CN109260176B - Tumor-specific cleavable PEG (polyethylene glycol) nanoparticle as well as preparation method and application thereof - Google Patents

Tumor-specific cleavable PEG (polyethylene glycol) nanoparticle as well as preparation method and application thereof Download PDF

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CN109260176B
CN109260176B CN201811223398.XA CN201811223398A CN109260176B CN 109260176 B CN109260176 B CN 109260176B CN 201811223398 A CN201811223398 A CN 201811223398A CN 109260176 B CN109260176 B CN 109260176B
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王冠海
林坚涛
张大威
于海兵
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Guangdong Medical University
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Abstract

The invention discloses a PEG nanoparticle with tumor specificity and breaking capacity, a preparation method and application thereof. The surface of the mesoporous silicon nanoparticle is modified with a cationic polymer layer containing amino and disulfide bonds, then PEG with end groups containing benzaldehyde is added, and a PEG shell layer is introduced to the surface of the nanoparticle to obtain the PEG nanoparticle with the tumor specificity and the fracture property. The carrier has the characteristics that PEG molecules in a tumor microenvironment can be dropped and can respond to oxidation reduction of glutathione in cells, has the advantages of stronger drug loading capacity, long blood circulation time, targeted drug delivery and the like, and is suitable for preparing antitumor drug carriers.

Description

Tumor-specific cleavable PEG (polyethylene glycol) nanoparticle as well as preparation method and application thereof
Technical Field
The invention belongs to the field of drug carriers, and particularly relates to a tumor-specific cleavable PEG nanoparticle, and a preparation method and application thereof.
Background
In recent years, the excellent performance of nanotechnology in a drug delivery system is receiving wide attention, the effect in tumor treatment is remarkable, and great advantages and potentials are shown. The nano-drug carrier loads the drug by a physical method, has higher drug loading capacity, can well maintain the activity of the drug, can improve the targeted delivery capacity and pharmacodynamic level of the drug to tumor tissues by enhancing the permeation and retention effects, realizes the fixed-point, timed and quantitative release of the drug, reduces the toxic and side effects of the drug to normal tissues, and fully exerts the curative effect of the drug, thereby achieving the aim of healing.
Mesoporous Silicon Nanoparticles (MSNs) are nanoparticles with a multi-pore-channel structure, have the characteristics of stable structure, adjustable pore diameter, easy surface modification and the like, and are widely researched in the aspect of transmission and controlled release of medicines and genes at present. However, due to the complex physiological environment in vivo, MSNs nanocarriers are easily eliminated by the immune system, resulting in low cellular internalization rate, low bioavailability of the drug, and the like. Polyethylene glycol (PEG) has good biocompatibility, good blood compatibility and good hydrophilicity, has no immunogenicity, and is widely applied in the field of biomedical materials. The MSNs are modified by PEG, so that the solution stability of the nano carrier can be improved, the elimination of an immune system can be reduced, and the blood circulation time of the carrier can be prolonged. However, PEG of the traditional nanocarrier is coupled to the surface of the nanoparticle through a covalent bond, and due to the stability of the covalent bond, the PEG layer is too stable and cannot fall off in tumor tissues, which is not beneficial to phagocytosis of the nanocarrier by tumor cells, and can reduce the targeting release and bioavailability of the drug at the focus part, so that the ideal pegylated nanocarrier can stably exist in the blood circulation process, the circulation time is prolonged, and after entering the tumor tissues, the PEG shell layer can fall off, and the phagocytosis of the nanocarrier by tumor cells is improved.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a preparation method of PEG nanoparticles with breakable tumor specificity.
Another object of the present invention is to provide tumor-specific cleavable PEGylated nanoparticles obtained by the above preparation method.
It is a further object of the present invention to provide the use of the above mentioned tumor-specific cleavable pegylated nanoparticles.
The purpose of the invention is realized by the following technical scheme: a preparation method of PEG nanoparticles with tumor specificity and breaking capacity comprises the following steps:
(1) dispersing mesoporous silicon nanoparticles in an organic solvent, adding gamma-Aminopropyltriethoxysilane (APTES), reacting, and purifying to obtain mesoporous silicon nanoparticles with amino groups on the surface;
(2) dispersing the mesoporous silicon nano particles with the amino groups on the surfaces, which are obtained in the step (1), in an organic solvent, adding succinic anhydride, reacting, and performing centrifugal separation to obtain carboxyl-containing mesoporous silicon nano particles;
(3) dispersing the mesoporous silicon nano particles containing carboxyl obtained in the step (2) in a solvent, adding cystamine dihydrochloride and a catalyst, reacting, and carrying out solid-liquid separation to obtain amino-and disulfide bond-modified mesoporous silicon nano particles;
(4) loading the amino-group-and-disulfide-bond-modified mesoporous silicon nanoparticles obtained in the step (3) with an anti-tumor drug, dispersing the loaded anti-tumor drug in a solvent, sequentially adding a cationic monomer and a crosslinking agent, reacting, and purifying to obtain mesoporous silicon nanoparticles with a cationic polymer layer introduced on the surface;
(5) dissolving PEG with end group benzaldehyde in a phosphate solution to obtain a solution A; dispersing the mesoporous silicon nano particles with the cationic polymer layer introduced to the surface prepared in the step (4) in a phosphate solution to obtain a solution B; and mixing the solution A and the solution B, reacting and purifying to obtain the PEG nanoparticles with the tumor specificity being fractured.
The mesoporous silicon nano particles in the step (1) are MCM-41 type; the mesoporous silicon nano-particles with the particle size of 100nm and the pore diameter of 2-10nm are preferably selected.
The mesoporous silicon nanoparticles described in the step (1) are preferably prepared by the following steps: ethyl Orthosilicate (TEOS) is used as a raw material, hexadecyl trimethyl ammonium bromide is used as a surfactant, 1,3, 5-trimethylbenzene is used as a pore-making agent, and the MCM-41 type mesoporous silicon nano particles are obtained through stirring reaction.
The dosage of the 1,3, 5-trimethylbenzene is 0.5-2 times of the molar weight of the TEOS.
The amount of the cetyl trimethyl ammonium bromide is 0.5-2 times of the molar weight of the TEOS.
The reaction temperature is preferably 65-75 ℃; more preferably 70 deg.c.
The reaction time is preferably 1.5-2.5 h; more preferably 2 h.
The organic solvent in step (1) is preferably anhydrous toluene.
The organic solvent in the step (1) is only used as a reaction medium and does not participate in the reaction, and the dosage of the organic solvent is preferably as follows: the mesoporous silicon nano particle is 50-150 mL: 1g, calculating; more preferably, the ratio of organic solvent: mesoporous silicon nanoparticles (100 mL): and 1g is calculated.
The mass usage amount of the gamma-aminopropyltriethoxysilane in the step (1) is calculated according to the proportion of the gamma-aminopropyltriethoxysilane to the mesoporous silicon nano particles (mass ratio) of 1-2: 1; more preferably, the ratio of the mesoporous silicon nanoparticles to the mesoporous silicon nanoparticles is 1: 1.
The reaction described in step (1) is preferably a heating reflux reaction.
