CN111249467A - Tumor self-targeting multi-stage response type mesoporous silicon drug delivery system and preparation method thereof - Google Patents
Tumor self-targeting multi-stage response type mesoporous silicon drug delivery system and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a tumor self-targeting multistage response type mesoporous silicon drug delivery system, which comprises mesoporous silicon nanoparticles, wherein antitumor drugs are loaded on the mesoporous silicon nanoparticles, and a polymer layer and a low-toxicity tumor targeting single-chain antibody are modified on the surfaces of the mesoporous silicon nanoparticles, so that the drug delivery system has breast cancer cell targeting property, temperature responsiveness and pH responsiveness. The invention also provides a corresponding preparation method. The tumor self-targeting multistage response type mesoporous silicon drug delivery system provided by the invention has good biocompatibility and drug controlled release performance of specific targeting and temperature and pH dual response of HER2 high-expression breast cancer cells, and can realize safe and controllable drug release while improving tumor specific recognition.
Description
Technical Field
The invention relates to the field of tumor targeted drug research and development and drug controlled release, in particular to a tumor self-targeted multistage response type mesoporous silicon drug delivery system and a preparation method thereof.
Background
In recent years, malignancy remains a significant threat that afflicts modern mankind, and chemotherapy remains a powerful means of combating malignancy. In chemotherapy applications, nanoparticles have been widely developed as drug carriers for achieving more efficient drug delivery due to their excellent tissue penetration and drug loading capabilities. The mesoporous silicon nanoparticles have the advantages of high loading efficiency, good biocompatibility, surface modification and the like, so that an ideal matrix is provided for the design of the functionalized drug carrier.
However, pure nanoparticles are often difficult to respond to changes in physical, chemical and physiological environments in vivo to achieve precise drug release. Therefore, by performing reasonable surface chemical modification and biological modification on the nanoparticles, drug release can be intelligently regulated according to known conditions. Based on this, a variety of "goalkeeper" molecules have been developed to modify nanoparticle surfaces to act as "gating" molecules in response to stimulus conditions such as pH, temperature, light, etc.
In practical applications, the drug carrier with single stimulation response often has premature release of the drug, resulting in great loss of the anti-tumor therapeutic effect, even damage to normal tissues due to drug leakage. Therefore, a simple and versatile response stimulation mode should be introduced to further achieve precise drug release.
Since the excessive metabolism of tumor cells often causes glycolysis to be up-regulated, the local tumor lesion tissues are caused to present higher temperature and a slightly acidic medium environment, and a potential trigger switch is provided for the controllable drug release. Based on the idea of multi-stimulation mode, the drug carrier with double responses of pH value and temperature can better fit with the tumor microenvironment, thereby realizing intelligent drug release.
In the selection of "gated" molecules, N-isopropylacrylamide is a temperature-sensitive polymer that tends to stretch above the critical temperature (32 ℃) while maintaining a tight conformation and being difficult to dissolve at the critical temperature. Furthermore, methacrylic acid (MAA) is another pH sensitive compound. The conformation is tight when the MAA is in neutral and basic environments, but at lower pH values it will protonate and relax. The combination of the two compounds can be used as a dual 'switch' for controlling the release of the drug, and has a certain application prospect in the application of tumor treatment.
