CN109364267B - Tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle and preparation method thereof - Google Patents
Tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle and preparation method thereof Download PDFInfo
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Abstract
The mesoporous silica nano drug-loaded particle with the tumor tissue and cell double targets provided by the invention is composed of a mesoporous silica nano particle with a modified surface and a drug, wherein the mesoporous silica nano particle with the modified surface is composed of a mesoporous silica nano particle with a modified surface by amino, a polyacrylic acid film layer of the mesoporous silica nano particle with the modified surface by amino through electrostatic action, and polyethylene glycol connected with polyacrylic acid in the polyacrylic acid film layer through an amido bond, the polyacrylic acid film layer is used as a plugging valve, the pore channel of the mesoporous silica nano particle with the modified surface by amino can be in a plugging or opening state in response to the change of pH value, and the drug is positioned in the pore channel structure of the mesoporous silica nano particle with the modified surface by amino. The drug-loaded nanoparticle can effectively avoid damage of targeted drugs or chemotherapeutic drugs with lymphocyte toxicity to lymphocytes, and improve the combined treatment effect of targeted therapy and immunotherapy on tumors.
Description
Technical Field
The invention belongs to the field of targeted drug-loaded materials, and relates to a tumor tissue and cell dual-targeted mesoporous silica nano drug-loaded particle and a preparation method thereof.
Background
Overactivation of the mitogen-activated protein kinase (MAPK) pathway by genetic alterations in various cancersIs common in all. Small molecule targeted therapy is considered to be one of the most promising approaches to the treatment of cancer and genetic lesions. About 60% of melanomas contain BRAFV600EMutation, specific targeting of BRAFV600EThe mutated targeted drug Vemurafenib (PLX 4032) showed an unprecedented response rate in the treatment of metastatic melanoma. In a series of clinical trials, the objective overall response rate of vemurafenib-treated patients was 48% -53%, and the average benefit time was 5.3-6.7 months. However, after a period of targeted therapy, the tumor develops resistance and subsequently the tumor recurs quickly. Studies have shown that one of the mechanisms of BRAF resistance is reactivation of the MAPK pathway, including NRAS mutations, BRAF gene amplification, BRAF alternative splicing, MEK mutations, NF1 down-regulation, and others. In addition, BRAF inhibitors (BRAFi) can also induce over-activation of wild-type BRAF, induce the development of Squamous Cell Carcinoma (SCC), and severely impact the quality of life of patients.
In order to solve the problems, a novel inhibitor of the MEK kinase which targets the downstream of the MAPK pathway, namely, sematinib, is developed, so that the response rate in treating drug resistance and melanoma is better, and the survival period of a patient can be remarkably prolonged. In clinical trials of combined therapy with BRAFi and MEK inhibitor (MEKi), the median progression-free survival in the combination treatment group was 9.4 months, while that in the BRAFi monotherapy group was 5.8 months; the incidence of SCC decreased from 19% in monotherapy to 7% in combination therapy. Therefore, BRAFi combined with MEKi double-drug therapy replaces BRAFi single-drug therapy, and becomes a first-line clinical scheme. Although combination therapy increases patient overall response rates, extends patient survival and alleviates side effects, disease progression remains prevalent. Therefore, there is an urgent need to find new long-term anti-tumor strategies.
Over the last few years, immunotherapy, especially antibody-mediated immune checkpoint blockade, has shown a long-lasting tumor-inhibiting effect in the treatment of metastatic melanoma. Representative drugs for antibody-mediated immune checkpoint blockade include Iplilimumab, which blocks CTLA-4, Pembrolizumab and Opdivo, which blocks the PD-1/PD-L1 pathway. In clinical studies, blocking PD-1/PD-L1 by Pembrolizumab and Opdivo antibodies significantly extended progression-free and overall survival with low toxicity. However, antibody-mediated PD-1/PD-L1 immunodetection point blockade has a low overall effector rate, with only about 10% -40% of patients being therapeutically effective against the anti-PD 1 antibody.
Both BRAFi and MEKi combined dual drug targeted therapy and immune checkpoint blockade cannot permanently benefit the majority of patients. Interestingly, their advantages and disadvantages are exactly complementary. Therefore, combining immunotherapy with targeted therapy for the treatment of tumors would be a promising anti-tumor strategy. However, the combination of the two has been hampered by the potential toxic side effects of the targeted drug on the immune system. On one hand, BRAFi and MEKi can promote immune recognition by promoting a tumor antigen presentation mechanism and promote the proportion of tumor infiltrating T cells; on the other hand, MEKi may inhibit its function by inhibiting the activated T cell MAPK pathway, inhibiting tumor immune response. In the clinical experiment of BRAFi and MEKi combined immune checkpoint blockade, the combined effect is not ideal. Although the role of MEKi in immune checkpoint blockade combination regimens is still controversial, the toxic side effects of MEKi on T cells need to be avoided.
The nano targeting vector has excellent biocompatibility, biological safety and precise nano pore size, so that the nano targeting vector is widely applied to a drug delivery system. Based on the characteristics of high permeability and retention effect (EPR effect) of the tumor and the difference between the tumor microenvironment and the normal tissue microenvironment, such as the pH value of the tumor tissue is lower than that of the normal tissue, the reducibility of the tumor tissue is higher than that of the normal tissue, the variety and content of enzyme are changed, the permeability of tumor cells is enhanced, and the like, the drug carrier with specific targeting and stimulation response of the tumor tissue and the tumor cells can be designed. As for carriers of anticancer drugs, nano-carriers represented by liposomes and micelles are currently most used. However, the further development of liposomes and micelles is limited by their disadvantages such as low drug loading and poor stability. In addition, the transmembrane drug transfer mediated by the liposome is promoted by membrane fusion, endocytosis and small molecule diffusion, so that the liposome can be endocytosed by tumor cells and also can be endocytosed by lymphocytes, and the function of the lymphocytes can be influenced. Therefore, the development of a novel nano drug delivery system which can realize accurate targeting of tumor tissues and tumor cells and can prevent lymphocytes from being damaged by targeted drugs has important significance for realizing the cooperative treatment of tumor targeted therapy and immunotherapy.
Disclosure of Invention
Aiming at the problem of immunotoxicity caused by targeted drugs, the invention provides a tumor tissue and cell dual-targeted mesoporous silica nano drug-loaded particle and a preparation method thereof, so as to avoid the targeted drugs from damaging lymph cells and improve the combined treatment effect of targeted therapy and immunotherapy on tumors.
The tumor tissue and cell dual-targeting mesoporous silica nano drug-carrying particle provided by the invention consists of a medicament and a mesoporous silica nano particle with the surface modified by polyacrylic acid and polyethylene glycol, the mesoporous silica nano-particles with the surfaces modified by polyacrylic acid and polyethylene glycol are prepared by modifying mesoporous silica nano-particles with the surfaces modified by amino, coating the polyacrylic acid film layer of the mesoporous silicon dioxide nano-particles with the surface modified by amino through electrostatic action, and polyethylene glycol connected with polyacrylic acid in the polyacrylic acid film layer through amido bond, the polyacrylic acid film layer is used as a plugging valve, which can respond to the change of pH value to make the pore channel of the mesoporous silicon dioxide nano-particle with the surface modified by amino group in a plugging or opening state, the drug is positioned in a pore channel structure of the mesoporous silica nanoparticle with the surface modified by the amino; the drug-loaded nanoparticle has the ability to avoid drug-induced lymphocytotoxicity.
