Composite nanometer silicon ball drug delivery system, preparation method and application thereof

Abstract

The application discloses a composite nano-silicon ball drug delivery system and a preparation method and application thereof. The system takes mesoporous silica nanoparticles as a carrier, loads gefitinib, and is coated with a PD-L1 antibody modified platelet membrane on the surface. The application prepares a composite multifunctional mesoporous silica nanoparticle which loads gefitinib and is coated with a PD-L1 antibody modified platelet membrane on the surface, so that the composite multifunctional mesoporous silica nanoparticle can be directed to reach a tumor surgery blood vessel damage site, kill residual tumor cells, reduce matrix hardness in the microenvironment, enhance the permeability and toxicity of immune cells, the two effects are interdependent and mutually enhanced, and the application overcomes the invalidity of colorectal tumor immunotherapy and prolongs the survival period of patients.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to a composite nanometer silicon ball drug delivery system, a preparation method and application thereof, which precisely targets tumor cells by virtue of a platelet membrane coated on the surface thereof. BACKGROUND

[0002] Colorectal cancer (CRC) has become a common digestive tract tumor with an increasing incidence worldwide, and its mortality rate is second only to lung cancer, ranking second in the global cancer death causes. At present, immunotherapy plays a crucial role in the treatment of colorectal cancer. However, for the microsatellite stable (MSS) colorectal cancer patients accounting for 85% to 90%, the single immune checkpoint inhibitor treatment strategy often has little effect. Recent studies have shown that the collagen matrix hardness in the tumor extracellular matrix (ECM) may constitute a physical barrier, hindering the effective action of immunotherapy drugs.

[0003] Mesoporous silica nanoparticles (MSN) as a new type of nanomaterial have attracted much attention due to their unique physical properties and great potential as drug carriers. Therefore, designing and constructing a nanometer silicon ball drug delivery system that can precisely target tumor cells, effectively reduce tumor matrix hardness, and remove immune checkpoint blockade has far-reaching significance for improving the efficacy of colorectal cancer immunotherapy.

[0004] Traditional drug delivery methods, such as intravenous injection or oral administration, are often limited by the pharmacokinetic properties of drugs in the body, uneven distribution, difficulty in predicting drug concentration at tumor sites, and possible systemic side effects, thereby affecting the anti-tumor effect. The emergence of nanometer drug delivery systems, with its unique advantages such as improving drug kinetic properties, achieving tumor targeted delivery, and tumor microenvironment responsive site-specific drug release, provides new possibilities for the targeted delivery of immune checkpoint drugs, and is expected to significantly improve the bioavailability of drugs, thereby achieving more ideal anti-tumor effect, and showing great clinical application prospects.

[0005] With the in-depth study of material science and pharmaceutics, nanotechnology has been developing unprecedentedly. Among them, mesoporous silica nanoparticles (MSNs) have attracted much attention due to their adjustable pore size, flexible amorphous framework, and large specific surface area. Although MSNs themselves do not have biological or chemical activity, the silanol groups on their inner surface provide a wealth of possibilities for further chemical modification. For example, through alkylation modification, various organic groups (such as amino, phenyl, thiol, and vinyl groups) can be combined, thereby introducing catalytically active centers. In addition, chemical modification can also effectively improve the dispersion performance of MSNs. Their unique void space and nanocage structure make it possible to efficiently load and chemically bind small molecule drugs, thereby significantly increasing drug loading and preventing premature drug leakage. These monodisperse silica nanoparticles have excellent performance in the field of biomedicine, indicating that they may play an important role in future individualized tumor therapy.

[0006] However, nanoparticles often face the problem of being wrapped by plasma proteins after entering the human body, and are then prematurely excreted through metabolic organs such as the liver or kidneys, which greatly reduces the bioavailability of nanomedicines. In recent years, delivery carriers coated with cell membranes have attracted much attention as a simple and effective biomimetic drug delivery method. This delivery method has the advantages of prolonging the circulation time, reducing immunogenicity, and achieving active targeting. Among them, platelets, as a special type of blood cells, have the ability to target vascular injury sites and recognize and adhere to tumor cells. The P-selectin-rich platelet membrane can specifically bind to the CD44 molecule on the surface of tumor cells, enabling platelet membrane-coated nanoparticles to have homologous targeting ability based on receptor membrane recognition molecules and excellent biocompatibility.