Breast cancer, one of the malignant cancers, remains a considerable health problem. Among them, human epidermal growth factor receptor 2(HER2) is a typical oncogene, and its overexpression is considered to be a major cause of disease progression. Among breast cancer patients, HER2 positive breast cancer patients account for approximately 20% to 30% of the patients as a whole, and are often associated with a risk of disease metastasis. In recent years, gene targeting strategies have been increasingly applied to address this severe condition to enhance general chemotherapy. After the specific antibody and the nano-carrier are subjected to conjugation modification, the nano-carrier can be efficiently transported to a tumor part and the drugs in the tumor part can be released by virtue of antigen-antibody specific recognition so as to enhance the anti-cancer treatment effect, and meanwhile, the nano-carrier is prevented from off-targeting in normal tissues so as to reduce non-specific tissue damage. In the antibody-mediated tumor targeting strategy, the humanized single-chain antibody has the characteristics of low cost, easy production, easy chemical modification, low immunogenicity, high biological activity and the like, can further keep stronger receptor recognition capability in practical application, simultaneously reduces the side effect on a normal organism, and achieves the tumor targeting effect of 'reduction and synergism'. Therefore, the gene targeting strategy modified by the HER2 single-chain antibody has certain potential and application prospect for accurately treating HER2 positive breast cancer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a preparation method of a tumor self-targeting multistage response type mesoporous silicon drug delivery system, has the characteristics of high biocompatibility, specific targeting and multiple responsiveness, improves the accuracy and controllability of delivering chemotherapy drugs by a nano carrier, improves the biological safety of the carrier, and overcomes the limitation of traditional single-response drug release.
In order to achieve the purpose, the invention provides a tumor self-targeting multistage response type mesoporous silicon drug delivery system, which comprises the following specific steps:
the drug delivery system comprises mesoporous silicon nanoparticles, the mesoporous silicon nanoparticles are loaded with anti-tumor drugs, and a polymer layer and a low-toxicity tumor-targeting single-chain antibody are modified on the surfaces of the mesoporous silicon nanoparticles, so that the drug delivery system has breast cancer cell targeting property, temperature responsiveness and pH responsiveness.
Preferably, the antitumor drug is a hydrophobic antitumor drug.
Preferably, the polymer in the polymer layer is a copolymer of N-isopropylacrylamide and methacrylic acid. The NIPAm/MAA polymer is a temperature and pH double response layer of the mesoporous silicon intelligent drug delivery system.
Preferably, the low toxicity tumor targeting single chain antibody is an anti-HER 2 single chain antibody. The surface of the mesoporous silicon intelligent drug delivery system provided by the invention is grafted with the anti-HER 2 single-chain antibody, has good biocompatibility, and can be specifically combined with the receptor protein HER2 highly expressed on the surface of HER2 positive breast cancer cells, thereby realizing the targeted targeting of tumors.
The invention also provides a preparation method of the tumor self-targeting multistage response type mesoporous silicon drug delivery system, which comprises the following steps:
(1) synthesizing monodisperse mesoporous silicon nanoparticle MSN;
(2) preparing a composite particle DOX @ MSN-MPS modified by methacryloxypropyltrimethoxysilane;
(3) preparing polymer layer modified composite particles DOX @ MSN-pNIPAm/MAA;
(4) preparing the tumor-targeted single-chain antibody modified composite particle DOX @ MSN-pNIPAm/MAA-HER 2.
Preferably, the step (1) is specifically:
the synthesis is carried out by a sol-gel method, and the monodisperse MSN is obtained by repeated washing after centrifugal collection.
Preferably, the step (2) is specifically:
dispersing MSN in ethanol, adding Methacryloxypropyltrimethoxysilane (MPS), reacting, centrifuging and collecting the MPS modified MSN;
dispersing MSN-MPS in ethanol, adding a hydrochloric acid solution, and reacting to obtain silicon-based MSN-MPS with the template removed;
dispersing the silicon-based MSN-MPS with the template removed and the anti-tumor drug in water, and collecting the composite particles DOX @ MSN-MPS which load the anti-tumor drug and remove the template after reaction.
Preferably, the step (3) is specifically:
dissolving the copolymer of N-isopropylacrylamide NIPAm and methacrylic acid MAA in water, adding DOX @ MSN-MPS and potassium persulfate, and reacting under the protection of nitrogen to obtain the polymer layer modified composite particle DOX @ MSN-pNIPAm/MAA.
Preferably, the step (4) is specifically:
adding N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into a HER2 single-chain antibody solution to activate carboxyl on a HER2 single-chain antibody for 1 hour, and ultrasonically dispersing DOX @ MSN-pNIPAm/MAA into a mixed solution to react to obtain the tumor targeting single-chain antibody modified composite particle DOX @ MSN-pNIPAm/MAA-HER 2.