In the technical scheme of the tumor tissue and cell dual-targeting mesoporous silica drug-loaded nanoparticle, the particle size of the drug-loaded nanoparticle is 100-200 nm.
In the technical scheme of the tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle, the content of polyacrylic acid in the nano drug-loaded particle is preferably 7.2 wt.% to 7.5 wt.%, but is not limited to this range, and the specific content can be adjusted according to actual application requirements.
In the technical scheme of the tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle, the content of polyethylene glycol connected with polyacrylic acid in a polyacrylic acid membrane layer through an amide bond in the nano drug-loaded particle is preferably 1.8 wt.% to 2.8 wt.%, but is not limited to this range, the specific content can be adjusted according to the actual application requirement, and the content of polyethylene glycol is further preferably 2.2 wt.% to 2.5 wt.%.
In the technical scheme of the tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle, the content of amino modifying groups on the surface of the mesoporous silica nano particle in the drug-loaded nanoparticle is such that the Zeta potential of the mesoporous silica nano particle with the surface modified by amino is preferably 10-30 mV.
In the technical scheme of the tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle, the mesoporous silica nano particle with the surface modified by amino is obtained by grafting a group R on the surface of the mesoporous silica nano particle, and the group R is preferably a group R
O connected with Si in the group R is connected with Si on the surface of the mesoporous silicon nano-particles to form Si-O bonds.
In the technical scheme of the mesoporous silica nano drug-loaded particle with tumor tissue and cell dual targeting, the drug is a targeting drug with lymphocytotoxicity or a chemotherapeutic drug with lymphocytotoxicity, and includes but is not limited to AZD6244, PLX4032, BEZ235 and AZD 8055. The content and the type of the drug in the silicon nano drug-loaded particles are determined according to the influence of practical application requirements, and the content of the drug is preferably 5 wt.% to 15 wt.%, but the content of the drug is not limited to the range.
In the technical scheme of the mesoporous silica nano drug-loaded particle with tumor tissue and cell dual targeting, the nano drug-loaded particle has the capability of passively targeting tumor tissue, has the capability of specifically targeting tumor cells and avoiding the endocytosis of the tumor cells by lymphocytes, has the capability of avoiding the lymphocytotoxicity caused by small molecular drugs in vitro and in vivo, and has the capability of cooperatively resisting tumor and stimulating an immune system with tumor immune drugs in vivo.
The invention also provides a preparation method of the tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle, which comprises the following steps:
(1) preparation of mesoporous silica nanoparticles
Adjusting the pH value of a hexadecyl trimethyl ammonium bromide aqueous solution to 11.0-11.5, heating to 78-82 ℃ under stirring, dropwise adding ethyl orthosilicate, reacting at 78-82 ℃ for 2-3 h, dispersing a reaction product in a concentrated hydrochloric acid-absolute ethanol solution, refluxing at 78-82 ℃ for 3-5 h, washing the obtained product with ethanol and water, and drying to obtain mesoporous silica nanoparticles;
(2) preparation of amino-modified mesoporous silica nanoparticles
Dispersing mesoporous silica nanoparticles in isopropanol or ethanol to form a dispersion liquid A, mixing the dispersion liquid A with 3-aminopropyltriethoxysilane, stirring and reacting at 78-82 ℃ for 12-16 h, washing a reaction product with ethanol and water, and drying to obtain amino-modified mesoporous silica nanoparticles;
(3) preparation of amino-modified mesoporous silica nano drug-loaded particles
Dispersing the amino-modified mesoporous silica nanoparticles in PBS buffer solution or water to form dispersion liquid B, mixing the dispersion liquid B with a solution of a medicament, reacting for 8-10 h, washing an obtained reaction product with the PBS buffer solution, and freeze-drying to obtain amino-modified mesoporous silica nanoparticle medicament-carrying particles;
(4) preparation of polyacrylic acid modified mesoporous silica nano drug-loaded particles
Dispersing the amino-modified mesoporous silica nano drug-loaded particles in PBS buffer solution or water to form dispersion solution C, adding polyacrylic acid aqueous solution, stirring and reacting for 2-4 h, washing a reaction product with the PBS buffer solution to obtain polyacrylic acid-modified mesoporous silica nano drug-loaded particles, and dispersing the polyacrylic acid-modified mesoporous silica nano drug-loaded particles in the PBS buffer solution or water to form dispersion solution D;
(5) preparation of mesoporous silica nano drug-loaded particles for dual targeting of tumor tissues and cells
Adding aminated polyethylene glycol into the dispersion liquid D, carrying out ultrasonic mixing, then stirring and reacting for 12-16 h, filtering by adopting an ultrafiltration membrane, collecting nanoparticles obtained by the reaction, and washing by using deionized water to obtain the mesoporous silica nano drug-loaded particles with double targets of tumor tissues and cells.
In the step (1) of the method, the concentration of the hexadecyl trimethyl ammonium bromide aqueous solution is preferably 1.8-2.5 mg/mL, and the volume ratio of the ethyl orthosilicate to the hexadecyl trimethyl ammonium bromide aqueous solution is preferably 1 (90-150).
In the step (2) of the above method, the concentration of the mesoporous silica nanoparticles in the dispersion A is preferably 8 to 15mg/mL, and the volume ratio of the 3-aminopropyltriethoxysilane to the dispersion A is preferably 1 (150 to 200).
In the step (3) of the method, the preferable mixing ratio of the dispersion liquid B and the solution of the drug is such that the mass ratio of the drug to the amino-modified mesoporous silica nanoparticles is 1 (1-10).
In the step (4) of the method, the preferable adding amount of the polyacrylic acid aqueous solution is that the mass ratio of the polyacrylic acid to the amino-modified mesoporous silica nano drug-loaded particles is (2.5-25): 100; the molecular weight of the polyacrylic acid is preferably 1500-2000 KD, but is not limited to the molecular weight range.
In the step (5) of the method, the mass ratio of the aminated polyethylene glycol to the polyacrylic acid-modified mesoporous silica nano drug-carrying particles in the dispersion liquid D is preferably (2-5): 100; the molecular weight of the aminated polyethylene glycol is preferably 4-6 KD, but is not limited to the molecular weight range.
In the step (1) of the method, the volume ratio of the concentrated hydrochloric acid to the absolute ethyl alcohol in the concentrated hydrochloric acid-absolute ethyl alcohol solution is preferably (1-2) to (8-9).
In the method, the concentration of the amino-modified mesoporous silica nanoparticles in the dispersion liquid B is preferably 2-6 mg/mL, the concentration of the amino-modified mesoporous silica drug-loaded nanoparticles in the dispersion liquid C is preferably 3-5 mg/mL, and the concentration of the polyacrylic acid-modified mesoporous silica drug-loaded nanoparticles in the dispersion liquid D is preferably 3-5 mg/mL.
The pH of the PBS buffer used in the above method was 7.4.
Compared with the prior art, the technical scheme of the invention has the following beneficial technical effects:
1. the mesoporous silica nano drug-loaded particle with tumor tissue and cell dual targets provided by the invention is composed of mesoporous silica nano particles and drugs, the surfaces of which are modified by polyacrylic acid and polyethylene glycol, and compared with amorphous silicon materials such as liposome and micelle, the mesoporous silica nano material has the advantages of excellent biocompatibility, better biological safety, larger specific surface area and pore volume, uniform and controllable mesoporous size, excellent stability, high drug loading capacity and the like.