The temperature of the heating reflux reaction is preferably 100-120 ℃; more preferably 110 deg.c.
The heating reflux reaction time is preferably 20-30 h; more preferably 24 h.
The purification steps described in step (1) are preferably as follows: suction filtration, washing with toluene and ethanol, and drying.
The organic solvent in the step (2) is preferably at least one of ethanol, tetrahydrofuran, ethyl acetate and acetone; more preferably acetone.
The organic solvent used in the steps (1) and (2) has the same function as that of the organic solvent used in the step (1).
The ratio of the consumption of the succinic anhydride in the step (2) to the mesoporous silicon nanoparticles with amino groups on the surface is 0.5-2: 1 (mass ratio); more preferably, the ratio of the mesoporous silicon nanoparticles to the mesoporous silicon nanoparticles containing amino groups on the surface is 2: 1.
The solvent in the step (3) and the step (4) is water or phosphate buffer solution.
The phosphate buffer solution is preferably a phosphate buffer solution with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M.
The catalyst described in step (3) is preferably 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).
The catalyst is used in a catalytic amount.
The mass amount of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) is calculated according to the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to the mesoporous silicon nano-particles containing carboxyl groups, which is 1: 1-5; preferably calculated according to the mass ratio of the mesoporous silicon nanoparticles containing carboxyl to the mesoporous silicon nanoparticles containing carboxyl of 1:2.
The mass usage amount of the N-hydroxysuccinimide (NHS) is calculated according to the mass ratio of the N-hydroxysuccinimide (NHS) to the carboxyl-containing mesoporous silicon nanoparticles of 1: 1-5; preferably calculated according to the mass ratio of the mesoporous silicon nanoparticles containing carboxyl to the mesoporous silicon nanoparticles containing carboxyl of 1: 2.5.
The mass usage amount of the cystamine dihydrochloride obtained in the step (3) is calculated according to the mass ratio of the cystamine dihydrochloride to the carboxyl-containing mesoporous silicon nano particles of 1: 0.5-2.5; preferably calculated according to the mass ratio of the mesoporous silicon nanoparticles containing carboxyl to the mesoporous silicon nanoparticles containing carboxyl being 1: 1.
Step (4) is preferably: loading the amino-group-and-disulfide-bond-modified mesoporous silicon nanoparticles obtained in the step (3) with an anti-tumor drug, dispersing the loaded mesoporous silicon nanoparticles into a solvent, sequentially adding a cationic monomer and a crosslinking agent under the protection of nitrogen, adding an initiator to initiate a polymerization reaction, and purifying to obtain mesoporous silicon nanoparticles with a cationic polymer layer introduced to the surface;
the initiator is preferably ammonium persulfate and N, N, N ', N' -tetramethyl ethylene diamine; more preferably ammonium persulfate and N, N, N ', N' -tetramethylethylenediamine in a mass ratio of 1:1 to obtain a mixture.
The addition amount of the initiator is 0 to 0.02 times of the mass of the cationic monomer.
The reaction condition is preferably 0-35 ℃ for 1-24 h.
The medicament in the step (4) is an anti-tumor medicament, and is preferably one or at least two of adriamycin, daunorubicin, adriamycin, demethoxydaunorubicin, epirubicin, taxol, vinblastine, vincristine, tamoxifen, formestane, anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene and tegafur.
The step (4) of loading the amino-containing and disulfide bond-modified mesoporous silicon nanoparticles obtained in the step (3) with the anti-tumor drug is preferably: preparing the drug into a solution, mixing the solution with the amino-containing and disulfide bond-modified mesoporous silicon nanoparticles obtained in the step (3), stirring to enable the drug to be fully adsorbed, and performing solid-liquid separation to obtain the antitumor drug-loaded mesoporous silicon nanoparticles.
The cationic monomer in the step (4) is a cationic monomer containing amino, or a mixture of the cationic monomer containing amino and other cationic monomers; preferably a mixture of the cationic monomer containing amino groups and other cationic monomers; more preferably, the molar ratio of the amino group-containing cationic monomer to the other cationic monomers is 9: 12 parts by weight of the obtained mixture. Other cationic monomers refer to cationic monomers that do not contain an amino group.
The amino group-containing cationic monomer is preferably at least one of N- (3-aminoethyl) methacrylamide and N- (3-aminopropyl) methacrylamide.
The other cationic monomer is preferably at least one of N, N-dimethylacrylamide and N, N-diethylacrylamide.
The dosage of the cationic monomer in the step (4) is calculated according to the mass ratio of the cationic monomer to the mesoporous silicon nano particles modified by amino and disulfide bonds or the mesoporous silicon nano particles modified by amino and disulfide bonds after loading the medicine, which is 1-2: 1-2; more preferably, the mass ratio of the mesoporous silicon nanoparticles to the amino-and disulfide-bond-containing modified mesoporous silicon nanoparticles or the drug-loaded mesoporous silicon nanoparticles is 1: 1.
The crosslinking agent in step (4) is a compound having a disulfide bond, preferably at least one of N, N' -bis (acryloyl) cystamine and cystamine.
The dosage of the cross-linking agent is calculated according to the molar ratio of the cross-linking agent to the cationic monomer of 1-2: 21.
The purification step described in step (4) is preferably as follows: centrifuging, washing with water and ethanol, and drying.
The end group benzaldehyde PEG in the step (5) is polyethylene glycol with the following characteristics: one end group is benzaldehyde, the other end group is methoxyl, and the molecular weight is 500-10000.
The end-group benzaldehydeated PEG described in step (5) is preferably prepared by: amino polyethylene glycol monomethyl ether and N-succinimide 4-formyl benzoate are mixed according to the mass ratio of 20: 3, mixing and dissolving in N, N-Dimethylformamide (DMF) according to a ratio, stirring and reacting for 4 hours at the temperature of 4 ℃, precipitating by using excessive cold ether which is 10 times of the N, N-dimethylformamide, and recrystallizing a crude product by using propylene glycol to obtain the end group benzaldehyde PEG.
The dosage of the end group benzaldehyde-modified PEG in the step (5) is calculated according to the molar ratio of the end group benzaldehyde-modified PEG to amino on the surface of the mesoporous silicon of 1.1-1.5: 1; preferably, the molar ratio of the mesoporous silicon surface amino groups to the mesoporous silicon surface amino groups is 1.1: 1.
The phosphate buffer solution in the step (5) is preferably a phosphate buffer solution with the pH value of 7.2-7.4 and the concentration of 0.01-0.1M.
The reaction time in step (5) is preferably 24 hours.
The purification step described in step (5) is preferably as follows: centrifuging, washing with water and ethanol, and drying.
A PEG nanoparticle with tumor specificity and breaking ability is prepared by the above preparation method.
The PEG nanoparticles with the tumor specificity and the fracture property have the advantages of good water dispersibility, good biocompatibility, stronger drug loading capacity, long blood circulation time, low toxicity, targeted drug delivery, 100-200nm of particle size, 2-10nm of pore size and uniform distribution.
The PEG nanoparticles with the tumor specificity and the ability of breaking are applied to the preparation of antitumor drugs.