The invention has the beneficial effects that:
1. the invention relies on the acidic high-temperature environment in the tumor cells to realize dual drug controlled release, greatly reduces the advanced leakage of the drug, reduces the damage to normal cells, realizes the stable and controllable drug release and improves the treatment effect;
2. according to the invention, the nanoparticle targets the tumor HER2 receptor at a fixed point, so that the endocytosis of the breast cancer cells to the nano-carrier is improved, and the drug delivery and anti-tumor treatment effects are enhanced;
3. the invention is constructed by a strategy of layer-by-layer assembly, and can be expanded to be used for other nano material drug carrying systems.
Drawings
Fig. 1A is a picture of a preparation process of the doxorubicin-loaded composite mesoporous silicon nanoparticles.
Fig. 1B is a diagram of the effect of the adriamycin-loaded composite mesoporous silicon nanoparticles on a human body.
Fig. 2A and 2B are Transmission Electron Microscope (TEM) images of the mesoporous silicon nanoparticles before and after modification, respectively.
Fig. 3A and 3B are respectively the cell compatibility and blood compatibility of the mesoporous silicon nanoparticles.
Fig. 4 is a cumulative drug release curve of the adriamycin-loaded composite mesoporous silicon nanoparticles under different temperature and pH conditions.
Fig. 5A and 5B are pictures of the uptake capacity of tumor cells and normal cells to the composite mesoporous silicon nanoparticles, respectively.
Fig. 6A and 6B are images of the killing ability of the doxorubicin-loaded composite mesoporous silicon nanoparticles to tumor cells and normal cells, respectively.
Fig. 7A and 7B are pictures of killing ability of the doxorubicin-loaded composite mesoporous silicon nanoparticles to tumor cells in mice, respectively.
Detailed Description
In order to more clearly describe the technical contents of the present invention, the following further description is given in conjunction with specific embodiments.
Example 1: preparation and characterization of temperature and pH dual-response and tumor-targeting mesoporous silicon drug delivery system
As shown in fig. 1A, the invention provides a high-biocompatibility tumor-targeted intelligent multi-response mesoporous silicon drug delivery system and a preparation method thereof, comprising the following steps: synthesizing and preparing mesoporous silicon nanoparticles, loading an antitumor drug after silicon-based modification, then covalently modifying a copolymer of N-isopropylacrylamide (NIPAm) and methacrylic acid (MAA), and grafting an anti-HER 2 single-chain antibody, thereby obtaining the nanoparticles with tumor targeting property and temperature and pH dual responsiveness.
Wherein, the surface of the synthesized mesoporous silicon nano-particles is modified with a silane coupling agent MPS, and the pore channels of the mesoporous silicon nano-particles are loaded with anti-tumor drugs; modifying a temperature and pH dual-response layer through high-molecular precipitation polymerization; and grafting anti-HER 2 single-chain antibody on the surface of the modified nanoparticle through condensation reaction to prepare the composite nanoparticle.
As shown in fig. 1B, the highly biocompatible tumor-targeted intelligent multi-response mesoporous silicon drug delivery system provided by the invention has good biocompatibility, specific targeting property of HER2 high-expression breast cancer cells, and drug controlled release property of temperature and pH dual response, and can realize safe and controllable drug release while improving tumor specific recognition.