2. In the nano drug-loaded particles provided by the invention, polyacrylic acid and mesoporous silica nano drug-loaded particles with positive charges and surfaces modified by amino groups are combined through electrostatic action to form a polyacrylic acid film layer to block the pore channels of the mesoporous silica, the polyacrylic acid is a polyanion polymer and has the property of pH controllability and can respond to pH stimulation, the polyacrylic acid is protonated and is converted from negative charges to positive charges in an acid environment (pH is less than 5.0), and meanwhile, when the pH value is reduced, the carboxyl protonation of the polyacrylic acid weakens the electrostatic interaction between the polyacrylic acid and the mesoporous silica nano drug-loaded particles with surfaces modified by amino groups, so that the polyacrylic acid shell film layer becomes loose, and the drugs are released from the pore channels of the mesoporous silica. Normal pH environment in vivo (pH ≈ 7.4)) is not sufficient to protonate polyacrylic acid, thereby ensuring that the drug is not prematurely released. When the drug-loaded nanoparticles are endocytosed by cells, polyacrylic acid is protonated by the acidic environment of lysosomes and inclusion bodies in the cells, and the polymer shell on the surfaces of the drug-loaded nanoparticles is loosened, so that the drugs are released.
3. According to the invention, through screening the amount of amino modifying groups of mesoporous silica nanoparticles with the surfaces modified by amino and the amount of polyacrylic acid, the covering thickness of a polyacrylic acid film layer used as a plugging valve is proper, and the particle size of the drug-loaded nanoparticles is proper, so that the drug-loaded nanoparticles provided by the invention have good capacity of being endocytosed by cancer cells, and meanwhile, the drug-loaded nanoparticles are ensured not to be endocytosed by lymphocytes, and the drug-loaded nanoparticles which can only be endocytosed by tumor cells but not by lymphocytes are obtained.
4. The preparation method connects the polyethylene glycol with the polyacrylic acid through the amido bond, the introduction of the polyethylene glycol can increase the biocompatibility of the nano particles, and simultaneously, the charge of the prepared polyacrylic acid modified mesoporous silica nano drug-loaded particles is changed from negative charge to neutral, so that the circulation time of the nano drug-loaded particles in blood can be prolonged, and the nano drug-loaded particles are prevented from being removed by reticuloendothelial cells and combined with plasma protein. Due to the EPR effect of the tumor, the drug-loaded nanoparticles provided by the invention can be enriched at the tumor part through passive targeting, so that the utilization rate of the drug in tumor tissues is improved, and the toxic and side effects of the drug in blood systems and other tissues are reduced.
5. Through the series of designs mentioned in the point 4, the tumor tissue and cell dual-targeting mesoporous silica nano drug-carrying particle is successfully constructed, and compared with the traditional mesoporous silica drug carrier and lipid nano carrier, the nano drug-carrying particle provided by the invention has higher tumor and cell targeting drug-carrying capacity, can effectively improve the drug utilization rate and reduce the toxic and side effects.
6. Experiments prove that the nano drug-loaded particles have the capabilities of passively targeting tumor tissues and specifically targeting tumor cells, can be prevented from being endocytosed by lymphocytes, have the capability of protecting the lymphocytes from being damaged by targeted drugs in vitro and in vivo, and have the capabilities of resisting tumors and stimulating an immune system in cooperation with tumor immune drugs in vivo when the loaded drugs are MEK inhibitors AZD 6244.
7. The invention also provides a preparation method of the mesoporous silica nano drug-carrying particle with tumor tissue and cell dual targets, the method is used for carrying out amination modification on the mesoporous silica nano particle, the process for coating the polyacrylic acid film layer and grafting polyethylene glycol is simple, the drug-carrying mode is simple and easy to implement, no special reagent and equipment are needed, and the method has the characteristic of easy popularization and application.
Drawings
Fig. 1 is a schematic diagram of the preparation process of the drug-loaded nanoparticles and the release of the drug from the drug-loaded nanoparticles in response to pH change in example 1.
Fig. 2 is a transmission electron microscope image of the mesoporous silica nanoparticles prepared in example 1.
FIG. 3 shows PAA, MSNP-NH in example 12And infrared spectrograms of MSNP-PAA and MSNP-PAA-PEG.
FIG. 4 is the results of nitrogen adsorption-desorption isotherms and surface pore size tests of MSNP-PAA-PEG and MSNP in example 1.
FIG. 5 shows MSNP and MSNP-NH in example 12Results of dynamic light scattering test of MSNP-PAA and MSNP-PAA-PEG.
FIG. 6 shows MSNP and MSNP-NH in example 12Zeta potential test results of MSNP-PAA and MSNP-PAA-PEG.
FIG. 7 is a transmission electron micrograph of MSNP-PAA-PEG prepared in example 1, wherein (A) (B) (C) are transmission electron micrographs at different magnifications, respectively.
FIG. 8 is the drug release profile of MSNP-PAA-PEG in example 2 at different pH values.
FIG. 9 shows the uptake of MSNP-PAA-PEG-FITC in example 3 by tumor cells in vitro.
FIG. 10 shows the lymphocyte uptake of MSNP-PAA-PEG-FITC in example 3 in vitro.
FIG. 11 is the results of quantitative analysis of the tumor and lymphocyte uptake levels of MSNP-PAA-PEG-FITC in example 3 in vitro.
FIG. 12 is a distribution diagram of MSNP-PAA-PEG-FITC in example 3 in each organ of mice.
FIG. 13 shows the results of cytotoxicity test on tumor cells and lymphocytes in example 4, in which A is a graph showing the results of cell survival test on tumor cells, B is a graph showing the effect on lymphocyte proliferation, and C and D are graphs showing the effect on secretion of IFN-. gamma.and IL-2.
FIG. 14 is the results of the in vivo antitumor effect test in example 5, wherein panel A is the time-course of tumor volume after injection and panel B is the results of the tumor volume test 26 days after injection.
FIG. 15 is the results of the effect on tumor-infiltrating lymphocytes in example 5, in which A is tumor-infiltrating CD8+T cell ratio, B Panel is activated CD8+T ratio, C diagram is memory type CD8+T ratio, D plot of tumor-infiltrated CD4+T cell ratio.
FIG. 16 shows the results of in vivo safety evaluation after treatment in example 5, wherein A is the results of HE staining and B-I is the results of biochemical analysis of blood.
FIG. 17 is the results of the in vivo antitumor effect test in example 6, wherein panel A is the time-course of tumor volume after injection and panel B is the results of the tumor volume test 26 days after injection.
FIG. 18 shows the results of the effect of example 6 on tumor-infiltrating lymphocytes, and graph A shows tumor-infiltrating CD8+T cell ratio, B Panel is activated CD8+T ratio, C diagram is memory type CD8+T ratio, D plot is CD25hiT cell ratio.
Detailed Description
The tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particles and the preparation method thereof provided by the invention are further described by the following embodiments. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make certain insubstantial modifications and adaptations of the present invention based on the above disclosure and still fall within the scope of the present invention.