[0007] Defactinib, as a small molecule inhibitor of FAK, exerts its effect by inhibiting the trans-autophosphorylation of focal adhesion protein tyrosine 397 (Y397). When the physical signals induced by tumor stroma are transmitted to tumor cells through integrins, the inhibition of FAK at the integrin aggregation site will block its function as a molecular scaffold to recruit SRC family phosphokinase domains, thereby inhibiting the transduction of downstream signaling pathways. This ultimately reduces the feedback of tumor cells to external physical stress. Therefore, by inhibiting FAK activity, the stiffness of the tumor stroma can be effectively regulated, promoting the deeper penetration of nanoparticles into tumor tissue and enhancing the ERP effect. In addition, the loosened tumor stroma also helps to improve the infiltration of cytotoxic T cells, thereby achieving the "warming" effect of the tumor immune microenvironment. SUMMARY

[0008] The technical problems solved by the present application are: the present application provides a composite nanometer silicon ball drug delivery system, a preparation method and application thereof, a FAK small molecule inhibitor gefitinib is loaded in the surface mesoporous by electrostatic adsorption, and a PD-L1 antibody is coupled on the PM surface, two drugs are delivered by intravenous injection, so as to make the biomimetic nanoparticles accurately target tumor cells and postoperative microresidual lesions, reduce the hardness of tumor stroma, promote the penetration of nanoparticles and killer T cells into tumor tissue, remove the immune checkpoint blockage of tumor cells, promote the efficient killing of T cells on tumor cells, inhibit the growth and recurrence of tumors, overcome the primary or secondary drug resistance of dMMR / MSI-H tumors, and improve the long-term survival and prognosis of patients with colorectal cancer.

[0009] Technical scheme: a composite nanometer silicon ball drug delivery system, the system uses mesoporous silica nanoparticles as a carrier, loads gefitinib, and is coated with a PD-L1 antibody modified platelet membrane on the surface; the morphology of the nanoparticles is analyzed by using a transmission electron microscope and a laser dynamic light scattering particle size analyzer: the nanoparticles are uniformly dispersed, solid spherical, the average hydration particle size is about 150 nm, and the surface is clear and regular mesoporous structure; the specific surface area and pore volume are ~765.7 m 2 / g and 1.12 cm 3 / g, and the pore size is about 5-7 nm; the absorption peaks of 803 cm -1 , 960 cm -1 , and 1093 cm -1 in the FTIR spectrum indicate that Si-O-Si exists, and the shrinkage vibration peak at 3440 cm -1 indicates that Si-OH exists; the zeta potential analysis result shows that the average potential of the nanoparticles is-32.8 mV.

[0010] The preparation method of the composite nanometer silicon ball drug delivery system comprises the following steps: (1) preparation of drug-loaded mesoporous silica nanoparticles (MSN@D): first, mesoporous silica nanoparticles are added to anhydrous ethanol, and ultrasonic treatment is performed at room temperature to make the mesoporous silica nanoparticles completely dispersed in the anhydrous ethanol to obtain a mesoporous silica nanoparticle suspension; Defactinib is added to the mesoporous silica nanoparticle suspension, and ultrasonic treatment is again performed to make the Defactinib uniformly dispersed in the mesoporous silica nanoparticles; the mixture is stirred at room temperature, and centrifugation is performed to remove the unloaded Defactinib, and the precipitate is the MSN@D nanoparticles; (2) preparation of platelet membrane-coated drug-loaded mesoporous silica nanoparticles (MSN@D@PM): the MSN@D nanoparticles are dispersed in a PBS solution containing platelet membranes, and ultrasonic treatment is performed on ice to obtain the MSN@D@PM; (3) preparation of MSN@D@PM@Ab nanoparticles: the surface of the MSN@D@PM is thiolated by using Traut reagent for 45 min, and then the excess Traut reagent is removed by washing with Tyrode buffer; PD-L1 antibody and a sulfo-SMCC linker are mixed at 4°C, the excess linker is removed by dialysis, and aPD-L1-sulfo-SMCC is obtained; the MSN@D@PM and the aPD-L1-sulfo-SMCC are mixed at a protein mass ratio of 1:10, and after reaction at room temperature, the supernatant is removed by centrifugation, and the precipitate is lyophilized to obtain the MSN@D@PM@Ab.

[0011] Preferably, the mass ratio of the MSN@D to the platelet membrane is 5:1.

[0012] Preferably, the molar ratio of the PD-L1 antibody to the sulfo-SMCC is 1:5.

[0013] Preferably, the molecular weight cutoff size of the membrane used for dialysis is 10 kDa.

[0014] Preferably, the optimal drug loading rate of the Defactinib in the system is 33.5%, and the drug loading amount is 25.1%.

[0015] The composite nanometer silicon ball drug delivery system prepared by the method.

[0016] The composite nanometer silicon ball drug delivery system in the preparation of a drug for treating colorectal tumors.

[0017] A drug for treating colorectal tumors, wherein the effective component comprises the composite nanometer silicon ball drug delivery system.