The invention provides a preparation method of a high-biocompatibility tumor-targeted intelligent multi-response mesoporous silicon drug delivery system, which comprises the following synthesis steps:
0.75g of cetyltrimethylammonium bromide (CTAB) is weighed into 360mL of deionized water, followed by the dropwise addition of 2.625mL of a 2mol/L sodium hydroxide solution; heating the mixed solution to 80 ℃, reacting for 2 hours, centrifuging for 15 minutes at 12100rpm, and collecting; discarding the supernatant of the collected solution, respectively washing the precipitate for 3 times by using deionized water and ethanol, and drying the obtained product in vacuum at 40 ℃ to obtain monodisperse Mesoporous Silicon Nanoparticles (MSN);
weighing 1.5g of MSN, and ultrasonically dispersing in 360mL of ethanol; subsequently, 0.6mL of MPS was added to the dispersion, reacted at 80 ℃ for 12 hours, centrifuged at 12100rpm for 15 minutes to collect the product, washed with deionized water and ethanol each for 3 times to precipitate, and the resulting product was vacuum-dried at 40 ℃ to obtain a silica-based MSN (MSN-MPS-CTAB);
dispersing 1.5g of MSN-MPS in 360mL of ethanol, adding 40mL of 2mol/L hydrochloric acid solution, reacting for 24 hours at 80 ℃ to remove a template CTAB, centrifugally collecting, repeatedly washing and precipitating by using deionized water and ethanol, and drying the obtained product in vacuum at 40 ℃ to obtain a template-removed silicon-based MSN (MSN-MPS); finally, ultrasonically dispersing the MSN-MPS and the adriamycin in deionized water, reacting for 24 hours in a dark place, centrifugally collecting the adriamycin-loaded nanoparticles (DOX @ MSN-MPS), measuring supernatant by using an ultraviolet visible light spectrophotometer, and determining the adsorption capacity of the adriamycin;
weighing 250mg of DOX @ MSN-MPS, 625mg of NIPAm, 11.5mg of Sodium Dodecyl Sulfate (SDS) and 30mg of N, N-Methylene Bisacrylamide (MBA), weighing 38 mu L of MAA, and dissolving in 200mL of deionized water; subsequently, adding 45mg of potassium persulfate, reacting for 4 hours at 70 ℃ under the protection of nitrogen, centrifugally collecting a product, repeatedly washing the product by using deionized water and ethanol, and drying the product in vacuum at 40 ℃ to obtain a composite nanoparticle DOX @ MSN-pNIPAm/MAA;
selecting pichia pastoris containing anti-HER 2 single-chain antibody transformant, inoculating the pichia pastoris into BMGY culture medium, inducing protein expression by using methanol, measuring the protein content and collecting the protein for later use. 80mg of MSN-pNIPAm/MAA, 182.5mg of N-hydroxysuccinimide (NHS) and 125mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were weighed and added to 100mL of deionized water. Then adding 100mL of HER2 single-chain antibody solution of 91 mu g/mL, reacting at room temperature for 24 hours, centrifuging, collecting, repeatedly washing with deionized water and ethanol, and drying in vacuum at 40 ℃ to obtain the antibody nanoparticles (DOX @ MSN-pNIPAm/MAA-HER 2).
The transmission electron microscope examination result shows that the monodisperse mesoporous silicon nanoparticles have uniform size and clear and visible pore channels (figure 2A); after the NIPAm/MAA layer is modified, an obvious shell structure can be seen on the outer layer of the mesoporous silicon nano-particles (fig. 2B).
Example 2: cell compatibility and blood compatibility
The experimental procedure was as follows:
subculturing human breast cancer cells (SK-BR-3) with high HER2 receptor expression, spreading on a 96-well plate according to 5 thousand per well, after 24 hours of incubation, respectively adding MSN or MSN-pNIPAm/MAA-HER2 to make the final concentration of particles be 0, 5, 10, 50, 100, 200, 500 μ g/mL, and continuing incubation for 24 hours;
subsequently, the well plate is removed andadding tetramethyl azocyclolan (MTT) into each well, and adding 5% CO at 37 deg.C2Culturing for 4 hours in an incubator;
finally, the well plate was taken out and the supernatant was aspirated, dimethyl sulfoxide (DMSO) was added, absorbance per well was measured in a microplate reader after shaking for 10 minutes, and the relative cell viability was 100% at a concentration of 0. mu.g/mL.
The results of the experiments, as shown in FIG. 3A, the cell viability of the MSN-pNIPAm/MAA-HER2 treated cells was higher than that of the MSN particles at the same particle concentration; the survival rate of SK-BR-3 cells in the MSN-pNIPAm/MAA-HER2 particle treated group is still maintained to be more than 80% at a high concentration of 500 mu g/mL.