The pH of the PBS buffer used in each of the following examples was 7.4, and the concentration of the PBS buffer was 10 mmol/L.
Example 1
In the embodiment, a small molecule targeted drug MEK inhibitor AZD6244 is used as a drug to be loaded to prepare the mesoporous silica nano drug-loaded particles with dual targeting of tumor tissues and cells, and the steps are as follows:
(1) preparation of Mesoporous Silica Nanoparticles (MSNP)
Dissolving 100mg of Cetyl Trimethyl Ammonium Bromide (CTAB) in deionized water to form a CTAB aqueous solution with the concentration of 2.1mg/mL, adjusting the pH value of the CTAB aqueous solution to 11.0 by adopting 2mol/L NaOH solution, stirring and heating in a water bath to 80 ℃, after the temperature is stable, dropwise adding tetraethoxysilane into the CTAB aqueous solution at the speed of 180 mu L/min according to the volume ratio of Tetraethoxysilane (TEOS) to the CTAB aqueous solution of 1:96, stirring and reacting at 80 ℃ for 3h to obtain white precipitate, then centrifuging at the speed of 10000rpm for 15min, collecting the precipitate, dispersing the obtained precipitate in 30mL of concentrated hydrochloric acid-absolute ethyl alcohol solution (the volume ratio of concentrated hydrochloric acid to absolute ethyl alcohol is 1:9), refluxing at 80 ℃ for 3h, alternately washing the obtained product for 3 times by using ethanol and deionized water, and drying at 60 ℃ for 12h to obtain the MSNP.
The morphology of MSNP was observed by a TEANAI-10 Transmission Electron Microscope (TEM), and as a result, as shown in fig. 2, fig. 2 (a) and (B) are transmission electron micrographs of MSNP at different magnifications, and as can be seen from fig. 2, the particle size of MSNP prepared in this step was about 100 nm.
(2) Preparation of amino-modified mesoporous silica nanoparticles (MSNP-NH)2)
Dispersing 200mg MSNP in 20mL ethanol to form a dispersion A, adding 100 mu L3-aminopropyl triethoxy silicon (APTES) alkane into the dispersion A, stirring in a water bath at 80 ℃ for reaction for 12h, cooling the obtained reaction solution to room temperature, centrifuging at 10000rpm for 15min, collecting precipitate, alternately washing the obtained precipitate with ethanol and deionized water for 3 times, and drying at 60 ℃ for 12h to obtain MSNP-NH2。
(3) Preparation of amino-modified mesoporous silica drug-loaded nanoparticles (MSNP-NH)2-AZD)
Mixing MSNP-NH2Dispersing in deionized water to form MSNP-NH2The dispersion B with a concentration of 5mg/mL, AZD6244 was dissolved in dimethyl sulfoxide (DMSO) to form a 5mg/mL AZD6244 solutionUniformly mixing 2mL of dispersion liquid B with 200 mu L of AZD6244 solution, reacting at room temperature for 8h, centrifuging at 10000rpm for 15min, collecting precipitate, washing the obtained precipitate with PBS buffer solution for 3 times, and freeze-drying to obtain MSNP-NH2-AZD。
(4) Preparation of polyacrylic acid modified mesoporous silica drug-loaded nanoparticles (MSNP-PAA)
Mixing 100mg of MSNP-NH2-AZD dispersed in deionized water to form MSNP-NH2-dispersion C with AZD concentration of 2.5mg/mL, adding 3mL of an aqueous solution of polyacrylic acid (PAA, molecular weight 1800KD) with concentration of 5mg/mL, stirring for reaction for 2h, centrifuging at 14800rpm for 5min, collecting the precipitate, washing the obtained precipitate with PBS buffer solution 3 times to obtain MSNP-PAA, and redispersing the MSNP-PAA in deionized water to form dispersion D of MSNP-PAA with concentration of 5 mg/mL.
(5) Preparation of mesoporous silica drug-loaded nanoparticles (MSNP-PAA-PEG) for dual targeting of tumor tissues and cells
Taking 200mL of dispersion D, adding 50mg of aminated polyethylene glycol (mPEG-5K-NH) with molecular weight of 5KD into the dispersion D2) I.e. mPEG-5K-NH2Performing ultrasonic treatment for 30min at a mass ratio of 5:100 with MSNP-PAA in the dispersion liquid D, stirring for reaction for 12h, filtering with an ultrafiltration membrane with a molecular weight cutoff of 100KD, collecting nanoparticles obtained by the reaction, and washing with deionized water for 3 times to obtain MSNP-PAA-PEG.
MSNP-NH2Is obtained by grafting a group R on the surface of the MSNP,
o connected with Si in the group R is connected with Si on the surface of the mesoporous silicon nano-particle to form a Si-O bond, and after medicine loading, a medicine molecule is positioned on MSNP-NH2In the pore canal structure, PAA coats MSNP-NH through electrostatic interaction2AZD forms a PAA membrane layer to obtain MSNP-PAA, the PAA in the MSNP-PAA forms MSNP-PAA-PEG through polyethylene glycol connected by amido bonds, the grafted PEG is used for increasing the biocompatibility of the nano drug-loaded particles, and the PAA membrane layer is used as a blocking valve and can respond the change of pH value to enable the MSNP-NH2The pore canal of (2) is in a blocked or an open state. MS prepared in this exampleIn NP-PAA-PEG, the content of PAA was 7.5 wt.%, and the content of polyethylene glycol linked to PAA in the PAA film layer through an amide bond was 2.3 wt.%. The content of AZD6244 in the MSNP-PAA-PEG prepared in this example was determined to be 10 wt.%.
In the process of preparing the MSNP-PAA-PEG, fourier transform infrared analysis (FTIR) and a nitrogen adsorption-desorption isotherm curve are used to monitor each step of reaction, and the results are respectively shown in fig. 3 to 4, and as can be seen from fig. 3, APTES, PAA and PEG are successfully connected with the mesoporous silica nanoparticle MSNP. FIGS. 5 and 6 are MSNP and MSNP-NH, respectively2Dynamic light scattering particle size and Zeta potential test results of MSNP-PAA and MSNP-PAA-PEG further show that APTES, PAA and PEG are successfully coupled to the mesoporous silica nanoparticle MSNP. The diameter of the nanoparticles can be measured and the mesoporous structure of the surface of the nanoparticles can be observed by a transmission electron microscope, a transmission electron microscope photo of the MSNP-PAA-PEG under different magnifications is shown in FIG. 7, and it can be known from FIG. 7 that the particle size of the MSNP-PAA-PEG is 100nm, and the edges of the MSNP-PAA-PEG are rough and have wrinkles.
Comparative example 1
In this comparative example, mesoporous silica nanoparticles loaded with Fluorescein Isothiocyanate (FITC) were prepared by the following steps:
(1) MSNP was prepared following the procedure of step (1) of example 1;
(2) preparation of MSNP-NH according to the procedure of step (2) of example 12；
(3) AZD6244 in step (3) of example 1 was replaced with FITC, and FITC-loaded MSNP-NH was prepared in accordance with the procedure in step (3) of example 12；
(4) MSNP-NH obtained in step (4) of example 12-AZD replacement by FITC-loaded MSNP-NH2Preparing a polyacrylic acid modified mesoporous silica nanoparticle (MSNP-PAA-FITC) loaded with FITC and a dispersion liquid thereof according to the operation of the step (4) in the embodiment 1;
(5) FITC-loaded mesoporous silica nanoparticles, denoted MSNP-PAA-PEG-FITC, were prepared according to the procedure of step (5) of example 1.