[0018] Beneficial effects: The application prepares a composite multifunctional mesoporous silica nanoparticle, which is loaded with Defactinib and coated with PD-L1 antibody modified platelet membrane on the surface, so as to be able to reach the tumor surgery blood vessel damage site in a targeted manner and kill residual tumor cells while reducing the matrix hardness in the microenvironment, enhancing the immune cell penetration and toxicity function, the two effects depend on and enhance each other, overcome the ineffective colorectal tumor immunotherapy and prolong the survival period of patients. Experiments show that the nanoparticle can significantly inhibit the growth of cecum orthotopic tumor in mice, reduce the collagen crosslinking of tumor tissue, and enhance the CD8 + T cell number in the tumor center and edge. In addition, the mice do not have significant weight loss during the treatment period, the spleen volume shrinks to normal, and the nanoparticle has no obvious toxic side effects on the main organs in the mouse body, indicating that it has great clinical transformation application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a preparation flowchart of the composite mesoporous silica nanoparticle.

[0020] Figure 2 It is the characterization of various distributed nanoparticles. (a) TEM images of MSN, MSN@D, MSN@D@PM and MSN@D@PM@Ab, scale bar: 100 nm; (b) Zeta potential and hydrodynamic size of MSN, platelet membrane, MSN@D, MSN@D@PM and MSN@D@PM@Ab; (c) Time and pH dependence of MSN@D@PM@Ab release Defactinib in PBS; (d) Time variation curve of MDPAs hydrodynamic size at 4℃ and 25℃; (e) Representative confocal microscope images of MDPAs, blue label PMs, red label PD-L1 antibody, scale bar: 10 μm; (f) SDS-PAGE analysis of protein expression profile in purified platelets, platelet membrane and MDPAs; (g) Western blot analysis of platelet-related membrane proteins in purified platelets, platelet membrane and MDPAs.

[0021] Figure 3In vivo therapeutic validation of MDPAs. (a) Schematic illustration of the cecal orthotopic tumor treatment (MC38-luc, luciferase-labeled MC38 cells; IVIS, in vivo imaging system; IHC, immunohistochemical staining; SRS, sirius red staining); (b) In vivo bioluminescence imaging of the cecal orthotopic tumor (n = 5); (c) Resected tumors (left) and tumor size (right) showing the in vivo therapeutic efficacy of MSN@D@PM@Ab and other control groups; (d) Survival curve of the treatment groups; (e-f) Representative sirius red staining and quantification of cecal sections of paraffin-embedded tissues after 22 and 48 days of treatment; (g-h) Immunohistochemical staining showing CD8+ T cells (brown) infiltrating the tumor core and margin, scale bar 200 pm. *P < 0.05, **P < 0.01 and ***P < 0.001 were determined by one-way ANOVA and multiple comparison tests.

[0022] Figure 4 In vivo targeting ability and pharmacokinetic analysis of MDPAs. (a) In vivo biodistribution at different time points after intravenous injection of Cy5-labeled MDPAs or equal dose of free Cy5 (1 mg kg -1 ) (n = 3); (b) Fluorescence imaging of orthotopic tumors and major organs at 6 h and 48 h, respectively (n = 3) (H: heart; Li: liver; S: spleen; Lu: lung; K: kidney; T: tumor); (c) Quantitative analysis of fluorescence intensity of orthotopic tumors and major organs at 6 h and 48 h (n = 3). ***P < 0.001 and ****P < 0.0001 compared with the control group.

[0023] Figure 5 Fourier transform infrared spectroscopy (FTIR) characterization of MSN nanoparticles.

[0024] Figure 6 In vivo toxicity analysis of MSN@D@PM@Ab. (a) Representative images of HE staining of major organs after treatment with different nanoparticles; (b) Comparison of the weight changes of major organs of mice in different nanoparticle treatment groups; (c) Trend graph of the body weight changes of mice; (d-e) Photographs of the spleen and quantitative analysis of the spleen weight of mice in different treatment groups. DETAILED DESCRIPTION

[0025] In order to better understand the present application, we will further elaborate the present application in combination with specific examples and drawings, but it is worth noting that the implementation of the present application is not limited to this.

[0026] The reagents and raw materials used in the present application are commercially available or can be prepared according to the literature method. The test method of the specific conditions not mentioned in the examples of the present application is according to the conventional condition, or according to the condition suggested by the manufacturer.

[0027] Example 1

[0028] 1.1 Preparation and characterization of mesoporous silica nanoparticles (MSNs)

[0029] 1) Take the template agent CTAB 200 mg into a round-bottom flask, add 100 mg TEAH3 and 100 mL deionized ultrapure water into the flask in turn, and stir in a 70°C constant temperature water bath for 1 h until completely dissolved. Take 100 mg TEOS and add it to the reaction solution. Stir in a 70°C constant temperature water bath for 4 h until the solution shows a light blue opalescent color. Mature at room temperature overnight.