The results show that the prepared composite nanoparticles have good cell compatibility due to the protection of the copolymer and the low toxicity of the modified HER2 single-chain strip antibody.
In addition, an amount of red blood cells was collected and resuspended in physiological saline, and the concentration was adjusted to 2%. Synthetic nanoparticles MSN and MSN-pNIPAm/MAA-HER2) were added thereto to give final concentrations of 0, 5, 10, 25, 50, 100, 200, 500 μ g/mL. Meanwhile, deionized water is used as a positive control, and physiological saline is used as a negative control, so that 2% erythrocyte solutions are prepared respectively.
Hemolysis is expressed as percent hemolysis and is calculated as follows: percent hemolysis (%) ═ as-A-)/(A+-A-) X 100, wherein AsRepresents the absorbance of the sample, A+Absorbance of a Positive control, A-Represents the absorbance of the negative control.
Experimental results, as shown in fig. 3B, the MSN particle treated group produced severe hemolysis as the particle concentration increased; whereas the percentage of hemolysis at a high concentration of 500. mu.g/mL was still below 5% for the MSN-pNIPAm/MAA-HER2 particle treated group.
The above results indicate that the copolymer layer on the prepared composite nanoparticles can prevent hemolysis caused by direct contact between pure MSN particles and erythrocytes, and thus have better blood compatibility than pure MSN particles.
Example 3: temperature and pH dual response drug release
The experimental steps are as follows:
preparing a phosphate buffer solution with pH 7.4 (simulating the pH value of human blood) and a phosphate buffer solution with pH 5.0 (simulating the intracellular pH value of human tumor tissues);
dispersing 2mg of DOX @ MSN-pNIPAm/MAA-HER2 in phosphate buffer, and reacting at 37 ℃ and 25 ℃;
and taking the supernatant at a certain time interval, detecting by using an ultraviolet-visible spectrophotometer, and drawing an accumulative release curve according to the ratio of the medicine quantity cumulatively released at each time point to the total loaded medicine quantity.
As shown in fig. 4, when two stimuli, namely high temperature (41 ℃) and acidic (pH 5.0), are used to trigger drug release together, the obtained drug release is faster and the release amount is higher than that of the case of only setting a single stimulus condition, which indicates that the modified mesoporous silicon nanoparticle has drug release behavior with dual responses of temperature and pH, and has better drug release efficiency than a single response, and is expected to respond to the high temperature and acidic environment of the tumor microenvironment. Meanwhile, under the conditions of 25 ℃ and pH 7.4, the cumulative release rate in 24 hours is lower than 10%, which shows that the modified mesoporous silicon nanoparticles can effectively encapsulate the drug and reduce the early leakage of the drug.
Example 4: cell uptake capacity assay
The experimental steps are as follows:
respectively subculturing a human breast cancer cell (SK-BR-3) with high HER2 receptor expression and a human normal hepatocyte cell (L-02) with HER2 receptor non-expression, paving the subculturing cells on a 96-well plate according to 1 ten thousand per hole, respectively adding composite mesoporous silicon nanoparticles (MSN-pNIPAm/MAA-HER2 or MSN-pNIPAm/MAA) marked by FITC, and continuously culturing for 4 hours;
subsequently, cell nucleus staining was performed using Hoechst 33258, and the intracellular nanoparticle content was observed with a laser scanning confocal fluorescence microscope.
As shown in FIG. 5A, the experimental results showed a time-dependent increase in the amount of DOX @ MSN-pNIPAm/MAA-HER2 entering SK-BR-3 cells.
As shown in FIG. 5B, after endocytosis for 4 hours under the same condition, the amount of the DOX @ MSN-pNIPAm/MAA-HER2 entering the SK-BR-3 cell is higher than that of the DOX @ MSN-pNIPAm/MAA-HER2 entering the L-02 cell, which indicates that the modified mesoporous silicon nanoparticle can specifically recognize a HER2 receptor, so that the endocytosis capacity of a HER2 high-expression breast cancer cell is enhanced.