Comparative example 2
In this comparative example, unloaded mesoporous silica nanoparticles (MSNP Nano) not loaded with any substance were prepared, and the operation of this comparative example was substantially the same as that of example 1 except that the step (3) of example 1 was removed.
Example 2
In this example, the MSNP-PAA-PEG prepared in example 1 was tested for drug release rate in different pH environments.
MSNP-PAA-PEG prepared in example 1 was placed in PBS buffer (pH 7.4, pH 6.8, pH 5.5, pH 4.5) under different pH conditions, sampled at intervals and centrifuged, and the supernatant was taken to measure the drug release rate by HPLC. The test conditions for HPLC were: the mobile phase, methanol and water (8: 2(v: v), the flow rate is 1mL/min, the detection wavelength is 257.8nm, and the injection volume is 20 muL.
The release curves of MSNP-PAA-PEG under different pH values are shown in FIG. 8, and it can be seen from FIG. 8 that the release rates of AZD6244 are 9.9%, 20.1%, 49.82% and 71.02% respectively under the pH values of 7.4, 6.5, 5.5 and 4.5, and the release rates of the drug are significantly increased with the decrease of the pH values. Because the pH value of the normal pH environment in vivo is about 7.4, the PAA in the PAA film layer of the drug-loaded nanoparticles provided by the invention is not enough to be protonated under the condition, so that the drug can not be released in advance; because the tumor cell hyperproliferation tumor microenvironment has larger difference with the normal tissue microenvironment, such as pH lower than the normal tissue, the permeability of the tumor cell is enhanced, when the drug-loaded nanoparticles are endocytosed by the cells, PAA is protonated by the acidic environment of lysosome and inclusion bodies in the cells, and then the polymer shell on the surface of the drug-loaded nanoparticles is loosened, thereby realizing the drug release.
Example 3
In this example, the FITC-loaded mesoporous silica nanoparticles MSNP-PAA-PEG-FITC prepared in comparative example 1 were tested for their cellular uptake levels in vitro and biodistribution in vivo.
Melanoma cells SMM101 and SMM103, lymphocytes EL4T, Jurkat and mouse spleen lymphocytes 24h, 48h and 72h were treated with MSNP-PAA-PEG-FITC at final concentrations of 50. mu.g/mL and 25. mu.g/mL, respectively, and then the uptake of MSNP-PAA-PEG-FITC in tumor cells and lymphocytes was detected by flow cytometry and confocal microscopy, and the results are shown in FIGS. 9-11, and FIG. 9 is the endocytosis ratio of MSNP-PAA-PEG-FITC in tumor cells detected by flow cytometry. FIG. 10 shows the ratio of the flow cytometry detection of the internalization of MSNP-PAA-PEG-FITC into lymphocytes. FIG. 11 is the results of quantitative analysis of MSNP-PAA-PEG-FITC in vitro tumor and lymphocyte uptake levels. As can be seen from FIGS. 9 to 11, the uptake efficiency of MSNP-PAA-PEG-FITC in tumor cells was significantly higher than that of lymphocytes, almost no uptake was observed in lymphocytes, the uptake efficiency in tumor cells SMM101 and SMM103 was 92.95% and 66.16%, respectively, and the uptake efficiency in lymphocytes EL4T, Jurkat and mouse spleen lymphocytes was 8.44%, 6.54% and 16.61%, respectively. The results show that the mesoporous silica nanoparticle has the specific capacity of being endocytosed by tumor cells but not by lymphocytes.
The method comprises the steps of intravenously injecting MSNP-PAA-PEG-FITC into a tumor-bearing mouse, killing the mouse after injecting for 3h, 6h, 12h, 24h and 48h respectively, taking mouse tumor, heart, liver, spleen, lung and kidney tissues, detecting the distribution of the MSNP-PAA-PEG-FITC in each organ, and obtaining a result as shown in figure 12.
Example 4
In this example, differential toxicity of PLX4032, AZD6244, MSNP-PAA-PEG (MSNP-AZD for short) prepared in example 1, MSNP Nano prepared in comparative example 2, PLX4032+ AZD6244(PLX4032 in combination with AZD 6244), PLX + MSNP-AZD (PLX4032 in combination with MSNP-AZD) on tumor cells and T cells was determined.
(1) Cytotoxicity to tumor cells
The toxicity of the above substances on tumor cells in vitro was detected by Methyltrichlorosilane (MTS) using melanoma cells SMM101, SMM102 and SMM103 as model cells.
Tumor cells were cultured at 2.0X 103cells/well were plated at density in 96-well plates with 50. mu.L of DMEM medium per well. At 37 deg.CAfter overnight incubation in the cell incubator, 50 μ L of PLX4032, AZD6244, MSNP-AZD, MSNP Nano, PLX4032+ AZD6244, PLX + MSNP-AZD at different concentrations (0,0.01,0.1,1 and 10 μ M) were added to each well for 72h treatment, with 5 replicate wells per concentration. After 72h of treatment, 20. mu.L of MTS solution was added to each well, incubated for 1h, and the absorbance (OD) at 490nm was measured on a microplate reader, and the cell viability was calculated according to the following formula:
(2) cytotoxicity to lymphocytes
Detecting the influence of drug-loaded nanoparticles on lymphocyte proliferation by fluorescent dye CFSE, extracting splenic lymphocytes of mice, adjusting cell density to 5 × 106cells/mL, add the final concentration of 2.5 u M fluorescent dye CFSE at room temperature for 8min, then use 5 times volume of RPMI medium to stop staining, washing twice, remove the residual fluorescent dye CFSE. Labeling the cells at 2.0X 106Inoculating cells/wells into a 24-well plate pre-coated with a CD3 antibody, adding PLX4032, AZD6244, MSNP-AZD, PLX4032+ AZD6244 and PLX + MSNP-AZD with different concentrations (0,0.01,0.1,1 and 10 mu M) for treating for 72h, collecting cells after 72h, and detecting the proliferation condition of lymphocytes by adopting a flow cytometer; supernatants were collected and tested for secretion levels of IFN-. gamma.and IL-2 by lymphocytes using ELISA.
FIG. 13 shows the results of cytotoxicity test on tumor cells and lymphocytes in example 4, in which A is a graph showing the results of cell survival test on tumor cells, B is a graph showing the effect on lymphocyte proliferation, and C and D are graphs showing the effect on secretion of IFN-. gamma.and IL-2.
As can be seen from the A diagram of FIG. 13, MSNP-nano shows very weak cell growth inhibition in three mouse melanoma cell lines, indicating that the drug-free nanoparticles of the present invention are non-toxic to cells, and when MSNP-AZD alone and MSNP-AZD in combination with PLX4032, the drug-loaded nanoparticles show stronger tumor cytotoxicity as free drug.
As can be seen from diagrams B to D of FIG. 13: PLX4032 at low doses (1. mu.M) promotes proliferation of lymphocytes and secretion of IFN-. gamma.and IL-2, while PLX4032 at high concentrations inhibits proliferation of lymphocytes and secretion of IFN-. gamma.and IL-2; the free AZD6244 inhibits the proliferation of lymphocytes and the secretion of IFN-gamma and IL-2 in a dose-dependent manner, and the MSNP entraps the AZD6244 to obviously reduce the toxicity of the AZD6244 to the lymphocytes. The results show that the mesoporous silica nano drug-loaded particles have the capability of avoiding lymphocyte toxicity caused by targeted drugs in vitro.