The principle of the invention is as follows: the invention takes mesoporous silicon nano particles as cores, loads anti-tumor drugs, modifies cationic polymer layers containing amino and disulfide bonds on the surfaces of the mesoporous silicon nano particles, then adds PEG with end group benzaldehyde, forms a benzal bond which can be broken under the weak acid condition through the reaction between benzaldehyde and amino, introduces PEG shells on the surfaces of the nano particles, and constructs a multifunctional drug delivery carrier which has the characteristics that PEG molecules in a tumor microenvironment can drop and can respond to oxidation and reduction of glutathione in cells. Due to the existence of the PEG shell layer, the nano-carrier can effectively resist the elimination of an immune system, and can keep stable circulation in blood for a long time; after entering the tumor tissue, in a weak acid environment of the tumor tissue, the benzylidene imino bond is broken, the PEG shell is separated from the surface of the nano carrier, the charge on the surface of the carrier is changed from neutral to positive, and the phagocytic efficiency of cells can be improved; after entering tumor cells, the concentration of high Glutathione (GSH) is rapidly increased, and disulfide bonds in the cationic polymer layer are broken, so that the loaded drug is specifically released in the cells, and the targeted delivery and bioavailability of the anti-tumor drug are improved. Through the design, the tumor targeted drug delivery carrier with good water dispersibility, good biocompatibility, stronger drug loading capacity, long blood circulation time and low toxicity is prepared.
Compared with the prior art, the invention has the following advantages and effects:
1. the PEG of the PEG-functionalized nanoparticle with the tumor specificity and the rupture function provided by the invention is coupled and modified on the surface through a dynamic covalent bond, and has the characteristics that a PEG shell layer can be dropped in a tumor microenvironment and responds to the oxidation reduction of glutathione in cells. After the nano-carrier enters a weak acid environment of tumor tissues, the benzylidene amino bond coupling the PEG and the polymer layer is broken, the PEG shell layer is separated from the surface of the nano-carrier, the charge on the surface of the carrier is changed from neutral to positive, and the phagocytic efficiency of cells is improved; after entering tumor cells, the polymer layer on the surface is broken and dissociated under the action of intracellular glutathione, and the loaded drug is released in the tumor cells in a targeted manner, so that the bioavailability of the drug can be effectively improved.
2. The tumor-specific breakable PEG nanoparticle provided by the invention has good water dispersibility, good biocompatibility, stronger drug loading capacity, long blood circulation time, low toxicity, targeted drug delivery, a particle size of 100-200nm, a pore size of 2-10nm and uniform distribution.
Drawings
FIG. 1 is a photograph of the nano-carriers prepared in examples 1, 4 and 6 by transmission electron microscopy; wherein, the graphs A to C respectively correspond to the nano-carriers prepared in the examples 1, 4 and 6 in sequence.
FIG. 2 is an XRD diffraction pattern of the nano-carrier prepared in example 1; wherein, curve 1 is the XRD diffraction pattern of the unmodified mesoporous silicon nanoparticles, and curve 2 is the XRD diffraction pattern of the mesoporous silicon nanocarrier prepared in example 1.
FIG. 3 is a comparison graph of the nitrogen adsorption method for measuring the adsorption effect of the inner pores of the nano-carrier prepared in example 1; wherein, curve 1 represents the unmodified mesoporous silicon nanoparticles, and curve 2 represents the mesoporous silicon nano-carrier prepared in example 1.
FIG. 4 is a bar graph showing the change in particle size of the nanocarrier prepared in example 1 in the interaction with protein at different times.
FIG. 5 is a graph of cumulative release of doxorubicin loaded with nanocarriers prepared in examples 1, 2 and 4 at GSH concentrations mimicking the blood GSH concentration (0.01mM) and the tumor cell microenvironment (10 mM); wherein, curve 1 is the cumulative release curve of the nano-vector prepared in example 1 in a pH5.0+10mM glutathione environment, curve 2 is the cumulative release curve of the nano-vector prepared in example 4 in a pH5.0+10mM glutathione environment, curve 3 is the cumulative release curve of the nano-vector prepared in example 1 in a pH7.4+10mM glutathione environment, curve 4 is the cumulative release curve of the nano-vector prepared in example 4 in a pH7.4+10mM glutathione environment, curve 5 is the cumulative release curve of the nano-vector prepared in example 2 in a pH5.0+10mM glutathione environment, and line 6 is the cumulative release curve of the nano-vector prepared in example 2 in a pH7.4+10mM glutathione environment.
FIG. 6 is a photograph showing the results of releasing doxorubicin in Hela cells under different conditions from the nanocarriers prepared in examples 1, 2, and 4; wherein, a graph A is a photograph of the change in fluorescence of the nanocarrier prepared in example 1 after staining for 2h and 4h, a graph B is a photograph of the change in fluorescence of the nanocarrier prepared in example 2 after staining for 2h and 4h, and a graph C is a photograph of the change in fluorescence of the nanocarrier prepared in example 4 after staining for 2h and 4 h.
FIG. 7 is a graph showing the results of measuring the effect of nanocarriers prepared in examples 1 and 4 on the apoptosis rate of Hela cells by flow cytometry; wherein, the graph A is the result graph of the effect of the nano-carrier prepared in the example 1 on the apoptosis rate of the Hela cells, and the graph B is the result graph of the effect of the nano-carrier prepared in the example 4 on the apoptosis rate of the Hela cells.
FIG. 8 is a nuclear magnetic hydrogen spectrum of α -methoxy- ω -benzaldehyde-PEG prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
(1) The pre-synthesized MCM-41 type mesoporous silicon nanoparticles (synthesized by the method in Colloids and Surfaces B: Biointerfaces,2017,155: 41-50, the particle size is 100nm, the pore diameter is 2-10 nm) are dried in vacuum at 60 ℃ for 24h, 0.50g is weighed and added into 50mL of anhydrous toluene, then 0.20mL of gamma-aminopropyltriethoxysilane is added, after uniform stirring, the temperature is raised to 110 ℃, and reflux reaction is carried out for 24h under the protection of nitrogen. And after the reaction is finished, carrying out suction filtration, washing twice by using toluene, washing once by using ethanol, and carrying out vacuum drying for 24h at the temperature of 60 ℃ to obtain the amino-containing mesoporous silicon nanoparticles. Dispersing 0.10g of mesoporous silicon nano particles containing amino in acetone, adding 0.20g of succinic anhydride, stirring and reacting for 24 hours at room temperature, and performing centrifugal separation to obtain the mesoporous silicon nano particles containing carboxyl. Dispersing 0.10g of mesoporous silicon nanoparticles containing carboxyl into a phosphate buffer solution (pH7.4, 0.1M), adding 0.10g of cystamine dihydrochloride, 0.05g of EDC and 0.04g of NHS, stirring at room temperature for reaction for 24h, performing centrifugal separation, and performing vacuum drying at 60 ℃ for 24h to obtain mesoporous silicon nanoparticles containing disulfide bonds and amino groups.