Example 5: in vitro tumor cell killing ability experiment
The experimental steps are as follows:
respectively subculturing a human breast cancer cell (SK-BR-3) with high HER2 receptor expression and a normal human liver cell (L-02) with HER2 receptor non-expression, paving the subculturing cells on a 96-well plate according to the density of 1 ten thousand per well, and dividing the subculturing cells into 3 groups of free adriamycin, DOX @ MSN-pNIPAm/MAA and DOX @ MSN-pNIPAm/MAA-HER2, wherein each group is provided with 5 concentration gradients of adriamycin with the concentration of 0.5, 1, 2, 4 and 8 mu g/mL;
adding the dispersion of free doxorubicin, DOX @ MSN-pNIPAm/MAA and DOX @ MSN-pNIPAm/MAA-HER2 particles at various concentrations to each cell well at 37 deg.C with 5% CO2Incubating for 4h in an incubator;
subsequently, the well plate was removed and tetramethylazocycloblue (MTT) was added to each well, followed by 5% CO at 37 ℃2Culturing for 4 hours in an incubator;
finally, the well plate is taken out and the supernatant is removed, dimethyl sulfoxide (DMSO) is added, and after oscillation for 10 minutes, the absorbance of each well is measured by a microplate reader.
As shown in FIG. 6A, the activity of SK-BR-3 cells treated by 3 kinds of particles decreases with increasing concentration, which indicates that both high concentration of drug and drug using carrier can kill tumor cells. In addition, the survival rate of the cells after the action of DOX @ MSN-pNIPAm/MAA-HER2 is lower than that of the cells only affected by DOX or affected by DOX @ MSN-pNIPAm/MAA, which shows that the particles modified with HER2 single-chain antibody are easier to be taken by the tumor cells highly expressed by HER2 receptor than the particles of unmodified antibody, and simultaneously, the high temperature and the acidic environment in the tumor cells are accompanied, the release of a large amount of internal medicine can be promoted, and the treatment effect is better than that of the single free medicine.
In addition, as shown in fig. 6B, the activity of L-02 cells after 3 kinds of particle treatment has no significant difference at each doxorubicin concentration, which indicates that the synthesized composite mesoporous silicon nanoparticles do not cause additional targeting injury to normal cells not expressing HER 2.
The results show that the tumor cell killing capability of the mesoporous silicon nanoparticles modified with the HER2 single-chain antibody and the NIPAm/MAA layer loaded with the adriamycin is stronger than that of unmodified mesoporous silicon nanoparticles, and the mesoporous silicon nanoparticles can not cause accidental injury to normal cells.
Example 6: in vivo tumor cell killing ability test
The experimental steps are as follows:
selecting SK-BR-3 cells to construct a breast cancer animal model, selecting healthy BALB/c nude mice with the age of 4-5 weeks, and dividing the healthy BALB/c nude mice into 5 groups of phosphate buffer (Control), MSN-pNIPAm/MAA, free DOX, DOX @ MSN-pNIPAm/MAA and DOX @ MSN-pNIPAm/MAA-HER2, wherein each group comprises 4 nude mice;
the SK-BR-3 cells are subcultured for 24h and then used for constructing a breast cancer animal model;
adjusting the cell density of the SK-BR-3 to 50 ten thousand per mL, injecting 0.2mL of SK-BR-3 cells into the right rear leg of the nude mouse by using an insulin syringe, and enabling the cell inoculation amount of each nude mouse to be 10 ten thousand;
when the tumor grows to about 150mm3Then, 0.1mL of experimental drug or drug-loaded particles are injected into the vein;
after 14 days, nude mice were euthanized and tumor tissues were examined histopathologically.