[0030] 2) Centrifuge the reaction solution at 15000 r·min -1 for 15 min, repeat 2 times, remove the residual CTAB, and dissolve the precipitate in hydrochloric acid ethanol solution (hydrochloric acid: anhydrous ethanol = 1:30) in a 50 mL round-bottom flask. Reflux at 80°C constant temperature for 8 h. Replace the new hydrochloric acid ethanol solution and continue to reflux for 16 h. Centrifuge the reaction solution at 12000 r·min -1 for 20 min, discard the supernatant, and wash the precipitate with deionized ultrapure water and 95% ethanol solution alternately to remove the residual CTAB and hydrochloric acid ethanol solution.

[0031] 3) Disperse the precipitate in 1 mL deionized ultrapure water, freeze at -80°C overnight, and vacuum dry for 3 days to obtain white MSNs lyophilized powder.

[0032] 4) Reconstitute the MSN lyophilized powder in PBS for use. It is observed that the mesoporous silica nanoparticles are relatively stable in PBS and do not show aggregation within one month.

[0033] 5) The characterization of nanoparticles mainly uses transmission electron microscopy to observe its morphological characteristics. Drop the nanoparticle suspension of different formulations on the copper transmission electron microscopy grid (Ted Pella, CA), perform negative staining with sodium phosphotungstate solution, and then dry at room temperature overnight. The electron micrograph is imaged at an acceleration voltage of 200 kV.

[0034] 6) Use laser dynamic light scattering particle size analyzer to detect the hydration particle size of mesoporous silica, use potential analyzer to detect the surface Zeta potential, and use Fourier transform infrared spectrometer to detect the chemical bonds and functional groups contained in mesoporous silica Figure 5 ).

[0035] 1.2 Extraction and purification of platelet membrane (PM)

[0036] 1) Pericardial puncture was performed to collect whole blood from C57BL / 6J mice, and then 1 / 9 volume of citric acid dextrose buffer (ACD buffer, containing 75 mM sodium citrate, 39 mM citric acid and 135 mM glucose, pH = 7.4) was added. The anticoagulated blood was then centrifuged at 120 g for 20 min with the deceleration set to 0, and after the end of the process, a clear layering was observed. Platelet-rich plasma (PRP) was collected 1 cm above the white blood cell layer and transferred to a new 15 mL centrifuge tube.

[0037] 2) Then, ACD anticoagulant and PGE1 were added to the PRP to prevent coagulation and platelet activation. The maximum deceleration was set to 800 g for 20 min to obtain the platelet pellet. The platelet pellet was resuspended in Tyrode buffer (12 mM NaHCO3, 10 mM HEPES, 134 mM NaCl, 1 mM MgCl2, 0.34 mM Na2HPO4, 2.9 mM KCl, pH = 7.4) and the protein content was quantified using a BCA kit.

[0038] 3) 1 μΜ of prostaglandin El (PGE1) and 1 mM of phenylmethylsulfonyl fluoride were added to the solution to prevent platelet activation or membrane protein degradation.

[0039] 4) After 5 cycles of freezing (-80 °C) and thawing (25 °C), the platelet suspension was centrifuged at 12000 g for 30 min at 4 °C, and the platelet membrane was collected.

[0040] 5) Finally, the platelet membrane was stored at -80 °C for the preparation of nanoparticles with different formulations.

[0041] 6) To characterize whether the main functional proteins of the platelet membrane on the surface of the nanoparticles (such as CD41, CD61 and CD62p) were preserved, SDS-PAGE and Western blot experiments were used to detect the protein profiles of platelets, PMs and MSN@D@PM@Ab. First, the different sample pellets were collected by centrifugation at 15000 g for 10 min, and then lysed in ice-cold RIPA buffer for 20 min. The supernatant was collected by centrifugation at 12000 g for 15 min, boiled in SDS buffer for 5 min, and then loaded onto a 10% polyacrylamide gel in 10 wells. Protein separation was performed by electrophoresis at 100 V and 140 mA for 70 min.

[0042] 7) After the separation, the protein gel was transferred to a PVDF membrane and blocked in 5% skim milk for 1 hour. The PVDF membrane was incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. All bands were visualized by a Bio-Rad imaging system.

[0043] 8) To obtain the protein expression profile of different samples, the gels after electrophoresis were placed in a container of appropriate size, 100 mL of deionized water was added, and the container was shaken on a shaker for 5 min, and a total of 3-5 times to remove impurities such as SDS in the gel. After washing the gel, the protein gel was placed in the coomassie brilliant blue protein staining solution for 8 hours, and the gel was stained in the staining solution overnight. Add about 100 mL of deionized water, and shake on a shaker. Every 5-15 min, carefully pour out the liquid, add 100 mL of deionized water, and continue to decolorize on the shaker. After 30 min, the gel was visualized by Bio-Rad imaging system.

[0044] 1.3 Preparation and characterization of drug-loaded mesoporous silica nanoparticles (MSN@D)

[0045] 1) First, weigh 5 mg of MSN into 5 mL of anhydrous ethanol, and ultrasonic at 20 KHz, 50 W for 30 min to 1 h at room temperature to make MSN completely dispersed in anhydrous ethanol.

