special immune microenvironment and unique tumor phenotype, the development of new treatment methods for GBM faces great challenges. In recent years, the research on immune checkpoint co-stimulatory and co-inhibitory receptors has been increasing. In glioblastoma, the expression of PD-L1 is related to tumor grade and prognosis. Therefore, PD-1 / PD-L1 has become an important target in glioma immunotherapy. With the development of RNA interference technology, siRNA has been increasingly used in glioblastoma. Its mechanism is to silence the target gene using RNA interference technology, thereby efficiently inhibiting the expression of related genes in glioblastoma, regulating tumor cell cycle, and inhibiting tumor cell proliferation.However, in the research of immunotherapy and precise targeted therapy, many patients still cannot obtain satisfactory curative effect and appear immune-related adverse reactions. The main obstacle of central nervous system drug delivery is the blood-brain barrier (BBB), which hinders more than 90% of small molecule drugs and almost 100% of large therapeutic drugs from reaching the tumor area. Although the vascular endothelial growth factor secreted by tumor cells can destroy tight junctions, leading to increased capillary permeability, ultimately resulting in severe damage to the BBB. The increase in interstitial pressure within the tumor and the presence of certain feeding blood vessels form the blood-brain tumor barrier (BBTB), limiting the application of drugs and carriers in tumors, and severely reducing the therapeutic effect of drugs. Therefore, exploring treatment methods that synergistically enhance the therapeutic effect of tumor drugs, improving the efficiency of drug penetration through the BBTB, increasing the local drug concentration in the brain tumor area, maximizing the therapeutic efficacy of drugs, and avoiding drug-related adverse reactions are the key to improving the therapeutic effect of brain tumors. Magnetic resonance guided focused ultrasound (MRgFUS) is a combination of MRI and FUS technology. It realizes accurate positioning of the target area through three-dimensional high-resolution imaging of MRI, and better displays the details of the target area with higher tissue resolution. During FUS treatment, MRI can be used for real-time non-invasive precise temperature measurement. Combined with computer-aided software, it can monitor the temperature rise and potential tissue changes in real time, and observe the opening of the BBTB under low-frequency ultrasound and make corresponding adjustments. It can open the BBTB under the monitoring of magnetic resonance, and visualize the transport and release of the carried drugs.

[0003] Nanogels form a three-dimensional structure by cross-linking hydrophilic polymers, which are prepared from natural proteins, polysaccharides and polymers. Its characteristic is that it can load macromolecules, polypeptides and protein-based drugs without affecting its own structure. The drug targeting delivery system based on nanogels is more safe and effective than traditional chemotherapy regimens to cross the blood-brain barrier, is preferentially taken up by tumor cells, and ultimately identifies and kills tumor cells without damaging healthy tissues. Nanogels prepared from PEI are very soft, so they are very easy to pass through physiological barriers and can also be easily phagocytosed by cells.

[0004] However, there is no nanogel material for the diagnosis and treatment of glioblastoma at present.

[0005] Therefore, the application takes nanogel as a carrier, loads super-small Fe3O4 nanoparticles, realizes MR imaging of tumors, and further loads first-line clinical chemotherapeutic drug Temozolomide (TMZ) and PD-L1 siRNA due to a large number of aminos on the surface, realizes tumor chemotherapy, and synergistically resists tumors, enhances the anti-tumor effect of glioblastoma. The preparation process of the material is relatively easy, the USINGs-TMZ / siRNA complex is relatively stable in the body, can protect the loaded gene from degradation by enzymes in the body during transportation, promotes the rapid endosome escape of siRNA, and enhances the tumor treatment effect. Subsequently, through in-vitro experiments of the USINGs-TMZ / siRNA gene complex, the silencing effect of PD-L1 and the anti-tumor effect of combination with TMZ are evaluated. SUMMARY

[0006] In order to solve the above problems, the application provides a kind of nanocomposite for realizing glioblastoma diagnosis and treatment integration.