(2) Adding 0.10g of mesoporous silicon nanoparticles containing disulfide bonds and amino groups into 2.0mL of acetone solution containing 10mg of adriamycin, stirring and adsorbing for 24h, performing centrifugal separation, performing vacuum drying at 60 ℃ for 24h to obtain adriamycin-loaded mesoporous silicon nanoparticles, and measuring the load of adriamycin in the nanoparticles by ultraviolet spectroscopy.
(3) 50mg of mesoporous silicon nanoparticles loaded with adriamycin (DOX) are dispersed in 2.0mL of PBS buffer (pH7.4, 0.1M), 50mg of cationic monomers (N, N-dimethylacrylamide and N- (3-aminoethyl) methacrylamide) are added under the protection of nitrogen, and after uniform stirring, N' -bis (acryloyl) cystamine is added as a cross-linking agent, and the molar ratio of N, N-dimethylacrylamide/N- (3-aminoethyl) methacrylamide/cross-linking agent is adjusted to 12:9: 1. Then adding 30 mu L of ammonium persulfate (100mg/mL of deoxygenated aqueous solution) and 3 mu of L N, N, N ', N' -tetramethylethylenediamine to initiate polymerization reaction, reacting for 1h at room temperature, centrifugally separating the product, washing with water and ethanol, centrifuging three times, and drying in vacuum at 60 ℃ overnight to obtain the mesoporous silicon nanoparticles with the cationic polymer layer introduced on the surface.
(4) Mixing amino polyethylene glycol monomethyl ether (mPEG-NH)2M.w. ═ 2000)0.2g and N-succinimide 4-formylbenzoate 0.03g were dissolved in 2.0mL DMF, reacted with stirring at 4 ℃ for 4h, precipitated with 10 fold excess cold ether, and the crude product was recrystallized from propylene glycol to give α -methoxy- ω -benzaldehyde-PEG (end group benzaldehyde-pegylated PEG), and the product was characterized by nuclear magnetism (see fig. 8).
(5) Dispersing 50mg of mesoporous silicon nanoparticles with the surface introduced into the cationic polymer layer in 5mL of PBS buffer solution (pH7.4, 0.1M) to obtain a solution A; 20mg of a-methoxy- ω -benzaldehyde-PEG with a molecular weight of 2000 was dissolved in 2mL of PBS (pH7.4, 0.1M) to give solution B. And mixing the solution A and the solution B, stirring for reacting for 24 hours, centrifugally separating a product, washing and centrifuging for three times by using water and ethanol, and carrying out vacuum drying at 60 ℃ overnight to obtain the surface-PEGylated adriamycin-loaded mesoporous silicon nanoparticles.
Example 2
Example 2 differs from example 1 mainly in that: the steps (4) and (5) of the present embodiment replace the amino polyethylene glycol monomethyl ether in example 1 with monomethoxy polyethylene glycol succinimide succinate, so that the PEG coupled to the surface can be converted into a stable covalent bond by cleavage under slightly acidic conditions.
(1) The method comprises the steps of drying synthesized MCM-41 type mesoporous silicon nanoparticles (Colloids and Surfaces B: Biointerfaces,2017,155: 41-50.) (the particle diameter is 100nm and the pore diameter is 2-10 nm) at 60 ℃ for 24 hours in vacuum, weighing 0.50g of the dried particles, adding the weighed particles into 50mL of anhydrous toluene, adding 0.20mL of gamma-aminopropyltriethoxysilane, stirring uniformly, heating to 110 ℃, and carrying out reflux reaction for 24 hours under the protection of nitrogen. And after the reaction is finished, carrying out suction filtration, washing twice by using toluene, washing once by using ethanol, and carrying out vacuum drying for 24h at the temperature of 60 ℃ to obtain the amino-containing mesoporous silicon nanoparticles. Dispersing 0.10g of mesoporous silicon nano particles containing amino in acetone, adding 0.20g of succinic anhydride, stirring and reacting for 24 hours at room temperature, and performing centrifugal separation to obtain the mesoporous silicon nano particles containing carboxyl. Dispersing 0.10g of mesoporous silicon nanoparticles containing carboxyl into a phosphate buffer solution (pH7.4, 0.1M), adding 0.10g of cystamine dihydrochloride, 0.05g of EDC and 0.04g of NHS, stirring and reacting for 24h at room temperature, centrifugally separating, and drying in vacuum at 60 ℃ for 24h to obtain the mesoporous silicon nanoparticles containing disulfide bonds and amino groups.
(2) Adding 0.10g of mesoporous silicon nanoparticles containing disulfide bonds and amino groups into 2.0mL of acetone solution containing 10mg of adriamycin, stirring and adsorbing for 24h, performing centrifugal separation, performing vacuum drying at 60 ℃ for 24h to obtain adriamycin-loaded mesoporous silicon nanoparticles, and measuring the load of adriamycin in the nanoparticles by ultraviolet spectroscopy.
(3) 50mg of mesoporous silicon nanoparticles loaded with adriamycin (DOX) are dispersed in 2.0mL of PBS buffer (pH7.4, 0.1M), 50mg of cationic monomers (N, N-dimethylacrylamide and N- (3-aminoethyl) methacrylamide) are added under the protection of nitrogen, and after uniform stirring, N' -bis (acryloyl) cystamine is added as a cross-linking agent, and the molar ratio of N, N-dimethylacrylamide/N- (3-aminoethyl) methacrylamide/cross-linking agent is adjusted to 12:9: 1. Then adding 30 mu L of ammonium persulfate (100mg/mL of deoxygenated aqueous solution) and 3 mu of L N, N, N ', N' -tetramethylethylenediamine to initiate polymerization reaction, reacting for 1h at room temperature, centrifugally separating the product, washing with water and ethanol, centrifuging three times, and drying in vacuum at 60 ℃ overnight to obtain the mesoporous silicon nanoparticles with the cationic polymer layer introduced on the surface.
(4) Dispersing 50mg of mesoporous silicon nanoparticles with the surface introduced into the cationic polymer layer in 5mL of PBS buffer solution (pH7.4, 0.1M) to obtain a solution A; 20mg of monomethoxypolyethylene glycol succinimide succinate having a molecular weight of 2000 was dissolved in 2mL of PBS solution (pH7.4, 0.1M) to obtain solution B. And mixing the solution A and the solution B, stirring for reacting for 24 hours, centrifugally separating a product, washing and centrifuging for three times by using water and ethanol, and carrying out vacuum drying at 60 ℃ overnight to obtain the PEG mesoporous silicon nano particles loaded with the adriamycin through surface covalent bond coupling.
Example 3
Example 3 differs from example 1 mainly in that: in step (3) of this example, N-diethylacrylamide was used in place of N, N-dimethylacrylamide in example 1.