[0046] 2) Weigh 5 mg of Defactinib drug and add it to the MSN suspension in small amounts several times, and ultrasonic again to make it uniformly dispersed with MSN.

[0047] 3) Stir the mixture at 500 rpm at room temperature for 6 h, take out 500 μL for HPLC detection, and centrifuge the remaining sample at 10000 rpm for 15 min to remove the unloaded drug, and the precipitate is the sample MSN@D.

[0048] 4) When the production yield is expanded, 50 mg of MSN and 50 mg of Defactinib drug are prepared according to the above steps, and the supernatant is removed after centrifugation, and the precipitate is freeze-dried into a powder for use.

[0049] 1.4 Preparation and characterization of platelet membrane-coated drug-loaded mesoporous silica nanoparticles (MSN@D@PM)

[0050] 1) First, explore the optimal ratio of PM to MSN@D, disperse 10 mg of MSN@D nanoparticles in 1 mL of PBS solution containing different amounts of platelet membranes, and ultrasonic (42 kHz, 100 W) on ice for 45 min. Then detect the mass of platelet membranes required for complete coverage of MSN by TEM and DLS. When no free MSN@D particles are observed under transmission electron microscope, and the surface potential of the product MSN@D@PM is the same as that of pure platelet membranes (PM), it is considered that all MSN are covered by PM. Through this identification method, the optimal formula is MSN@D:PM = 5:1 (mass ratio).

[0051] 2) Next, the experiment was carried out with the optimal mass ratio of m (MSN@D:PM) = 5:1, 5 mg of MSN@D was dissolved in 5 mL of anhydrous ethanol, and ultrasonic was performed until MSN@D was completely dispersed in anhydrous ethanol, 1 mg of PM was added to the above-mentioned MSN@D dispersion, and ultrasonic dispersion was performed for 30 min, then 500 rpm magnetic stirring was performed at 4-10°C (ice bag was added in the water bath) for 15 min, then the dispersion was squeezed back and forth 10 times through a 200 nm filter membrane, 1 mL of the filtered sample was taken for subsequent characterization, and the remaining sample was centrifuged, and MSN@D@PM powder was obtained after freeze-drying.

[0052] 3) When the production yield was expanded, 75 mg of MSN@D was dissolved in 20 mL of anhydrous ethanol, and ultrasonic was performed until MSN@D was completely dispersed in anhydrous ethanol, 15 mg of PM was added to the above-mentioned MSN@D suspension, and ultrasonic dispersion was performed for 30 min, then 500 rpm stirring was performed at 4-10°C for 15 min, then the sample was centrifuged, and MSN@D@PM powder was obtained after freeze-drying.

[0053] 1.5 Preparation and characterization of MSN@D@PM@Ab nanoparticles

[0054] 1) In order to couple the PD-L1 antibody to the MSN@D@PM nanoparticles, the membrane surface was thiolated with Traut reagent for 45 min, and then washed with Tyrode buffer (800 g, 5 min) for three times to remove the excess Traut reagent.

[0055] 2) The PD-L1 antibody was mixed with sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) at a molar ratio of 1:5 at 4°C for 2 h. The excess linker was removed by dialysis (molecular weight cutoff size of 10 kDa) to obtain aPD-L1-sulfo-SMCC.

[0056] 3) The MSN@D@PM was mixed with aPD-L1-sulfo-SMCC at a protein mass ratio of 1:10, and after reaction at room temperature for 1 h, the unbound PD-L1 antibody in the supernatant was removed by centrifugation at 10000 g for 10 min, and the precipitate was freeze-dried to obtain MSN@D@PM@Ab.

[0057] 4) The characterization of the nanoparticles mainly used ultraviolet-visible spectrophotometer to detect the absorption spectrum of the sample at 280 nm, and used laser confocal microscope to observe the fluorescence protein co-localization to determine the success of antibody coupling.

[0058] 5) In the preparation of samples for laser confocal microscopy experiments, in order to connect fluorescein (cy5.5-NHS) on PD-L1 antibody, 0.7 mL of 700 ug cy5.5-NHS (DMSO dissolved) was added to 0.89 mL of a solution containing 3 mg of PD-L1 antibody, and the mixture was gently shaken and mixed, then the reaction tube was placed in the dark, and the reaction was mixed at room temperature for 60 min, and the two reactants were mixed by gently inverting the tube every 10-15 min. After the reaction was completed, the supernatant was removed by centrifugation at 3000 rpm for 3 min, and the precipitate was cy5.5-NHS (red light) labeled PD-L1 antibody.

[0059] 6) In order to connect another fluorescein (coumarin-6) on PM, 1 mL of 3 mg / mL coumarin-6 (ethanol dissolved) was added to 3 mg of PM, and the mixture was gently shaken and mixed, and the above steps were repeated to obtain coumarin-6 (blue light) labeled PM.