[0007] The nanocomposite of the application takes nanogel as a carrier, loads super-small Fe3O4 nanoparticles, realizes magnetic resonance imaging of central nervous system malignant tumors. Because of a large number of aminos on the surface, it can further load first-line clinical chemotherapeutic drug Temozolomide (TMZ) and PD-L1 siRNA, and selectively release drugs in the brain tumor area by using MRgFUS technology, thereby reducing the systemic toxic side effects of chemotherapeutic drugs, combining gene therapy to promote further apoptosis of tumor cells, revealing the molecular biological mechanism of immunotherapy, and providing a new means for the treatment of refractory malignant brain tumors. The preparation process of the material is relatively easy, the USINGs-TMZ / siRNA complex is relatively stable in the body, can protect the loaded gene from degradation by enzymes in the body during transportation, promotes the rapid endosome escape of siRNA, and enhances the tumor treatment effect. Subsequently, through in-vitro experiments of the USINGs-TMZ / siRNA gene complex, the silencing effect of PD-L1 and the anti-tumor effect of combination with TMZ are evaluated. Magnetic resonance phased array ultrasound system is used to realize accurate positioning and scaling of the target area, clarify the experimental parameters for optimizing BBTB opening, guide the nanomaterials to reach the tumor area through BBTB, improve the safety of magnetic resonance visualization monitoring, and explore a new method for stably increasing the drug concentration in brain tumor tissue.

[0008] In order to achieve the above purpose, the application provides the following technical scheme:

[0009] In a first aspect of the application, a nanocomposite is provided, comprising nanogel, the nanogel loading super-small Fe3O4 nanoparticles, Temozolomide and PD-L1 siRNA.

[0010] In a second aspect, the present application provides a method for preparing a nanocomposite, comprising the following steps:

[0011] S1. preparing sodium citrate stabilized ultra-small Fe3O4 nanoparticles;

[0012] S2. dissolving Span 80 and Tween 60 in cyclohexane and ultrasonically treating to form a water-in-oil emulsion;

[0013] S3. dissolving the sodium citrate stabilized ultra-small Fe3O4 nanoparticles formed in step S1 in ultrapure water, and then dissolving bis and PEI 25000, respectively, to obtain an aqueous phase solution;

[0014] S4. adding the aqueous phase solution obtained in step S3 to the water-in-oil emulsion obtained in step S2 and ultrasonically treating to form a uniform microemulsion;

[0015] S5. adding ethylenediamine to the microemulsion obtained in step S4, reacting, centrifuging the product, resuspending the precipitate with anhydrous ethanol, and then dialyzing with ultrapure water to obtain nanogel USIONGs;

[0016] S6. dissolving TMZ in methanol and then adding it to the nanogel USIONGs and stirring for 10 to 20 hours, centrifuging, taking out the supernatant and dialyzing for 2 to 3 days to obtain nanogel USIONGs-TMZ;

[0017] S7. incubating the aqueous solution of USIONGs-TMZ with PD-L1 siRNA for 15 to 20 minutes to obtain a USIONGs-TMZ / siRNA complex.

[0018] Preferably, in step S6, the mass ratio of the nanogel USIONGs to TMZ is 1:0.2-0.5.

[0019] Preferably, in step S6, the mass ratio of the nanogel USIONGs to TMZ is 1:0.4.

[0020] Preferably, in step S7, the nitrogen to phosphorus ratio during incubation is 4-8.

[0021] Preferably, the preparation of sodium citrate stabilized ultra-small Fe3O4 nanoparticles is as follows:

[0022] S11. dissolving ferric chloride and sodium citrate in diethylene glycol and stirring at 75 to 85°C in an air environment for 0.5 to 1.5h;

[0023] S12. adding anhydrous sodium acetate and then transferring to a high-pressure reaction kettle and reacting at 200 to 220°C for 4 to 6h;

[0024] S13. Cool the reaction product to room temperature, then centrifuge, discard the supernatant, and vacuum dry the precipitate to obtain ultrasmall Fe3O4 nanoparticles (USIO NPs).

[0025] A third aspect of the invention also provides applications of the aforementioned nanocomposite for integrated diagnosis and treatment of glioblastoma. MRgFUS technology is used to temporarily open the blood-brain barrier (BBTB), increasing the enrichment of nanoparticles in the tumor region and enhancing the imaging effect of the nanocomposite. The nanogel composite USIO NGs-TMZ / siRNA, loaded with TMZ and PD-L1, is injected into tumor-bearing mice. Ultrasound-based blood-brain barrier opening technology is used to enhance drug concentration in the brain tumor region, achieving combined chemotherapy / immunotherapy for glioma. By blocking the PD-1 / PD-L1 signaling pathway, anti-tumor immunity in the tumor microenvironment is activated, jointly enhancing the therapeutic effect on the tumor.