(1) The pre-synthesized MCM-41 type mesoporous silicon nano particles are dried in vacuum at 60 ℃ for 24 hours, 0.50g of the pre-synthesized MCM-41 type mesoporous silicon nano particles are weighed and added into 50mL of anhydrous toluene, then 0.20mL of gamma-aminopropyltriethoxysilane is added, the mixture is stirred uniformly, the temperature is increased to 110 ℃, and the reflux reaction is carried out for 24 hours under the protection of nitrogen. And after the reaction is finished, carrying out suction filtration, washing with toluene twice, washing with ethanol once, and carrying out vacuum drying at 50 ℃ for 24h to obtain the amino-containing mesoporous silicon nanoparticles. Dispersing 0.10g of mesoporous silicon nano particles containing amino in acetone, adding 0.20g of succinic anhydride, stirring and reacting for 24 hours at room temperature, and performing centrifugal separation to obtain the mesoporous silicon nano particles containing carboxyl. Dispersing 0.10g of mesoporous silicon nanoparticles containing carboxyl into a phosphate buffer solution (pH7.4, 0.1M), adding 0.10g of cystamine dihydrochloride, 0.05g of EDC and 0.04g of NHS, stirring and reacting for 24h at room temperature, centrifugally separating, and drying in vacuum at 50 ℃ for 24h to obtain the mesoporous silicon nanoparticles containing disulfide bonds and amino groups.
(2) Adding 0.10g of mesoporous silicon nanoparticles containing disulfide bonds and amino groups into 2.0mL of acetone solution containing 10mg of adriamycin, stirring and adsorbing for 24h, performing centrifugal separation, and performing vacuum drying at 50 ℃ for 24h to obtain the adriamycin-loaded mesoporous silicon nanoparticles.
(3) 50mg of mesoporous silicon nano particles loaded with adriamycin are dispersed in 2.0mL of PBS buffer solution (pH7.4, 0.1M), 50mg of cationic monomers (N, N-diethylacrylamide and N- (3-aminoethyl) methacrylamide) are added under the protection of nitrogen, and after uniform stirring, N' -bis (acryloyl) cystamine is added as a cross-linking agent, and the molar ratio of N, N-diethylacrylamide/N- (3-aminoethyl) methacrylamide/cross-linking agent is adjusted to 12:9: 1. Then adding 30 mu L of ammonium persulfate (100mg/mL of deoxidized aqueous solution) and 3 mu of L N, N, N ', N' -tetramethylethylenediamine to initiate polymerization reaction, reacting for 1h at room temperature, centrifugally separating the product, washing with water and ethanol, centrifuging for three times, and drying in vacuum at 50 ℃ overnight to obtain the mesoporous silicon nano-particles with the cationic polymer layer introduced on the surface.
(4) Dispersing 50mg of mesoporous silicon nanoparticles with the surface introduced into the cationic polymer layer in 5mL of PBS (pH7.4, 0.1M) to obtain a solution A; 20mg of a-methoxy- ω -benzaldehyde-PEG (prepared according to step (4) of example 1) having a molecular weight of 2000 was dissolved in 2mL of PBS solution (pH7.4, 0.1M) to obtain solution B. And mixing the solution A and the solution B, stirring for reacting for 24 hours, centrifugally separating a product, washing and centrifuging for three times by using water and ethanol, and carrying out vacuum drying at 50 ℃ overnight to obtain the surface-PEGylated adriamycin-loaded mesoporous silicon nanoparticles.
Example 4
Example 4 differs from example 1 mainly in that: the crosslinking agents used are different and the crosslinking reaction conditions are different.
(1) The pre-synthesized MCM-41 type mesoporous silicon nano particles are dried in vacuum at 60 ℃ for 24 hours, 0.50g of the pre-synthesized MCM-41 type mesoporous silicon nano particles are weighed and added into 50mL of anhydrous toluene, then 0.20mL of gamma-aminopropyltriethoxysilane is added, the mixture is stirred uniformly, the temperature is increased to 110 ℃, and the reflux reaction is carried out for 24 hours under the protection of nitrogen. And after the reaction is finished, carrying out suction filtration, washing toluene twice, washing ethanol once, and carrying out vacuum drying at 60 ℃ for 24h to obtain the amino-containing mesoporous silicon nanoparticles. Dispersing 0.10g of mesoporous silicon nano particles containing amino in acetone, adding 0.20g of succinic anhydride, stirring and reacting for 24 hours at room temperature, and performing centrifugal separation to obtain the mesoporous silicon nano particles containing carboxyl. Dispersing 0.10g of mesoporous silicon nanoparticles containing carboxyl into a phosphate buffer solution (pH7.4, 0.1M), adding 0.10g of cystamine dihydrochloride, 0.05g of EDC and 0.04g of NHS, stirring and reacting for 24h at room temperature, centrifugally separating, and vacuum-drying for 24h at 60 ℃ to obtain the mesoporous silicon nanoparticles containing disulfide bonds and amino groups.
(2) Adding 0.10g of mesoporous silicon nanoparticles containing disulfide bonds and amino groups into 2.0mL of acetone solution containing 10mg of adriamycin, stirring and adsorbing for 24h, performing centrifugal separation, and performing vacuum drying at 60 ℃ for 24h to obtain the adriamycin-loaded mesoporous silicon nanoparticles.
(3) Dispersing 50mg of doxorubicin-loaded mesoporous silicon nanoparticles into 2.0mL of PBS buffer (pH7.4, 0.1M), cooling to 0 ℃, adding 50mg of cationic monomers (N, N-dimethylacrylamide and N- (3-aminoethyl) methacrylamide) under the protection of nitrogen, uniformly stirring, adding cystamine as a cross-linking agent, adjusting the molar ratio of N, N-dimethylacrylamide/N- (3-aminoethyl) methacrylamide/cross-linking agent to be 12:9:2, reacting at 0 ℃ for 24 hours, centrifugally separating a product, washing and centrifuging with water and ethanol for three times, and drying in vacuum at 60 ℃ overnight to obtain the mesoporous silicon nanoparticles with the cationic polymer layer introduced on the surface.
(4) Dispersing 50mg of mesoporous silicon nanoparticles with the surface introduced into the cationic polymer layer in 5mL of PBS buffer solution (pH7.4, 0.1M) to obtain a solution A; 20mg of a-methoxy- ω -benzaldehyde-PEG (prepared according to step (4) of example 1) having a molecular weight of 2000 was dissolved in 2mL of PBS solution to obtain solution B. And mixing the solution A and the solution B, stirring for reacting for 24 hours, centrifugally separating a product, washing and centrifuging for three times by using water and ethanol, and carrying out vacuum drying at 60 ℃ overnight to obtain the surface-PEGylated adriamycin-loaded mesoporous silicon nanoparticles.
Example 5
Example 5 differs from example 1 mainly in that: the molecular weight of the used a-methoxy-w-benzaldehyde-PEG is different, and the dosage is different.
(1) The pre-synthesized MCM-41 type mesoporous silicon nano particles are dried in vacuum at 60 ℃ for 24 hours, 0.50g of the pre-synthesized MCM-41 type mesoporous silicon nano particles are weighed and added into 50mL of anhydrous toluene, then 0.20mL of gamma-aminopropyltriethoxysilane is added, the mixture is stirred uniformly, the temperature is increased to 110 ℃, and the reflux reaction is carried out for 24 hours under the protection of nitrogen. And after the reaction is finished, carrying out suction filtration, washing toluene twice, washing ethanol once, and carrying out vacuum drying at 60 ℃ for 24h to obtain the amino-containing mesoporous silicon nanoparticles. Dispersing 0.10g of mesoporous silicon nano particles containing amino in acetone, adding 0.20g of succinic anhydride, stirring and reacting for 24 hours at room temperature, and performing centrifugal separation to obtain the mesoporous silicon nano particles containing carboxyl. Dispersing 0.10g of mesoporous silicon nanoparticles containing carboxyl into a phosphate buffer solution (pH7.4, 0.1M), adding 0.10g of cystamine dihydrochloride, 0.05g of EDC and 0.04g of NHS, stirring and reacting for 24h at room temperature, centrifugally separating, and vacuum-drying for 24h at 60 ℃ to obtain the mesoporous silicon nanoparticles containing disulfide bonds and amino groups.