[0060] 7) The coumarin-6 labeled PM was dispersed in 1 mL of PBS containing 1 mM prostaglandin E1. To 1 mL of cy5.5-NHS labeled aPD-L1 solution, 200 μL of 1 mM Sulfo-SMCC linker PBS solution (molar ratio of aPD-L1 to Sulfo-SMCC was 1:1.2) was added, and the mixture was stirred at 300 rpm at 4°C for 2 h. The solution was centrifuged at 8000 rpm for 10 min in an ultrafiltration tube to remove unbound linker. The sample obtained by reacting 1 mL of SMCC / cy5.5-PD-L1 with 1 mL of coumarin-6-PM solution at room temperature for 2 h was observed by laser confocal photography.

[0061] 8) In order to evaluate the stability of the final product at different temperatures and time points, 5 mg of MSN@D@PM@Ab sample was resuspended in 5 mL of PBS, and after complete dispersion, it was placed on a magnetic stirrer at room temperature and the time was recorded. Then 1 mL of solution was taken out at 7 days, 14 days, 21 days, and 28 days for laser dynamic light scattering particle size analysis, and was labeled as D1, D2, D3, and D4, respectively. The same experiment was repeated at 4°C, and the samples were labeled as D1', D2', D3', and D4'.

[0062] 9) In order to characterize the drug release of the nanoparticles, 10 mg of MSN@D@PM@Ab samples were respectively resuspended in 5 mL of pH=5, pH=6.5, pH=7.4 pH buffer, and made into a dispersion of 2 mg / mL, placed on a magnetic stirrer at 500 rpm, and the time was recorded. At the 3rd hour, the 6th hour, the 9th hour, the 12th hour, the 24th hour, the 48th hour, 500 μL samples were taken, labeled as H5 / 6.5 / 7.4_1-5, and the removed samples were immediately frozen in the refrigerator to prevent recording of the release, and HPLC detection was performed. The final results are as follows Figure 2 -c.

[0063] Example 2

[0064] The MSN@D@PM@Ab nanoparticles prepared in conjunction with Example 1 were verified as follows:

[0065] 1. Characterization of the composite nanosilicon ball drug delivery system

[0066] First, the present application characterizes the nanoparticles of each distributed product by transmission electron microscopy (TEM) and laser dynamic light scattering particle size analyzer (DLS). As shown in Figure 2 , the uniformly dispersed MSN is a solid sphere with uniform size, with an average hydration particle size of about 150 nm, and the surface can see clear and regular mesoporous structure. The zeta potential analysis results ( Figure 2 -b) show that the average potential of the MSN nanoparticles is -22.8 mV, and after multiple repeated experiments, it is found that the sample error between different batches is small. Compared with MSN and MSN@D, the MSN@D@PM nanoparticles are coated with a thin film-like material with a thickness of 5-7 nm, showing a typical core-shell structure, with an average particle size of 155 nm, which is close to the theoretical result, proving that the MSN@D nanoparticles coated with platelet membranes are successfully synthesized. Since the average potential of the surface of the simple platelet membrane is -43.5 mV, after being coated on the surface of the negatively charged MSN@D, the overall surface potential of the MSN@D@PM rises to -24.1 mV, which is consistent with the results reported in the previous literature. This change is speculated to be due to the platelet covering part of the negative charge on the surface of the MSN. The above results further prove the coating synthesis of MSN@D@PM.

[0067] Since the surface proteins of platelets have thiol-SH, by coupling the PD-L1 antibody with the SMCC linker and mixing with the MSN@D@PM, the MSN@D@PM@Ab is obtained, and various characterizations are performed. First, in order to confirm the successful connection of the antibody, the nanoparticles after staining were observed by fluorescence confocal microscope. As shown in Figure 2-e shows that the nanoparticles are uniformly distributed in the bright field, blue represents the platelet membrane, red represents the PD-L1 antibody, and the antibody connected to the surface of the platelet membrane after channel superposition shows pink light, indicating that the antibody has been successfully connected to the platelet surface.

[0068] Second, in order to characterize the integrity of the tumor-specific targeting protein of MSN@D@PM@Ab, the results of protein electrophoresis experiment (Fig. Figure 2 -f) shows that compared with pure platelets and platelet membranes, MDPA NPs express higher levels of CD41, CD61 and CD62P platelet-specific antigens, which indirectly indicates that they have stronger targeting binding ability with tumor cell surface receptors. In addition, the results of Coomassie blue staining (Fig. Figure 2 -g) found that the protein spectrum integrity of the three was consistent, and there was no band loss or expression reduction, which proved that the synthesis process did not cause loss of characteristic proteins of the product, and the membrane protein had good integrity and could inherit the biological interface function of the platelet membrane. Through the results of morphology and particle size analysis, it was found that MDPA NPs still had clear mesoporous and membrane-coated characteristics, with an average particle size of about 155 nm. It may be affected by the uniformity of membrane coating and the mutual adhesion of nanoparticles, so the particle size is slightly larger than that of empty MSN; the average surface potential is-32.7 mV, which indicates that the coupling of the antibody increases the negative value of the potential and enhances the colloidal stability.