[0026] The present invention has the following advantages:

[0027] This invention uses a nanogel as a carrier to load ultra-small Fe3O4 nanoparticles. Utilizing the abundant amino groups on the nanogel surface, temozolomide and PD-L1 siRNA are further loaded, achieving synergistic anti-tumor immunity during tumor chemotherapy and enhancing the anti-tumor effect of glioblastoma. Previous work crosslinked polyethyleneimine (PEI) and N,N'-bis(acryloyl)cysteine ​​(BAC) via a reverse microemulsion method to obtain nanogel materials suitable for loading USIO, subsequently achieving tumor targeting through cell membrane biomimetic technology. The bis crosslinking agent used in this invention has excellent activity and is widely used in biological sample analysis and protein research. The preparation method is simple and easy, and the synthesized nanogel USIO NGs-TMZ / siRNA complex is relatively stable in vivo. During transport, it can protect the loaded gene from degradation by in vivo enzymes, promote rapid endosome escape of siRNA, and enhance the therapeutic effect on tumors. The therapeutic nanogel complex prepared in this invention requires no additional biomimetic or other targeting methods. It innovatively employs MRgFUS technology combined with microbubbles to deliver first-line chemotherapy drugs and gene therapy drugs to gliomas in situ, effectively increasing drug concentration in the brain tumor region and achieving combined chemotherapy and immunotherapy. Immunotherapy is currently a hot topic and a promising approach in cancer treatment. By blocking the PD-1 / PD-L1 signaling pathway, it partially restores the immune function of tumor tissue. Combined with the first-line chemotherapy drug TMZ, it promotes tumor cell apoptosis, breaking the limitations of traditional single-treatment modalities and potentially pushing research into the treatment of nervous system tumors to a new stage. Attached Figure Description

[0028] Figure 1 These are the results of transmission electron microscopy (TEM) testing;

[0029] Figure 2 Zeta potential test results;

[0030] Figure 3 PDI test results;

[0031] Figure 4 UV-Vis test results;

[0032] Figure 5 FT-IR test results;

[0033] Figure 6 MR performance test results;

[0034] Figure 7 In vitro cell viability test results;

[0035] Figure 8 In vitro cell uptake experiment results;

[0036] Figure 9 Silencing efficiency of USINGs-TMZ / siRNA complex on PD-L1 in GL261 cells test results;

[0037] Figure 10 MRI imaging of BBB opening range under different sound pressure conditions;

[0038] Figure 11 MRI imaging of glioblastoma by nanocomposite USINGs-TMZ / siRNA;

[0039] Figure 12 Anti-tumor effect of chemotherapy / immunotherapy combination in in vivo treatment experiment;

[0040] Figure 13 Immunofluorescence staining to detect the expression of PD-L1 and PD-1 in tumor area. DETAILED DESCRIPTION

[0041] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0042] Embodiment 1

[0043] The preparation method of the nanocomposite USIO NGs-TMZ / siRNA provided by the embodiment is as follows:

[0044] Step 1, sodium citrate stabilized ultra-Fe3O4 nanoparticles (USIONPs) are prepared by a solvothermal method. The specific steps are as follows: 0.6488 g of ferric chloride (FeCl3) and 0.47 g of sodium citrate (Na3Cit) are dissolved in 40 mL of diethylene glycol (DEG), stirred at 80°C in an air environment for 1 h; after stirring, 1.312 g of anhydrous sodium acetate is added, and then transferred to a high-pressure reaction kettle, reacted at 200°C for 4 h; after the reaction is completed, the reaction product is cooled to room temperature, then transferred to a 50 mL centrifuge tube, and the centrifugal speed is set to 13000 rpm; after the centrifugal operation is completed, the supernatant is discarded, and the centrifugal operation is repeated 3 times; the precipitate is dried in a vacuum drying oven to obtain ultra-small Fe3O4 nanoparticles (USIONPs).

[0045] Step 2, nanogel USIO NGs are synthesized by a reverse microemulsion method. The specific steps are as follows: first, 420.6 mg of Span 80 and 79.4 mg of Tween 60 are weighed and dissolved in cyclohexane (20 mL) to form a clear water-in-oil (W / O) emulsion by ultrasonic treatment for 15 min. Sodium citrate stabilized ultra-small Fe3O4 nanoparticles (15 mg, 20 mg, 30 mg, 40 mg) are dissolved in 2 ml of ultrapure water, followed by dissolving bis (10 mg) and PEI 25000 (81.08 mg), respectively. Then the prepared microemulsion is placed on a magnetic stirrer and stirred, and the mixed aqueous solution is added and dispersed by ultrasonic treatment for 20-30 min to form a uniform microemulsion. The initiator ethylenediamine is added and reacted for 12 hours. Transfer to a 50 ml centrifuge tube and centrifuge at room temperature (13000 rpm, 10 min). Discard the supernatant and resuspend with 10 ml of anhydrous ethanol. Choose a 8000-14000 dialysis bag and dialyze with ultrapure water for 2-3 days.