As shown in fig. 7A and 7B, the tumor killing ability of DOX @ MSN-pNIPAm/MAA-HER2 group was higher than that of phosphate buffer blank group and than that of DOX @ MSN-pNIPAm/MAA group, indicating that the composite nanoparticle containing HER2 single-chain antibody has stronger therapeutic effect due to its high targeting to SK-BR-3 cells highly expressing HER2, compared to the composite nanoparticle without antibody. Meanwhile, the tumor killing capacity of the DOX @ MSN-pNIPAm/MAA-HER2 group is superior to that of the free adriamycin treatment group, and further, compared with single adriamycin treatment, the composite mesoporous silicon nanoparticle loaded with adriamycin has high HER2 receptor targeting and tumor microenvironment dual responsiveness, so that a stronger killing effect is generated on HER2 high-expression breast cancer cells.
The results show that the temperature and pH dual-response and tumor targeting mesoporous silicon drug delivery system has good temperature and pH dual-response drug release behavior. Furthermore, the composition has excellent in-vivo and in-vitro killing effect on breast cancer cells with high HER2 expression, and is expected to realize targeted and controllable anti-malignant breast cancer treatment.
In this specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.
Claims (9)
1. The tumor self-targeting multi-response type mesoporous silicon drug delivery system is characterized by comprising mesoporous silicon nanoparticles, wherein antitumor drugs are loaded on the mesoporous silicon nanoparticles, and a polymer layer and a low-toxicity tumor targeting single-chain antibody are modified on the surfaces of the mesoporous silicon nanoparticles, so that the drug delivery system has breast cancer cell targeting property, temperature responsiveness and pH responsiveness.
2. The tumor self-targeting multi-level response mesoporous silicon drug delivery system according to claim 1, wherein the anti-tumor drug is a hydrophobic anti-tumor drug.
3. The tumor self-targeting multistage-response mesoporous silicon drug delivery system according to claim 1, wherein the polymer in the polymer layer is a copolymer of N-isopropylacrylamide and methacrylic acid.
4. The tumor self-targeting multistage-response mesoporous silicon drug delivery system according to claim 1, wherein the low-toxicity tumor-targeting single-chain antibody is an anti-HER 2 single-chain antibody.
5. A preparation method of the tumor self-targeting multistage response type mesoporous silicon drug delivery system according to any one of claims 1 to 4, wherein the preparation method comprises the following steps:
(1) synthesizing monodisperse mesoporous silicon nanoparticle MSN;
(2) preparing a composite particle DOX @ MSN-MPS modified by methacryloxypropyltrimethoxysilane;
(3) preparing polymer layer modified composite particles DOX @ MSN-pNIPAm/MAA;
(4) preparing the tumor-targeted single-chain antibody modified composite particle DOX @ MSN-pNIPAm/MAA-HER 2.
6. The preparation method according to claim 5, wherein the step (1) is specifically:
the synthesis is carried out by a sol-gel method, and the monodisperse MSN is obtained by repeated washing after centrifugal collection.
7. The preparation method according to claim 5, wherein the step (2) is specifically:
dispersing MSN in ethanol, adding Methacryloxypropyltrimethoxysilane (MPS), reacting, centrifuging and collecting the MPS modified MSN;
dispersing MSN-MPS in ethanol, adding a hydrochloric acid solution, and reacting to obtain silicon-based MSN-MPS with the template removed;
dispersing the silicon-based MSN-MPS with the template removed and the anti-tumor drug in water, and collecting the composite particles DOX @ MSN-MPS which load the anti-tumor drug and remove the template after reaction.
8. The preparation method according to claim 5, wherein the step (3) is specifically:
dissolving the copolymer of N-isopropylacrylamide and methacrylic acid in water, adding DOX @ MSN-MPS and potassium persulfate, and reacting under the protection of nitrogen to obtain the composite particle DOX @ MSN-pNIPAm/MAA modified with a polymer layer.
9. The preparation method according to claim 5, wherein the step (4) is specifically:
adding N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride into a HER2 single-chain antibody solution, activating carboxyl on a HER2 single-chain antibody, and then ultrasonically dispersing DOX @ MSN-pNIPAm/MAA into a mixed solution to react to obtain the tumor targeting single-chain antibody modified composite particle DOX @ MSN-pNIPAm/MAA-HER 2.
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