Example 5
In this example, saine, PLX4032, AZD6244, PLX4032+ AZD6244(PLX4032 in combination with AZD 6244), MSNP-PAA-PEG (MSNP-AZD for short) prepared in example 1, and PLX4032+ MSNP-AZD (PLX4032 in combination with MSNP-AZD) were tested for in vivo anti-tumor effects and immunomodulatory effects.
The SMM103 melanoma cells of the mice are inoculated to the subcutaneous back of C57BL/6 to establish a melanoma transplantation tumor model of the mice, and the anti-tumor effect and the immunoregulation effect of the MSNP-AZD are evaluated in vivo. When the tumor volume grows to 80-100 mm3In this case, Saline, PLX4032, AZD6244, PLX4032+ AZD6244, MSNP-AZD and PLX4032+ MSNP-AZD were administered intravenously at doses of 5mg/kg PLX4032, 5mg/kg AZD6244 and 5mg/kg MSNP-AZD, respectively. Tumor volumes were measured every three days during dosing and mouse tumor growth curves were plotted. After 7 times of treatment, tumor tissues of mice were taken to evaluate the antitumor effect and the effect on tumor-infiltrating lymphocytes (TILs) of the above substances, and heart, liver, spleen, lung, kidney and blood of the mice were taken to evaluate the safety of the above substances. FIG. 14 is the results of the in vivo antitumor effect test in example 5, wherein panel A is the time-course of tumor volume after injection and panel B is the results of the tumor volume test 26 days after injection. As can be seen in fig. 14, all the single-drug and dual-drug combination treatment groups significantly inhibited the growth of tumors in mice. The tumor growth inhibition effect of the PLX4032 and the AZD6244 combined with the AZD6244 is better than that of the PLX4032 and the AZD6244 alone, and the tumor growth inhibition effect of the MSNP-AZD single treatment group and that of the combined PLX4032 treatment group are better than that of the free AZD6244 treatment group.
Swelling the miceThe tumor tissue is digested into single cell suspension, and the influence of each treatment group on tumor-infiltrated lymphocytes is detected by a flow cytometer. Results as shown in FIG. 15, FIG. 15 shows the effect on tumor-infiltrating lymphocytes in example 5, in which A is the tumor-infiltrating CD8+ T cell ratio and B is activated CD8+T ratio, C diagram is memory type CD8+T ratio, D plot of tumor-infiltrated CD4+T cell ratio. FIG. 16 shows the results of in vivo safety evaluation after treatment in example 5, wherein A is the results of HE staining and B-I is the results of biochemical analysis of blood. As can be seen from FIG. 15, free AZD6244 inhibited tumor-infiltrating cytotoxic lymphocytes (CTL, CD 3)+CD8+) In which activated CTLs (CD 8)+CD44+CD69+) And memory CTLs (CD 8)+CD44+CD62L-) The vaccine is more sensitive to AZD6244, and MSNP-AZD has no influence on tumor-infiltrated lymphocytes and has an immunoprotection effect. The results show that the mesoporous silica nano drug-loaded particles have the capability of avoiding lymphocyte toxicity caused by targeted drugs in vivo.
After treatment, the heart, liver, spleen, lung and kidney of the mouse are taken for HE staining, the blood of the mouse is taken for hematopoiesis analysis, and the in vivo safety of the substances is detected, and the result is shown in figure 16.
Example 6
In this example, Saline, anti-PD-1, PLX4032+ AZD6244 (combination of PLX4032 and AZD 6244), PLX4032+ MSNP-AZD (combination of PLX4032 and MSNP-AZD), PLX4032+ AZD6244+ anti-PD-1 (combination of PLX4032, AZD6244 and anti-PD-1), and PLX4032+ MSNP-AZD + anti-PD-1 (combination of PLX4032, MSNP-AZD and anti-PD-1) were tested for in vivo synergistic anti-tumor effects.
The SMM103 melanoma cells of the mice are inoculated to the subcutaneous back of C57BL/6 to establish a melanoma transplantation tumor model of the mice, and the synergic anti-tumor effect and the immunoregulation effect of the MSNP-AZD and the PD-1 antibody are evaluated in vivo. When the tumor volume grows to 80-100 mm3At the same time, the veins are separatedSaline, anti-PD-1, PLX4032+ AZD6244, PLX4032+ MSNP-AZD, PLX4032+ AZD6244+ anti-PD-1, PLX4032+ MSNP-AZD + anti-PD-1 were injected at doses of 5mg/kg PLX4032, 5mg/kg AZD6244, 5mg/kg MSNP-AZD and 2.5mg/kg anti-PD-1. Tumor volume was measured every three days and mouse tumor growth curves were plotted. After 7 times of treatment, the mice are sacrificed, and tumor tissues of the mice are taken and weighed to evaluate the anti-tumor effect of the medicament; the tumor tissue is digested into single cell suspension by collagenase, and the influence of the drug on the tumor-infiltrated lymphocytes is detected by a flow cytometer.
The results are shown in fig. 17-18, and it can be seen from fig. 17 that both the MAPK pathway targeted therapy and the anti-PD-1 immunotherapy can significantly inhibit tumor growth in mice; the anti-tumor effect of the combination of MSNP-AZD and PLX3032 is slightly better than that of the combination of free AZD6244 and PLX 3032; the combination of PLX4032+ AZD and anti-PD-1 has a synergistic anti-tumor effect, and the synergistic effect is more obvious after the AZD6244 is loaded into MSNP nanoparticles.
As can be seen in FIG. 18, tumor CD3 was found in the PLX4032+ AZD6244+ anti-PD-1 treated group+The lymphocyte proportion was significantly lower than that of anti-PD-1 monotherapy group, indicating that PLX4032 in combination with AZD6244 inhibited tumor-infiltrating lymphocytes, whereas AZD6244 could avoid injury to lymphocytes by MSNP encapsulation of AZD 6244. In addition, cytotoxic lymphocytes (CTL, CD 3) in tumor infiltration+CD8+) Activated CTLs (CD 8)+CD44+CD69+) And memory CTLs (CD 8)+CD44+CD62L-) The same lymphocyte protection effect is also shown.
In conclusion, the mesoporous silica nano drug-loaded particles provided by the invention can avoid damage of small molecule targeted drugs to lymphocytes, and have the capability of resisting tumors and stimulating an immune system in cooperation with tumor immune drugs.