[0046] Step 3, different amounts of TMZ are dissolved in methanol according to different feeding ratios (mass ratios are about 1:0.05, 1:0.1, 1:0.2, and 1:0.4), respectively, and then added to the USIO NGs material in the previous step and stirred for 12 h. After the methanol is completely volatilized, centrifuge at 2000 rpm for 10 min. Take out the supernatant and dialyze for 2-3 days. The encapsulation efficiency (%) is calculated by the following formula:

[0047] Encapsulation efficiency (%) = (amount of loaded drug / amount of input drug) x 100%; (1)

[0048] Drug loading rate = total amount of drug loaded / total amount of NGs (2) Calculate the encapsulation efficiency and drug loading rate. When the mass ratio is 1:0.4, the drug loading rate and encapsulation efficiency are the highest, among which the drug loading rate is 0.28 and the encapsulation efficiency is 0.97. The mass ratio of carrier / TMZ used in subsequent experiments is 1:0.4.

[0049] Table 1 encapsulation efficiency and drug loading rate of TMZ at different mass ratios

[0050]

[0051]

[0052] The USIO NGs (20 mg, 30 mg, 40 mg) synthesized in the previous step were loaded with TMZ at a mass ratio of 1:0.4, and the drug encapsulation efficiency and drug loading rate were measured in the same way. The results were similar. By calculating the concentration of Fe in the solution, the mass fraction of Fe was calculated, and the mass fraction of Fe in USIO NGs (40 mg) was 7%. In subsequent experiments, USIO NGs with 40 mg of USIO were used for experiments.

[0053] Table 2 encapsulation efficiency and drug loading rate of TMZ loaded by different nanocarriers

[0054] Encapsulation efficiency Drug loading efficiency USINGs (40 mg) + TMZ 97％ 28％ USINGs (30 mg) + TMZ 97％ 28％ USINGs (20 mg) + TMZ 98％ 28％

[0055] Step 4, determine the compression ability of USIO NGs-TMZ to siRNA gene by agarose gel retardation experiment, use ultrapure water to configure USIO NGs-TMZ into 2mg / mL solution, prepare 8-hole agarose gel containing 4S Green Nucleic Acid Stain (1.0% w / v), the siRNA quantity of each hole is 1ug, incubate the above USIO NGs-TMZ solution and siRNA according to different nitrogen phosphorus ratio (N / P=0.25, 0.5, 1, 2, 4, 6, 8) for 15-20 minutes, after incubation, mix the USIONGs-TMZ / siRNA complex with the loading buffer and add it to the hole of the agarose gel, the control group is 1ug siRNA, set the voltage to 90V and the time to 30 minutes, after electrophoresis, observe the migration of the gene in the gel by gel imaging instrument.

[0056] Test the reaction products involved in the preparation method provided in Example 1, and the specific test methods and results are described as follows:

[0057] (1) Transmission electron microscopy (TEM) test

[0058] USIONGs, USIONGs-TMZ were configured into 1 mg / mL solution, 10 μL was taken out respectively and dropped on the surface of clean copper mesh, and then naturally air dried under air atmosphere for TEM test to observe the morphological characteristics of the materials. As shown in Figure 1 , the TEM results show that USIONGs-TMZ is spherical, uniformly distributed, uniform in size, and good in dispersibility. By observation at a higher magnification, USIONGs-TMZ has uniform size distribution, and clear USIONPs can be seen. By analyzing the particle size of USIONGs-TMZ through Image J software, it can be known from Figure 1 that the average particle size of USIONGs-TMZ is about 132.35 nm.

[0059] (2) Zeta potential test

[0060] USIO, NGs, USIONGs, USIONGs-TMZ were configured into 1 mg / mL solution, and the solution was diluted with ultrapure water, and then tested for Zeta potential, hydrodynamic diameter and dispersion index (PDI) by dynamic light scattering analysis (DLS). Referring to the results of agarose gel blocking experiment and the method of configuring USIONGs-TMZ / siRNA complex, USIONGs-TMZ / siRNA complexes with different N / P ratios (N / P = 2, 4, 6, 8) were configured, and the final volume was quantified as 1 mL using PBS buffer solution. The surface Zeta potential and hydrodynamic diameter were determined by Nano-ZS nanoparticle size analyzer.