(2) Adding 0.10g of mesoporous silicon nanoparticles containing disulfide bonds and amino groups into 2.0mL of acetone solution containing 10mg of adriamycin, stirring and adsorbing for 24h, performing centrifugal separation, and performing vacuum drying at 60 ℃ for 24h to obtain the adriamycin-loaded mesoporous silicon nanoparticles.
(3) 50mg of mesoporous silicon nano particles loaded with adriamycin are dispersed in 2.0mL of PBS buffer solution (pH7.4, 0.1M), 50mg of cationic monomers (N, N-dimethylacrylamide and N- (3-aminoethyl) methacrylamide) are added under the protection of nitrogen and stirred uniformly, then N, N' -bis (acryloyl) cystamine is added as a cross-linking agent, and the molar ratio of N, N-dimethylacrylamide/N- (3-aminoethyl) methacrylamide/cross-linking agent is adjusted to be 12:9: 1. Then adding 30 mu L of ammonium persulfate (100mg/mL of deoxidized aqueous solution) and 3 mu of L N, N, N ', N' -tetramethylethylenediamine to initiate polymerization reaction, reacting for 1h at room temperature, centrifugally separating the product, washing with water and ethanol for three times, and carrying out vacuum drying at 60 ℃ overnight to obtain the mesoporous silicon nano-particles with the cationic polymer layer introduced on the surface.
(4) Dispersing 50mg of mesoporous silicon nanoparticles with the surface introduced into the cationic polymer layer in 5mL of PBS buffer solution (pH7.4, 0.1M) to obtain a solution A; 5mg of alpha-methoxy-omega-benzaldehyde-PEG with a molecular weight of 500 (prepared according to step (4) of example 1, with the only difference being the mPEG-NH used2Molecular weight of 500) is dissolved in PBS solution, stirred and reacted for 24 hours, the product is centrifugally separated, washed and centrifuged for three times by water and ethanol, and vacuum dried overnight at 60 ℃ to obtain the mesoporous silicon nano particle with the surface being PEG-linked and loaded with the adriamycin. 20mg of a-methoxy- ω -benzaldehyde-PEG with a molecular weight of 2000 was dissolved in 2mL of PBS (pH7.4, 0.1M) to give solution B. And mixing the solution A and the solution B, stirring for reacting for 24 hours, centrifugally separating a product, washing and centrifuging for three times by using water and ethanol, and carrying out vacuum drying at 60 ℃ overnight to obtain the surface-PEGylated adriamycin-loaded mesoporous silicon nanoparticles.
Example 6
Example 6 differs from example 1 mainly in that: the molecular weight of the used a-methoxy-w-benzaldehyde-PEG is different, and the dosage is different.
(1) The pre-synthesized MCM-41 type mesoporous silicon nano particles are dried in vacuum at 60 ℃ for 24 hours, 0.50g of the pre-synthesized MCM-41 type mesoporous silicon nano particles are weighed and added into 50mL of anhydrous toluene, then 0.20mL of gamma-aminopropyltriethoxysilane is added, the mixture is stirred uniformly, the temperature is increased to 110 ℃, and the reflux reaction is carried out for 24 hours under the protection of nitrogen. And after the reaction is finished, carrying out suction filtration, washing toluene twice, washing ethanol once, and carrying out vacuum drying at 60 ℃ for 24h to obtain the amino-containing mesoporous silicon nanoparticles. Dispersing 0.10g of mesoporous silicon nano particles containing amino in acetone, adding 0.20g of succinic anhydride, stirring and reacting for 24 hours at room temperature, and performing centrifugal separation to obtain the mesoporous silicon nano particles containing carboxyl. Dispersing 0.10g of mesoporous silicon nanoparticles containing carboxyl into a phosphate buffer solution (pH7.4, 0.1M), adding 0.10g of cystamine dihydrochloride, 0.05g of EDC and 0.04g of NHS, stirring and reacting for 24h at room temperature, centrifugally separating, and vacuum-drying for 24h at 60 ℃ to obtain the mesoporous silicon nanoparticles containing disulfide bonds and amino groups.
(2) Adding 0.10g of mesoporous silicon nanoparticles containing disulfide bonds and amino groups into 2.0mL of acetone solution containing 10mg of adriamycin, stirring and adsorbing for 24h, performing centrifugal separation, and performing vacuum drying at 60 ℃ for 24h to obtain the adriamycin-loaded mesoporous silicon nanoparticles.
(3) Dispersing 50mg of doxorubicin-loaded mesoporous silicon nanoparticles in 2.0mL of PBS buffer solution (pH7.4, 0.1M), adding 50mg of cationic monomers (N, N-dimethylacrylamide and N- (3-aminoethyl) methacrylamide) under the protection of nitrogen, stirring uniformly, then adding N, N ' -bis (acryloyl) cystamine as a cross-linking agent, adjusting the molar ratio of N, N-dimethylacrylamide/N- (3-aminoethyl) methacrylamide/cross-linking agent to be 12:9:1, then adding 30 muL of ammonium persulfate (100mg/mL of deoxidized aqueous solution) and 3 mu L N, N, N ', N ' -tetramethylethylenediamine to initiate polymerization, reacting for 1h at room temperature, centrifugally separating the product, washing with water and ethanol for three times, carrying out vacuum drying at 60 ℃ overnight, obtaining the mesoporous silicon nano-particles with the surface introduced with the cationic polymer layer.
(4) Dispersing 50mg of mesoporous silicon nanoparticles with the surface introduced into the cationic polymer layer in 5mL of PBS buffer solution (pH7.4, 0.1M) to obtain a solution A; alpha-methoxy-omega-benzaldehyde-PEG (prepared according to example 1 step (4) with the only difference being the mPEG-NH used) having a molecular weight of 100002Molecular weight of (4) 10000)50mg was dissolved in 2mL of PBS solution (pH7.4, 0.1M) to obtain solution B. Mixing solution A and solution B, stirring for reaction for 24 hr, centrifuging, washing with water and ethanol, centrifuging for three times at 60 deg.CAnd (4) drying the mixture overnight to obtain the mesoporous silicon nano particles with the surfaces being PEG-modified and loaded with the adriamycin.