Example 7
In the embodiment, a micromolecular targeted drug mTOR inhibitor AZD8055 is used as a drug to be loaded to prepare the mesoporous silica nano drug-loaded particles with dual targets of tumor tissues and cells, and the steps are as follows:
(1) preparation of Mesoporous Silica Nanoparticles (MSNP)
Cetyl Trimethyl Ammonium Bromide (CTAB) is dissolved in deionized water to form a CTAB aqueous solution with the concentration of 1.8mg/mL, the pH value of the CTAB aqueous solution is adjusted to 11 by adopting 2mol/L NaOH solution, the CTAB aqueous solution is heated to 78 ℃ in a water bath under stirring, after the temperature is stable, tetraethoxysilane is dripped into the CTAB aqueous solution at the speed of 180 mu L/min according to the volume ratio of Tetraethoxysilane (TEOS) to the CTAB aqueous solution of 1:90, a white precipitate is obtained after stirring and reacting for 3h at 78 ℃, then the white precipitate is centrifuged for 15min at the rotating speed of 10000rpm, the precipitate is collected and dispersed in 30mL concentrated hydrochloric acid-absolute ethyl alcohol solution (the volume ratio of concentrated hydrochloric acid to absolute ethyl alcohol is 2:8), the precipitate is refluxed for 3h at 78 ℃, the obtained product is alternately washed for 3 times by using ethanol and deionized water, and then dried for 12h at 60 ℃ to obtain MSNP.
(2) Preparation of amino-modified mesoporous silica nanoparticles (MSNP-NH)2)
Dispersing MSNP in isopropanol to form a dispersion liquid A with the concentration of 8mg/mL, adding 3-aminopropyl triethoxy silicon (APTES) alkane into the dispersion liquid A, stirring and reacting in a water bath at 78 ℃ for 16h with the volume ratio of APTES to the dispersion liquid A being 1:180, cooling the obtained reaction liquid to room temperature, centrifuging at 10000rpm for 15min, collecting precipitate, alternately washing the obtained precipitate with ethanol and deionized water for 3 times, and drying at 60 ℃ for 12h to obtain MSNP-NH2。
(3) Preparation of amino-modified mesoporous silica drug-loaded nanoparticles (MSNP-NH)2-AZD8055)
Mixing MSNP-NH2Dispersing in deionized water to form MSNP-NH2Dissolving AZD8055 in dimethyl sulfoxide (DMSO) to obtain AZD8055 solution with concentration of 5mg/mL, mixing the dispersion B with AZD8055 solution, and controlling MSNP-NH in AZD8055 and dispersion B2The mass ratio of the components is 1:1, the reaction is carried out for 10h at room temperature, then the centrifugation is carried out for 15min at the rotating speed of 10000rpm, the precipitate is collected, the obtained precipitate is washed for 3 times by PBS buffer solution, and the MSNP-NH is obtained after freeze drying2-AZD8055。
(4) Preparation of polyacrylic acid modified mesoporous silica drug-loaded nanoparticles (MSNP-PAA)
Mixing 100mg of MSNP-NH2-AZD8055 dispersed in deionized water to form MSNP-NH2Adding 5mL of polyacrylic acid (PAA with the molecular weight of 2000KD) aqueous solution with the concentration of 5mg/mL into the dispersion C with the concentration of AZD8055 for stirring reaction for 4 hours, centrifuging for 5min at the rotating speed of 14800rpm, collecting precipitates, washing the obtained precipitates for 3 times by using PBS buffer solution to obtain MSNP-PAA, and redispersing the MSNP-PAA in deionized water to form a dispersion D with the concentration of the MSNP-PAA of 4 mg/mL.
(5) Preparation of mesoporous silica drug-loaded nanoparticles (MSNP-PAA-PEG) for dual targeting of tumor tissues and cells
To dispersion D was added aminated polyethylene glycol having a molecular weight of 6K (mPEG-6K-NH)2) Reacting mPEG-6K-NH2Performing ultrasonic treatment for 20min at the mass ratio of the nano particles to the MSNP-PAA in the dispersion liquid D of 3:100, stirring for reaction for 16h, filtering by adopting an ultrafiltration membrane with the molecular weight cutoff of 100KD, collecting nano particles obtained by the reaction, and washing for 3 times by using deionized water to obtain the MSNP-PAA-PEG.
MSNP-NH2Is obtained by grafting a group R on the surface of the MSNP,
o connected with Si in the group R is connected with Si on the surface of the mesoporous silicon nano-particle to form a Si-O bond, and after medicine loading, a medicine molecule is positioned on MSNP-NH2In the pore canal structure, PAA coats MSNP-NH through electrostatic interaction2AZD8055 forms a PAA membrane layer to obtain MSNP-PAA, the PAA in the MSNP-PAA forms MSNP-PAA-PEG through polyethylene glycol connected by amido bonds, the grafted PEG is used for increasing the biocompatibility of the nano drug-carrying particles, the PAA membrane layer is used as a blocking valve and can respond the change of pH value to enable the MSNP-NH2The pore canal of (2) is in a blocked or an open state.
Example 8
In the embodiment, a small molecule targeted drug PI3K/mTOR inhibitor BEZ235 is used as a drug to be loaded to prepare the mesoporous silica nano drug-loaded particles with dual targeting of tumor tissues and cells, and the steps are as follows:
(1) preparation of Mesoporous Silica Nanoparticles (MSNP)
Cetyl Trimethyl Ammonium Bromide (CTAB) is dissolved in deionized water to form a CTAB aqueous solution with the concentration of 2.5mg/mL, the pH value of the CTAB aqueous solution is adjusted to 11.5 by adopting 2mol/L NaOH solution, the CTAB aqueous solution is heated to 82 ℃ in a water bath under stirring, after the temperature is stable, ethyl orthosilicate is dripped into the CTAB aqueous solution according to the volume ratio of 1:150 of The Ethyl Orthosilicate (TEOS) to the CTAB aqueous solution at the speed of 180 mu L/min, white precipitate is obtained after stirring and reaction for 2h at 82 ℃, then the white precipitate is centrifuged for 15min at the rotating speed of 10000rpm, the precipitate is collected and dispersed in 30mL concentrated hydrochloric acid-absolute ethyl alcohol solution (the volume ratio of the concentrated hydrochloric acid to the absolute ethyl alcohol is 1:9), the precipitate is refluxed for 2h at 82 ℃, the obtained product is alternately washed for 3 times by using ethanol and deionized water, and then dried for 12h at 60 ℃ to obtain the MSNP.
(2) Preparation of amino-modified mesoporous silica nanoparticles (MSNP-NH)2)
Dispersing MSNP in ethanol to form a dispersion liquid A with the concentration of 15mg/mL, adding 3-aminopropyltriethoxy silicon (APTES) alkane into the dispersion liquid A, stirring and reacting in a water bath at 78 ℃ for 16h with the volume ratio of APTES to the dispersion liquid A being 1: 150-200, cooling the obtained reaction liquid to room temperature, centrifuging for 15min at the rotating speed of 10000rpm, collecting precipitate, alternately washing the obtained precipitate for 3 times by using ethanol and deionized water, and drying at 60 ℃ for 12h to obtain MSNP-NH2。
(3) Preparation of amino-modified mesoporous silica drug-loaded nanoparticles (MSNP-NH)2-BEZ235)
Mixing MSNP-NH2Dispersing in PBS buffer to form MSNP-NH2Dissolving BEZ235 in methanol to form a BEZ235 solution with the concentration of 5mg/mL, uniformly mixing the BEZ235 solution with the BEZ235 solution, and controlling MSNP-NH in the BEZ235 solution and the dispersion B2The mass ratio of the components is 1:2, the reaction is carried out for 9h at room temperature, then the centrifugation is carried out for 15min at the rotating speed of 10000rpm, the precipitate is collected, the obtained precipitate is washed for 3 times by PBS buffer solution, and the MSNP-NH is obtained after freeze drying2-BEZ235。
(4) Preparation of polyacrylic acid modified mesoporous silica drug-loaded nanoparticles (MSNP-PAA)
Mixing 100mg of MSNP-NH2-BEZ235 is dispersed in PBS buffer to form MSNP-NH2-BEZAdding 4mL of polyacrylic acid (PAA, molecular weight 1500KD) aqueous solution with the concentration of 5mg/mL into the dispersion C with the concentration of 235 mg/mL, stirring and reacting for 3 hours, centrifuging for 5min at the rotating speed of 14800rpm, collecting precipitates, washing the obtained precipitates for 3 times by using PBS buffer solution to obtain MSNP-PAA, and re-dispersing the MSNP-PAA in deionized water to form dispersion D with the concentration of the MSNP-PAA of 3 mg/mL.