[0061] The test results are shown in Figure 2 A, the Zeta potential of USIONPs is -42.7±0.2 mV, the Zeta potential of hydrogel NGs is 42.1±0.17 mV, the Zeta potential of USIONGs is 25.1±0.25 mV, the Zeta potential of USIONGs-TMZ is 21±0.6 mV, and the Zeta potential of USIONGs-TMZ / siRNA is 18.6±1.1 mV. The results are shown in Figure 2 B, the hydrodynamic diameter of USIONPs is 87.3±6.9 nm, the hydrodynamic diameter of NGs is 162.5±4.5 nm, the hydrodynamic diameter of USIONGs is 263.0±3.2 nm, the hydrodynamic diameter of USIONGs-TMZ is about 193.3±7.4 nm, indicating that USIONGs-TMZ has good solubility.

[0062] The zeta potential of the USIONGs-TMZ / siRNA complex was about 18.6±1.10 mV when the nitrogen-phosphorus ratio was 2, and the zeta potential of the USIONGs-TMZ / siRNA complex ranged from 31-38 mV when the nitrogen-phosphorus ratio was 4, 6, and 8. Figure 2 C), which showed that the USIONGs-TMZ / siRNA complex had a positive potential, which was conducive to entering the cell membrane with a negative potential; at the same time, the results showed that the hydrated particle size of the complex ranged from 202-227 nm Figure 2 D), which was conducive to the uptake of the nanocomplex by cells.

[0063] (3) Polydispersity index (PDI) test

[0064] To further explore the stability of USIONGs-TMZ in different solutions, USIONGs-TMZ was dissolved in PBS and DMEM / F12 medium containing 10% fetal bovine serum, respectively, and the hydrated kinetic diameter of USIONGs-TMZ dispersed in different solutions was tested on days 1-7 to evaluate the change in particle size of the nanomaterial within 7 days. The test results are shown in Figure 3 A, and the hydrodynamic diameter of USIONGs-TMZ changed little over time, without producing a sedimentation phenomenon, indicating that the synthesized USIONGs-TMZ material had good colloidal stability.

[0065] The test results of the kinetic diameter were further calculated by software Malvern Zetasizer Software v7.13 PSS0012-39EN JP to obtain the polydispersity index, and the calculation results are shown in Figure 3 B, the polydispersity index of USIONPs was 0.239±0.03, the polydispersity index of NGs was 0.229±0.013, the polydispersity index of USIONGs was 0.199±0.006, and the polydispersity index of USIONGs-TMZ was 0.179±0.012, indicating that the synthesized nanomaterial had good solubility.

[0066] (4) Ultraviolet-visible light absorption spectrum (UV-Vis) characterization

[0067] NGs, TMZ, USIONGs, and USIONGs-TMZ were configured into a 0.5 mg / mL solution, and the ultraviolet absorption value in the 200-800 nm waveband was detected by ultraviolet-visible spectrophotometry (UV-Vis). Figure 4 The results showed that absorption peaks appeared at 260 nm and 327 nm, and this characteristic absorption proved the successful modification of Fe and TMZ, indicating that USIONGs-TMZ was successfully modified.

[0068] (5) Fourier Transform Infrared (FT-IR) Test

[0069] To verify the successful modification of organic molecules, Fourier transform infrared spectroscopy was used to characterize the chemical composition and characteristic functional groups of the material. For example... Figure 5 As shown, the curve is at 3416cm. -1 The absorption peaks near the free hydroxyl group (-OH) are from 3000 to 2853 cm⁻¹. -1 The nearby peak is the absorption peak of the stretching vibration of the methylene group in sodium citrate, at 1738 cm⁻¹. -1 The nearby peaks are due to the stretching vibrations of the carbonyl group (C=O), and the TMZ modification did not alter the structure of USIO NGs.