Effects of the embodiment
And (3) analyzing the prepared PEG multiple response nano-carrier:
1. the sample powders of examples 1, 4 and 6 were dispersed in ethanol, and an appropriate amount was dropped onto a copper mesh, and the morphology of the nanoparticles was observed by transmission electron microscopy. FIG. 1 is a transmission electron micrograph of the nanocarriers prepared in examples 1, 4, and 6 (corresponding to FIG. A, B, C, respectively). As can be seen from the electron micrograph, the particle size of the nano-carrier is about 100nm, and certain adhesion is generated between particles, which is a polymer layer on the surface of the particles. Meanwhile, parallel bands of the nanoparticles can be observed on the surfaces of the nanoparticles, which shows that the nano-carrier can effectively maintain the mesoporous structure of the nanoparticles.
2. XRD diffraction of the structure was performed on the nano-carrier particles prepared in example 1. The XRD diffractogram is shown in fig. 2, curve 1 is the unmodified mesoporous silicon nanoparticle, and curve 2 is the mesoporous silicon nanocarrier obtained in example 1. Three well-resolved reflection peaks of 100, 110 and 200 are observed from the figure, and the nanoparticles are proved to have ordered mesoporous structures with characteristic Bragg peaks.
3. The specific surface area and the internal pore structure of the nanocarrier prepared in example 1 were determined by a nitrogen adsorption method. The results are shown in FIG. 3 at relative pressure (P/P)0) When the temperature is 0.2-0.4, the nano-carrier has a typical IV-type isotherm. Adsorption is carried out by multilayer adsorption accompanied by capillary condensation. Second adsorption Process (P/P)00.90) helps to study the internal spatial morphology of the freeze-dried nanoparticles. Calculated by Barrett-Joyner-Halenda (BJH) model, the surface area is about 300m2g-1。
4. Particle size variation as determined by particle size analyzer. Fig. 4 shows that the nano-carrier prepared in example 1 interacts with protein at different times, and it can be seen from the change of particle size that the nano-particle with amino groups on the surface has a strong adsorption effect with protein, and the particle size gradually increases with time, while the particle size of the pegylated nano-particle basically does not change with time and is always kept around 100nm, which indicates that PEG can effectively resist the adsorption of protein, and is beneficial to long-term circulation in blood without being removed by immune system.
5. The cumulative release amount of the drug-loaded nanocarriers prepared in examples 1, 2, and 4 in the environment of simulated blood and tumor cells was determined. As shown in FIG. 5, it can be seen that the cumulative release of the nanocarriers prepared in examples 1 and 4 is less than or equal to about 20% over the 24h assay time range in the simulated blood environment (pH7.4 + 2. mu.M glutathione). The result shows that the carrier can effectively avoid the premature release of the drug in the blood circulation process; in a microenvironment environment simulating tumor cells (pH 5.0+10mM glutathione), the cumulative release within 24h exceeds 60%, and the drug release amount is not less than 40% in the first 6 h. Compared with the nano-carriers prepared in examples 1 and 4, the nano-carrier prepared in example 2 has a cumulative release amount of less than 10% regardless of whether the pH is 5.0 or 7.4, because the PEG shell layer is firmly attached to the surface of the nano-carrier, which prevents the smooth release of the drug from the carrier.
6. The results of the drug release of the nanocarriers prepared in examples 1, 2, and 4 in Hela cells were observed by confocal laser microscopy. Hela cells were seeded on 6-well plates (1X 10)4Cells/well) for 24h (the culture medium is DEME), the nano-carriers prepared in examples 1, 2 and 4 are added into the cell wells, the DOX concentration in the culture dish is maintained to be 2 mug/mL, Hoechst 33342 is added to stain the cell nucleus after continuous culture for 3h, and the fluorescence change of the adriamycin (red is adriamycin fluorescence, and blue is fluorescence emitted after cell nucleus staining) is observed under a laser confocal microscope after 2h and 4h respectively. The results are shown in fig. 6 (panel a is the nanocarrier prepared in example 1, panel B is the nanocarrier prepared in example 2, and panel C is the nanocarrier prepared in example 4): as can be seen from FIGS. 6A and 6C, the red fluorescence in the cells increased with the increase of time at different times (2h and 4h), which indicates that the nanocarriers prepared in examples 1 and 4 can successfully release doxorubicin in Hela cells; in contrast, the nano-carrier prepared in fig. 6B prevents the drug because the PEG shell layer is firmly attached to the surface of the nano-carrierThe red fluorescence was released smoothly from the carrier, and thus the intensity of red fluorescence in the cells was weak and was not substantially changed after 2 hours and 4 hours of culture. Thus, from a comparison of examples 1 and 4 with example 2, a cleavable PEG shell in a tumor can increase both the stability of the vector in blood and the phagocytic efficiency of cells and the cumulative release of intracellular drug.
7. The results of measuring the effect of the nanocarriers prepared in examples 1 and 4 on the apoptosis rate of Hela cells by flow cytometry. Hela cells at 1X 105Perwell Density in 24-well plates, the nanocarriers prepared in examples 1 and 3 were added to each well (2. mu.g/well) at 37 ℃ with 5% CO2Culturing for 72h under the environment, and then quantitatively detecting the Hela cell apoptosis by adopting a flow cytometer. As a result, as shown in fig. 7, fig. 7A is the nano-carrier prepared in example 1, and fig. 7B is the nano-carrier prepared in example 4. As can be seen from the figure, the nanocarriers prepared in examples 1 and 4 can cause very obvious apoptosis after being loaded with adriamycin, the apoptosis rate of example 1 reaches 40.2%, and the apoptosis rate of example 3 also reaches 39.1%, thus proving the effectiveness of the designed nanocarriers.
8. Nuclear magnetic hydrogen spectroscopy was performed on α -methoxy- ω -benzaldehyde-PEG (2000) prepared in example 1. The result is shown in FIG. 8, and the spectrum shows that the proton absorption peak of benzene ring appears at 8.0-8.4ppm, which indicates that benzaldehyde group is successfully introduced at the end of PEG molecular chain.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A preparation method of PEG nanoparticles with tumor specificity and breaking capacity is characterized by comprising the following steps:
(1) dispersing mesoporous silicon nanoparticles in an organic solvent, adding gamma-aminopropyltriethoxysilane, reacting, and purifying to obtain mesoporous silicon nanoparticles with amino groups on the surface;
(2) dispersing the mesoporous silicon nano particles with the amino groups on the surfaces, which are obtained in the step (1), in an organic solvent, adding succinic anhydride, reacting, and performing centrifugal separation to obtain carboxyl-containing mesoporous silicon nano particles;
(3) dispersing the mesoporous silicon nano particles containing carboxyl obtained in the step (2) in a solvent, adding cystamine dihydrochloride and a catalyst, reacting, and carrying out solid-liquid separation to obtain amino-and disulfide bond-modified mesoporous silicon nano particles;
(4) loading the amino-group-and-disulfide-bond-modified mesoporous silicon nanoparticles obtained in the step (3) with an anti-tumor drug, dispersing the loaded anti-tumor drug in a solvent, sequentially adding a cationic monomer and a crosslinking agent, reacting, and purifying to obtain mesoporous silicon nanoparticles with a cationic polymer layer introduced on the surface;
(5) dissolving PEG with end group benzaldehyde in a phosphate solution to obtain a solution A; dispersing the mesoporous silicon nano particles with the cationic polymer layer introduced to the surface prepared in the step (4) in a phosphate solution to obtain a solution B; mixing the solution A and the solution B, stirring for reaction, and purifying to obtain PEG nanoparticles with tumor specificity being fractured;
the cationic monomer in the step (4) is a cationic monomer containing amino, or a mixture of the cationic monomer containing amino and other cationic monomers;
the cationic monomer containing amino is at least one of N- (3-aminoethyl) methacrylamide and N- (3-aminopropyl) methacrylamide;
the other cationic monomer is at least one of N, N-dimethylacrylamide and N, N-diethylacrylamide;
the crosslinking agent described in the step (4) is a compound having a disulfide bond.