[0069] Finally, in order to characterize the drug release of MDPA NPs in different pH environments and the stability at different temperatures, Figure 2 -d shows that at 4℃ and 25℃, the particle size of MDPA NPs is about 150 nm after 28 days, which indicates that the nano material has colloidal stability at different temperatures and at different time during storage, and can exist stably in the complex blood environment rich in proteins, and prolong the circulation and drug release time. In addition, from Figure 2 -c shows that under the conditions of pH 5, pH 6.5 and pH 7, the drug release of MDPA NPs can still reach 95% after 72 hours, which indicates that the nanoparticles can tolerate changes in the acid-base environment of the body. At the same time, under the acidic conditions of pH=5, the release rate of defactinib drug is faster, which indicates that the acidic environment of the tumor microenvironment is more conducive to the release of the loaded drug, and will not cause the drug to leak in normal tissues in advance, and has certain biological safety.

[0070] 2. In vivo functional verification of composite nanosilicon sphere drug delivery system

[0071] To further investigate whether MSN@D@PM@Ab could inhibit tumor growth, we injected the Luc-MC38 cell line into the subcecal serosa of 25 C57BL6 mice. On day 10, bioluminescence was observed via in vivo imaging, confirming successful orthotopic tumor bearing in all mice. On the same day, the mice were divided into five groups and treated intravenously with the same dose of MSN, MSN@D, MSN@D@PM, MSN@PM plus free defatinib, and MSN@D@PM@Ab. In vivo imaging was then performed on days 16, 22, and 28 to observe and quantify the growth of the orthotopic cecal tumor. Figure 3 As shown in -b, the size of the orthotopic tumors in all mice was basically the same on day 10. On day 6 after administration, compared with the blank saline group, the cecal tumors continued to grow, while the growth of cecal tumors in the other three treatment groups (MSN@D, MSN@D@PM, and MSN@PM+D) was inhibited to some extent, and the tumor bioluminescence intensity decreased. In particular, the tumors in the MSN@D@PM@Ab treatment group decreased significantly, with the fluorescence intensity per unit area decreasing exponentially until the tumors in this group almost completely disappeared on day 28 after administration. On day 48, all mice were sacrificed, the orthotopic cecal tumors were dissected, and photographed for analysis. The average tumor volume of the mice treated with MSN@D@PM@Ab was 16 mm. 3 Smaller than the saline group (200mm) 3 MSN@D (180mm) 3 MSN@PM+D (90mm) 3 ) and MSN@D@PM (65mm) 3 The average tumor volume of mice treated with this method () Figure 3 -c), indicating that MDPA NPs have a significant inhibitory effect on the growth of colorectal in situ tumors. For example... Figure 3 As shown in Figure 1, the composite nanoparticles MDPAs significantly prolong the survival time of mice.

[0072] To further investigate the effects of MSN@D@PM@Ab on collagen fibers in cecal tumors of mice in different treatment groups while inhibiting tumor growth, we paraffin-fixed and embedded mouse tumors dissected on days 22 and 36, and stained them with Sirius red. Figure 3 As shown in -e, red represents collagen fibers. Compared to the saline control group, the collagen fiber content decreased in a decreasing trend in the four treatment groups: MSN@D, MSN@PM+D, MSN@D@PM, and MSN@D@PM@Ab, especially in the MDPA group where the level was the lowest. Furthermore, comparisons in mouse tumors at 22 and 36 days showed that the collagen fiber content in the tumors decreased with the extension of the drug release time from the nanoparticles. Figure 3- f), which indicated that MSN@D@PM@Ab nanoparticles could significantly reduce the collagen content in the mouse cecal orthotopic tumor, which helped to further study the effect of collagen on immune cell infiltration.

[0073] To further explore whether the reduction of tumor collagen fibers after nanoparticle treatment could affect immune cell infiltration, we performed immunohistochemical staining on the above paraffin sections, as described above Figure 3 - g, the CD8 + T cells in the tumor center and edge were quantitatively analyzed, and we found that compared with the saline, MSN@D, and MSN@PM+D groups, the MSN@D@PM and MSN@D@PM@Ab treatment groups significantly infiltrated more CD8 + T cells in both the tumor center and edge. In addition, the PD-L1 antibody connected to the surface of the MDPA nanoparticles could antagonize the PD-1 antigen signal on the surface of tumor cells, further enabling the T cells that reached the "battlefield" to exert tumor-killing effects. In summary, we concluded that the MDPA nanoparticles not only reduced the collagen content in the tumor to promote CD8+ T cell infiltration, but also activated T cell tumor-killing function by blocking checkpoint molecules on the surface of tumor cells, significantly inhibiting the proliferation and growth of colorectal cancer orthotopic tumors.