[0070] (6) Magnetic Resonance Imaging (MR) Performance Testing

[0071] First, the Fe content in USIO NPs, USIO NGs, and USIONGs-TMZ was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES), and 1 mL solutions of the materials were prepared with different iron concentrations (0.1, 0.2, 0.4, 0.8, 1.2, and 1.6 mM). Nuclear magnetic resonance imaging (NMR) was used to image the materials and measure their relaxation times. The relaxation rates of different materials were calculated using linear fitting, denoted as r1. The test results are as follows: Figure 6 As shown, the T1 imaging performance of the synthesized nanomaterials gradually improves with increasing iron concentration, and the relaxation rate of USIO NPs is approximately 1.06 mM. -1 s -1 The relaxation rate of USIO NGs is approximately 1.62 mM. -1 s -1 The relaxation rate of USIO NGs-TMZ is approximately 1.71 mM. -1 s -1 This indicates that the synthesized nanomaterials have good T1 imaging performance.

[0072] The following in vitro cell experiments demonstrate the therapeutic application of the USIO NGs-TMZ / siRNA nanocomposite in glioblastoma:

[0073] Experiment 1: In vitro cell viability detection.

[0074] The cytotoxicity of USIO NGs and the USIO NGs-siRNA complex was assessed using the CCK-8 colorimetric assay. Results are as follows: Figure 7As shown, at the same Fe concentration, the cytotoxicity of USIONGs-siRNA complex is lower, indicating that the negative charge on the surface of siRNA neutralizes part of the positive charge carried by the amino group on the surface of USIONGs, so that the surface potential of USIONGs-siRNA complex is reduced, thereby reducing the cytotoxicity. When the concentration is less than 2000 nM, the survival rate of GL261 cells can reach more than 71%, and the experimental results prove that the nano-complex can be used for subsequent cell and animal experiments.

[0075] Experiment 2, in vitro cell uptake experiment.

[0076] The gene delivery efficiency of USIONGs-TMZ complex was quantitatively analyzed by flow cytometry. Cy3-labeled siRNA was used to configure USIONGs-TMZ / siRNA complex with different nitrogen-phosphorus ratios (N / P=2, N / P=4, N / P=6, N / P=8). After different groups of nanomaterials were co-incubated with GL261 for 4h, the red fluorescence intensity was analyzed to quantitatively analyze the phagocytosis of GL261 to nanomaterials. The results are shown in Figure 8 : When N / P=2, the red fluorescence intensity is the strongest, and with the increase of N / P, the red fluorescence intensity decreases, and the subsequent experiment selects USIONGs-TMZ / siRNA complex with N / P=2. The influence of different time (2h, 4h, 6h, 8h) on cell phagocytosis was evaluated in the same way, and the results showed that when the nanomaterials were co-cultured with GL261 cells for more than 4h, the phagocytosis efficiency of the cells was higher, and the phagocytosis efficiency of the cells did not increase significantly at 6h and 8h.

[0077] Experiment 3, in vitro cell immunofluorescence detection of PD-L1 expression

[0078] Confocal fluorescence microscope was used to observe the silencing efficiency of USIONGs-TMZ / siRNA complex on PD-L1 in GL261 cells. Red fluorescence is USIONGs-TMZ / siRNA complex, green fluorescence is the expression of PD-L1, and blue fluorescence is the cell nucleus. Image J software was used for quantitative analysis of fluorescence. The experimental results are shown in Figure 9 : The green fluorescence intensity in the blank control group (PBS) is the highest, and compared with naked gene (Free siRNA) and negative control (USIONGs-TMZ / siNC), the experimental group (USIONGs-TMZ / siRNA complex with N / P=2) has the best silencing effect on PD-L1 protein level (p<0.001), so the USIONGs-TMZ / siRNA complex can complete gene delivery and effectively silence the expression of PD-L1 in GL261 cells.

[0079] The MRI imaging and anti-tumor effect evaluation of glioma by using nano-complex USINGs-TMZ / siRNA were carried out in vivo animal experiments:

[0080] Experiment 1: GL261 cells were used to establish subcutaneous tumor and orthotopic tumor models in male C57BL / 6 mice (6-8 weeks old) for MRI imaging of USINGs-TMZ / siRNA.

[0081] GL261 cells were cultured in a 37℃, 5% CO2 incubator, and the cells were trypsinized, centrifuged, washed with PBS, and resuspended to obtain a cell suspension. BALB / c nude mice were injected subcutaneously with 100 μL of cell suspension (about 2*10 6 cells) in the right leg to prepare a subcutaneous tumor model; in the mouse head, a microdrill was used to drill a hole in the right brain (2 mm from the sagittal suture, 2 mm behind the fontanel), and a microsyringe was used to inject 5 μL of cell suspension (about 1*10 5 cells) to prepare an orthotopic tumor model.