(5) Preparation of mesoporous silica drug-loaded nanoparticles (MSNP-PAA-PEG) for dual targeting of tumor tissues and cells
To dispersion D was added aminated polyethylene glycol having a molecular weight of 4K (mPEG-4K-NH)2) Reacting mPEG-4K-NH2Performing ultrasonic treatment for 40min at a mass ratio of 2:100 with MSNP-PAA in the dispersion liquid D, stirring for reaction for 14h, filtering with an ultrafiltration membrane with a molecular weight cutoff of 100KD, collecting nanoparticles obtained by the reaction, and washing with deionized water for 3 times to obtain MSNP-PAA-PEG.
MSNP-NH2Is obtained by grafting a group R on the surface of the MSNP,
o connected with Si in the group R is connected with Si on the surface of the mesoporous silicon nano-particle to form a Si-O bond, and after medicine loading, a medicine molecule is positioned on MSNP-NH2In the pore canal structure, PAA coats MSNP-NH through electrostatic interaction2-BEZ235 forms a PAA membrane layer to obtain MSNP-PAA, the PAA in the MSNP-PAA forms MSNP-PAA-PEG through polyethylene glycol connected by amido bonds, the grafted PEG is used for increasing the biocompatibility of the nano drug-loaded particles, the PAA membrane layer is used as a blocking valve and can respond the change of pH value to enable the MSNP-NH2The pore canal of (2) is in a blocked or an open state.
Claims (5)
1. The mesoporous silica nano drug-carrying particle with double targeting of tumor tissues and cells is characterized in that, the drug-loaded nanoparticles consist of mesoporous silica nanoparticles with surfaces modified by polyacrylic acid and polyethylene glycol and drugs, the drug is a targeted drug with lymphocyte toxicity, the mesoporous silica nanoparticles with the surfaces modified by polyacrylic acid and polyethylene glycol are prepared from mesoporous silica nanoparticles with the surfaces modified by amino, coating the polyacrylic acid film layer of the mesoporous silicon dioxide nano-particles with the surface modified by amino through electrostatic action, and polyethylene glycol connected with polyacrylic acid in the polyacrylic acid film layer through amido bond, the polyacrylic acid film layer is used as a plugging valve, which can respond to the change of pH value to make the pore channel of the mesoporous silicon dioxide nano-particle with the surface modified by amino group in a plugging or opening state, the drug is positioned in a pore channel structure of the mesoporous silica nanoparticle with the surface modified by the amino; the nano drug-loaded particles have the capability of avoiding drug-induced lymphocyte toxicity;
the particle size of the nano drug-loaded particles is 100-200 nm; in the drug-loaded nanoparticle, the content of polyacrylic acid is 7.2-7.5 wt.%, the content of polyethylene glycol connected with polyacrylic acid in a polyacrylic acid film layer through an amide bond is 1.8-2.8 wt.%, and the content of amino modifying groups on the surface of the mesoporous silica nanoparticle is such that the Zeta potential of the mesoporous silica nanoparticle with the surface modified by amino is 10-30 mV.
2. The tumor tissue and cell dual-targeting mesoporous silica nano drug-carrying particle according to claim 1, wherein the mesoporous silica nano particle with the surface modified by amino is obtained by grafting a group R on the surface of the mesoporous silica nano particle,
o connected with Si in the group R is connected with Si on the surface of the mesoporous silicon nano-particles to form Si-O bonds.
3. The tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle according to claim 1, wherein the content of the drug in the silicon nano drug-loaded particle is 5-15 wt.%.
4. The mesoporous silica nano drug-loaded particle for tumor tissue and cell dual targeting according to claim 1, wherein the nano drug-loaded particle has the ability of passively targeting tumor tissue, has the ability of specifically targeting tumor cells and avoiding the endocytosis of the tumor cells by lymphocytes, has the ability of avoiding the toxicity of the lymphocytes caused by small molecule drugs in vitro and in vivo, and has the ability of cooperating with tumor immune drugs in vivo to resist tumor and stimulate immune system.
5. The preparation method of the tumor tissue and cell dual-targeting mesoporous silica nano drug-loaded particle as claimed in any one of claims 1 to 4, is characterized by comprising the following steps:
(1) preparation of mesoporous silica nanoparticles
Adjusting the pH value of a hexadecyl trimethyl ammonium bromide aqueous solution to 11.0-11.5, heating to 78-82 ℃ under stirring, dropwise adding ethyl orthosilicate, reacting at 78-82 ℃ for 2-3 h, dispersing a reaction product in a concentrated hydrochloric acid-absolute ethanol solution, refluxing at 78-82 ℃ for 3-5 h, washing the obtained product with ethanol and water, and drying to obtain mesoporous silica nanoparticles;
(2) preparation of amino-modified mesoporous silica nanoparticles
Dispersing mesoporous silica nanoparticles in isopropanol or ethanol to form a dispersion liquid A, mixing the dispersion liquid A with 3-aminopropyltriethoxysilane, stirring and reacting at 78-82 ℃ for 12-16 h, washing a reaction product with ethanol and water, and drying to obtain amino-modified mesoporous silica nanoparticles;
(3) preparation of amino-modified mesoporous silica nano drug-loaded particles
Dispersing the amino-modified mesoporous silica nanoparticles in PBS buffer solution or water to form dispersion liquid B, mixing the dispersion liquid B with a solution of a medicament, reacting for 8-10 h, washing an obtained reaction product with the PBS buffer solution, and freeze-drying to obtain amino-modified mesoporous silica nanoparticle medicament-carrying particles;
(4) preparation of polyacrylic acid modified mesoporous silica nano drug-loaded particles
Dispersing the amino-modified mesoporous silica nano drug-loaded particles in PBS buffer solution or water to form dispersion solution C, adding polyacrylic acid aqueous solution, stirring and reacting for 2-4 h, washing a reaction product with the PBS buffer solution to obtain polyacrylic acid-modified mesoporous silica nano drug-loaded particles, and dispersing the polyacrylic acid-modified mesoporous silica nano drug-loaded particles in the PBS buffer solution or water to form dispersion solution D;
(5) preparation of mesoporous silica nano drug-loaded particles for dual targeting of tumor tissues and cells
Adding aminated polyethylene glycol into the dispersion liquid D, carrying out ultrasonic mixing, then stirring and reacting for 12-16 h, filtering by adopting an ultrafiltration membrane, collecting nanoparticles obtained by the reaction, and washing by using deionized water to obtain the mesoporous silica nano drug-loaded particles with dual targets of tumor tissues and tumor cells.
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