[0074] 3. Tumor targeting and pharmacokinetic analysis of composite nanosilica sphere drug delivery system

[0075] To further study the tumor targeting ability of MSN@D@PM@Ab in the MC38 cecal orthotopic tumor mouse model, we excised the tumor and major organs after injecting Cy5 fluorescently labeled nanoparticles for 6 h and 24 h, respectively. The in vitro fluorescence image results showed that compared with the simple Cy5 fluorescent dye control group, the accumulation of MSN@D@PM@Ab at the tumor site significantly increased. At 6 h and 48 h, the fluorescence signal of the MDPAs group was 6.78 times and 3.10 times higher than that of the control group, respectively Figure 4 -c). We also observed strong fluorescence signals in the lungs and livers of MDPAs-treated mice. The liver fluorescence signal suggests that part of the nanoparticles were captured by the reticuloendothelial system after entering the systemic circulation, resulting in prolonged residence time; the fluorescence signal in the lungs suggests that nanoparticles may experience delayed residence during gas exchange in the pulmonary capillary network and alveolar cavity. In addition, the signal in the kidneys may be due to the release of Cy5 fluorescent dye from the degradation of nanoparticles through the glomerular capillary network, which is also one of the rate-limiting steps before being removed from the body Figure 4 -a / b).

[0076] 4. In vivo toxicity and side effects analysis of the composite nano-silicon sphere drug delivery system

[0077] Finally, the in vivo biosafety of MSN@D@PM@Ab nanoparticles was investigated. H&E staining of vital organs in C57BL6 mice from different nanoparticle treatment groups revealed no significant hemorrhage, necrosis, or other pathological damage in any of the major organs, including the heart, liver, spleen, lungs, and kidneys. Figure 6 (ab). Furthermore, there were no significant changes in the weight of a few vital organs in each treatment group. These results indicate that MSN@D@PM@Ab has no significant organ toxicity and exhibits good biosafety characteristics. Next, during the observation of tumor recurrence and growth after administration, the mice were weighed every other day. Figure 6 c) It was found that the weight of mice in both the treatment group and the control group increased from 19g to 21g, with no significant weight loss. Therefore, it can be concluded that the nanomaterial has no toxic side effects in vivo.

[0078] Tumor-bearing mice often exhibit compensatory splenomegaly due to a dysfunctional immune response. Spleen harvesting on day 23 and assessment of its enlargement are potential markers of tumor progression. Mice in the saline, MSN@D, MSN@PM+D, and MSN@PM@D groups showed splenomegaly, but mice treated with MDPA NPs exhibited spleen morphology close to normal. Figure 6 (de), further demonstrating the good efficacy of MDPA NPs in immunotherapy.

Claims

1. The use of a composite nanometer silicon ball drug delivery system in the preparation of a drug for treating cecal orthotopic tumors by reducing the content of tumor collagen fibers, characterized in that, The composite nanometer silicon ball drug delivery system is prepared by the following steps: (1) preparation of drug-loaded mesoporous silica nanoparticles: first, mesoporous silica nanoparticles are added to anhydrous ethanol, and ultrasonic treatment is performed at room temperature to make the mesoporous silica nanoparticles completely dispersed in the anhydrous ethanol to obtain a mesoporous silica nanoparticle suspension; defactin is added to the mesoporous silica nanoparticle suspension, and ultrasonic treatment is again performed to make the defactin uniformly dispersed in the mesoporous silica nanoparticles; the mixture is stirred at room temperature, and centrifugation is performed to remove the unloaded drug, and the precipitate is the MSN@D nanoparticles; (2) preparation of platelet membrane-coated drug-loaded mesoporous silica nanoparticles: the MSN@D nanoparticles are dispersed in a PBS solution containing platelet membranes, the mass ratio of the MSN@D to the platelet membranes is 5:1, and ultrasonic treatment is performed on ice to obtain the MSN@D@PM; (3) preparation of MSN@D@PM@Ab nanoparticles: the surface of the MSN@D@PM is thiolated by using Traut reagent for 45 min, and then the excess Traut reagent is removed by washing with Tyrode buffer to obtain thiolated MSN@D@PM; a PD-L1 antibody is mixed with a sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker at 4°C, the molar ratio of the PD-L1 antibody to the sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate is 1:5, the excess linker is removed by dialysis, and aPD-L1-sulfo-SMCC is obtained, the molecular weight cutoff size of the membrane used for dialysis is 10 kDa; the thiolated MSN@D@PM is mixed with the aPD-L1-sulfo-SMCC at a protein mass ratio of 1:10, and after reaction at room temperature, the supernatant is removed by centrifugation, and the precipitate is lyophilized to obtain the MSN@D@PM@Ab.

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