2. The method of claim 1 for the preparation of tumor-specific cleavable pegylated nanoparticles characterized in that: the step (4) is as follows: and (3) loading the amino-group-and-disulfide-bond-modified mesoporous silicon nanoparticles obtained in the step (3) with an anti-tumor drug, dispersing the loaded mesoporous silicon nanoparticles into a solvent, sequentially adding a cationic monomer and a crosslinking agent under the protection of nitrogen, adding an initiator to initiate a polymerization reaction, and purifying to obtain the mesoporous silicon nanoparticles with the cationic polymer layer introduced on the surface.
3. The method of preparing tumor-specific cleavable pegylated nanoparticles according to claim 2, characterized in that:
the step of loading the amino-and disulfide-bond-modified mesoporous silicon nanoparticles obtained in the step (3) with the anti-tumor drug is preferably as follows: preparing a drug into a solution, mixing the solution with the amino-and disulfide bond-modified mesoporous silicon nanoparticles obtained in the step (3), stirring to enable the drug to be fully adsorbed, and performing solid-liquid separation to obtain the antitumor drug-loaded mesoporous silicon nanoparticles;
the initiator is ammonium persulfate and N, N, N ', N' -tetramethyl ethylene diamine;
the addition amount of the initiator is 0-0.02 times of the mass of the cationic monomer;
the reaction condition is 0-35 ℃ for 1-24 h.
4. The method for the preparation of tumor-specific cleavable pegylated nanoparticles according to claim 1 or 2, characterized in that: the mesoporous silicon nano particles in the step (1) are MCM-41 type;
the organic solvent in the step (1) is anhydrous toluene;
the organic solvent in the step (2) is at least one of ethanol, tetrahydrofuran, ethyl acetate and acetone;
the solvent in the step (3) and the step (4) is water or phosphate buffer solution;
the catalyst in the step (3) is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;
the medicament in the step (4) is one or at least two of adriamycin, daunorubicin, adriamycin, demethoxydaunorubicin, epirubicin, taxol, vinblastine, vincristine, tamoxifen, formestane, anastrozole, flutamide, 5-fluorouracil, methotrexate, cisplatin, carboplatin, oxaliplatin, carmustine, toremifene and tegafur;
the end group benzaldehyde PEG in the step (5) is polyethylene glycol with the following characteristics: one end group is benzaldehyde, the other end group is methoxyl, and the molecular weight is 500-10000.
5. The method of preparing tumor-specific cleavable pegylated nanoparticles according to claim 4, characterized in that:
the mesoporous silicon nano-particles in the step (1) are prepared by the following steps: taking tetraethoxysilane as a raw material, cetyl trimethyl ammonium bromide as a surfactant and 1,3, 5-trimethylbenzene as a pore-making agent, and stirring for reaction to obtain MCM-41 type mesoporous silicon nanoparticles;
the phosphate buffer solution has a pH value of 7.2-7.4 and a concentration of 0.01-0.1M;
the mass usage of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is calculated according to the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the carboxyl-containing mesoporous silicon nano-particles = 1: 1-5;
the mass usage amount of the N-hydroxysuccinimide is calculated according to the mass ratio of the N-hydroxysuccinimide to the carboxyl-containing mesoporous silicon nano particles of 1: 1-5;
the cationic monomer in the step (4) is a cationic monomer containing amino groups and other cationic monomers according to a molar ratio of 9: 12 to obtain a mixture;
the cross-linking agent in the step (4) is at least one of N, N' -bis (acryloyl) cystamine and cystamine;
the end group benzaldehyde PEG in the step (5) is prepared by the following steps: amino polyethylene glycol monomethyl ether and N-succinimide 4-formyl benzoate are mixed according to the mass ratio of 20: 3, mixing and dissolving in N, N-dimethylformamide according to a ratio, stirring and reacting for 4 hours at the temperature of 4 ℃, precipitating by using excessive cold ether which is 10 times of the N, N-dimethylformamide, and recrystallizing a crude product by using propylene glycol to obtain the PEG with the end group benzaldehyde.
6. The method for the preparation of tumor-specific cleavable pegylated nanoparticles according to claim 1 or 2, characterized in that:
the dosage of the organic solvent in the step (1) is as follows: mesoporous silicon nanoparticles = 50-150 mL: 1g, calculating;
the mass usage amount of the gamma-aminopropyl triethoxysilane in the step (1) is calculated according to the mass ratio of the gamma-aminopropyl triethoxysilane to the mesoporous silicon nano particles = 1-2: 1;
the mass ratio of the consumption of the succinic anhydride in the step (2) to the mesoporous silicon nanoparticles with amino groups on the surface = 0.5-2: 1 is calculated;
the mass usage amount of the cystamine dihydrochloride obtained in the step (3) is calculated according to the mass ratio of the cystamine dihydrochloride to the carboxyl-containing mesoporous silicon nano particles of 1: 0.5-2.5;
the dosage of the cationic monomer in the step (4) is calculated according to the mass ratio = 1-2: 1-2 of the cationic monomer to the amino-and disulfide bond-containing modified mesoporous silicon nanoparticles or the drug-loaded amino-and disulfide bond-containing modified mesoporous silicon nanoparticles;
the dosage of the cross-linking agent in the step (4) is calculated according to the molar ratio of the cross-linking agent to the cationic monomer of 1-2: 21;
the dosage of the end group benzaldehyde-modified PEG in the step (5) is calculated according to the molar ratio of the end group benzaldehyde-modified PEG to amino on the surface of the mesoporous silicon of 1.1-1.5: 1.
7. The method for the preparation of tumor-specific cleavable pegylated nanoparticles according to claim 1 or 2, characterized in that:
the reaction in the step (1) is a heating reflux reaction;
the purification steps in step (1) are as follows: performing suction filtration, washing by using toluene and ethanol, and drying;
the purification steps in the step (4) are as follows: centrifuging, washing with water and ethanol, and drying;
the reaction time in the step (5) is 24 hours;
the purification steps in the step (5) are as follows: centrifuging, washing with water and ethanol, and drying.
8. The method of preparing tumor-specific cleavable pegylated nanoparticles according to claim 7, characterized in that: the conditions of the heating reflux reaction are as follows: reacting for 20-30 h at 100-120 ℃.
9. A tumor-specific cleavable pegylated nanoparticle characterized by: the preparation method of any one of claims 1 to 8.
10. Use of the tumor-specific cleavable pegylated nanoparticles according to claim 9 for the preparation of an antitumor drug.
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