[0082] The BBTB was temporarily opened with the help of the MRgFUS device, and the opening degree of the BBTB was evaluated by MRI T1WI imaging. First, the coronal, sagittal, and transverse images of the mouse were scanned by magnetic resonance, and after the position was determined in three directions, 80 μL of microbubbles were injected through the tail vein, and focused ultrasound was used to process the right brain of the mouse. The ultrasound pulse repetition frequency was selected as 1 Hz, the pulse length was 100 ms, the ultrasound irradiation time was 60 s, and the sound pressure conditions were changed (0.2, 0.3, 0.4 MPa). After the ultrasound treatment was completed, Evans blue (EB) was injected through the tail vein for staining, and the permeability of the blood-brain barrier was detected, and 100 μL of 2% EB staining solution was injected. The results are shown in Figure 10 As the sound pressure gradually increased, the opening range of the BBB increased, and when the sound pressure was 0.4 Mpa, the opening range was the largest, and no obvious damage was found in the surrounding normal tissue.

[0083] MRI T1WI imaging was used to evaluate the imaging effect of USIONPs, USINGs, and USINGs-TMZ / siRNA. The signal of the tumor region was detected at the same time point after injection of nano-materials, and the T1WI imaging results are as follows: Figure 11As shown in AB, USIO NPs showed the best imaging effect 30 minutes after injection, while USIO NGs and USIO NGs-TMZ / siRNA showed the best imaging effect 60 minutes after injection. Based on the imaging effect, USIO NGs and USIO NGs-TMZ / siRNA showed better imaging effect than USIO NPs, and USIO NGs and USIO NGs-TMZ / siRNA had a longer circulation time in vivo.

[0084] The imaging effects of USIO NGs-TMZ / siRNA before and after BBTB opening were evaluated using T1WI MRI. Signal intensity in the tumor region was detected at the same time point after nanomaterial injection. The T1WI imaging results are as follows: Figure 11 As shown in the CD, after injection of USIONGs-TMZ / siRNA, the signal intensity in the tumor area increased over time, reaching its optimal value 60 minutes after injection. Furthermore, the imaging effect after BBTB opening was better than before opening, indicating that MRgFUS technology enhanced the enrichment of nanomaterials in the tumor area of ​​the brain.

[0085] Experiment 2: In the in vivo treatment experiment, the efficacy of each experimental group was evaluated by calculating the relative tumor proliferation rate, plotting tumor growth curves and mouse weight change curves.

[0086] To evaluate the antitumor efficacy of combined chemotherapy / immunotherapy, PBS, USIO NGs-TMZ, USIO NGs-siRNA, and USIO NGs-TMZ / siRNA were administered via tail vein injection, respectively. In group 5, USIO NGs-TMZ / siRNA was injected after MRgFUS technique to open the BBTB. T1WI MRI imaging was performed at 0, 3, 6, 9, and 12 days during treatment. Figure 12 In section A), weigh the mouse ( ). Figure 12 (B in the diagram), calculate tumor volume and plot tumor proliferation rate ( Figure 12 (C) Experimental results showed that the tumor growth rate was fastest in the PBS group. Both the USIO NGs-TMZ and USIO NGs-siRNA groups could inhibit tumor growth. The USIO NGs-TMZ / siRNA combined treatment group was more effective than the single treatment group. The USIO NGs-TMZ / siRNA+FUS group had the lowest tumor proliferation rate, indicating that both chemotherapy and immunotherapy can inhibit tumor growth. Chemotherapy combined with immunotherapy can synergistically enhance the therapeutic effect of tumor treatment, induce anti-tumor immunity, and exert a long-term therapeutic effect. The application of MRgFUS technology further enhances the enrichment of nanomaterials in intracranial orthotopic tumors, targets and improves drug delivery, and enhances the efficacy of nanomaterials.

[0087] At the same time, the body weight of mice was recorded during the treatment process, and the body weight of mice in all treatment groups had no significant change, indicating that USIONGs-TMZ, USIONGs-siRNA and USIONGs-TMZ / siRNA had good biological safety.

[0088] Experiment 3: The expression of PD-L1 and PD-1 in tumor area was detected by immunofluorescence staining, the efficiency of siRNA blocking pathway was evaluated, and the expression of CD4 + T and CD8 + T cells in tumor area was detected, and the synergistic treatment effect of USIONGs-TMZ / siRNA chemotherapy and anti-tumor immunity was evaluated.

[0089] In the experiment of evaluating the treatment effect of tumor in vivo by MR imaging, it was observed that the tumor volume of USIONGs-TMZ / siRNA and USIONGs-TMZ / siRNA+FUS treatment groups was reduced most significantly. In order to further evaluate the silencing of PD-L1 caused by nano-drugs and the related anti-tumor immunity, the expression of PD-L1 / PD-1, CD4 + / CD8 + T cells in tumor area was evaluated by immunofluorescence staining. The experimental results are shown in Figure 13 , which proves that the expression of PD-L1 in USIONGs-siRNA, USIONGs-TMZ / siRNA and USIONGs-TMZ / siRNA+FUS treatment groups is decreased, and the degree of decrease in USIONGs-TMZ / siRNA and USIONGs-TMZ / siRNA+FUS treatment groups is more significant. Compared with the PBS control group, the number of tumor-infiltrating CD8 + T cells in USIONGs-TMZ / siRNA and USIONGs-TMZ / siRNA+FUS treatment groups is increased, the recognition and killing effect on tumor is restored, and the anti-tumor effect is increased. Therefore, the results prove that USIONGs-TMZ / siRNA can effectively block the combination of PD-L1 and PD-1, inhibit the interaction of PD-L1 / PD-1, thereby enhancing the attack ability of T cells to target and resisting the immunosuppression of tumor microenvironment.

[0090] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A nanocomposite comprising a nanogel, characterized in that, The nanogel carries ultra-small Fe3O4 nanoparticles, temozolomide and PD-L1 siRNA.

2. The method for preparing a nanocomposite as described in claim 1, characterized in that, The method comprises the following steps: S1. Preparation of sodium citrate stabilized ultra-small Fe3O4 nanoparticles; S2. Dissolve Span 80 and Tween 60 in cyclohexane, and ultrasonic treatment to form a water-in-oil emulsion; S3. Dissolve the sodium citrate stabilized ultra-small Fe3O4 nanoparticles formed in step S1 in ultrapure water, and then dissolve bis and PEI 25000 respectively to obtain an aqueous phase solution; S4. Add the aqueous phase solution obtained in step S3 to the water-in-oil emulsion obtained in step S2, and ultrasonic treatment to form a uniform microemulsion; S5. Add ethylenediamine to the microemulsion obtained in step S4, react, centrifuge the product, resuspend the precipitate with anhydrous ethanol, and then dialyze with ultrapure water to obtain nanogel USIONGs; S6. Dissolve TMZ in methanol, then add it to the nanogel USIONGs and stir for 10 to 20 hours, centrifuge, take out the supernatant and dialyze for 2-3 days to obtain nanogel USIONGs-TMZ; S7. Incubate the aqueous solution of USIONGs-TMZ and PD-L1 siRNA for 15-20 minutes to obtain USIONGs-TMZ / siRNA complex.

3. The method for preparing a nanocomposite according to claim 2, characterized in that, In step S6, the mass ratio of the nanogel USIONGs to TMZ is 1:0.2-0.

5.

4. The method for preparing a nanocomposite according to claim 2, characterized in that, In step S6, the mass ratio of the nanogel USIONGs to TMZ is 1:0.

4.

5. The method for preparing a nanocomposite according to claim 2, characterized in that, In step S7, the nitrogen to phosphorus ratio during incubation is 4-8.

6. The method for preparing a nanocomposite according to claim 2, characterized in that, In step S2, the ultrasonic treatment time is 10 to 20 minutes.

7. The method for preparing a nanocomposite according to claim 2, characterized in that, In step S4, the ultrasonic treatment time is 20 to 30 minutes.

8. The method for preparing a nanocomposite according to claim 2, characterized in that, In step S5, the reaction time is 10 to 20 hours.

9. The method for preparing a nanocomposite according to claim 2, characterized in that, The preparation steps of sodium citrate stabilized ultra-small Fe3O4 nanoparticles are as follows: S11. Dissolve ferric chloride and sodium citrate in diethylene glycol, stir at 75 to 85°C in an air environment for 0.5 to 1.5h; S12. Add anhydrous sodium acetate, then transfer to a high-pressure reaction kettle, and react at 200 to 220°C for 4 to 6h; S13. Cool the reaction product to room temperature, then centrifuge, discard the supernatant, and vacuum dry the precipitate to obtain ultra-small Fe3O4 nanoparticles.

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