Preparation method of pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers

Abstract

The invention relates to a method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecular imprinting polymers. The invention belongs to the field of functional material preparation and relates to a method for preparing functional monomers prepared from biomaterials and biodegradable molecular imprinting polymer nanoparticles with transferrin and tumor markers as dual-template molecules. The invention solves the problems of poor biocompatibility of molecular imprinting technology, difficulty in degradation, low drug loading and encapsulation rate, few types of functional monomers, unclear degradation mechanism or difficulty in degradation, and insufficient interaction with target molecules. Methods: 1. Preparation of SS-DMONs; 2. Loading of anticancer drugs; 3. Preparation of Cys-DA; 4. Preparation of dual-template molecular imprinting nanopolymers. The invention increases the types of functional monomers of molecular imprinting polymers, has better safety and biocompatibility, higher drug loading capacity and stronger targeting effect.

Description

Technical Field

[0001] The invention belongs to the field of functional material preparation, and in particular relates to a method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecular imprinting polymers. Background Art

[0002] In clinical practice, chemotherapy remains the main anti-cancer approach. However, chemotherapy often has serious toxic side effects and is non-selective. The main challenge is to selectively kill tumor cells without affecting normal tissues to significantly improve drug utilization and treatment efficiency. The development of targeted drug delivery systems (TDDS) triggered by the tumor microenvironment (TME) is a promising approach that can control drug concentrations at safe levels in healthy tissues, reduce toxic side effects, and improve drug utilization and treatment efficacy.

[0003] Nanoparticles have been widely studied due to their excellent properties. Enhanced Permeability and Retention Effect (EPR) endows nanoparticles with passive targeting ability, achieves selective distribution, and reduces toxicity to normal cells. Among all carriers, mesoporous silica nanoparticles (MSNs) is a promising carrier, which has been widely studied due to its unique properties such as large pore volume and specific surface area, adjustable pore structure, high biocompatibility and thermal stability. However, due to the stable -Si-O-Si- structure and saturated state of MSNs, it is difficult to degrade in the biological environment, which limits its application in the field of biology. A large number of reports have shown that disulfide bonds (-SS-) can be responsively broken in a reductive TME. Therefore, directly embedding -SS- into the traditional MSNs skeleton will most likely achieve reductive responsive degradation of nanocarriers.

[0004] Compared with passive targeting through the EPR effect, active targeted therapy can target tumor cells more accurately and carry more anticancer drugs to the TME. Therefore, in order to protect the drugs inside SS-DMONs from premature diffusion and provide active targeting capabilities for SS-DMONs, molecularly imprinted polymers (MIPs) can be prepared on the surface of SS-DMONs using molecularly imprinted technique (MIT). MIT is widely used to produce economical and stable MIPs, which have highly selective binding sites and can be used to recognize various biological substances such as amino acids, glycans, genetic materials, cells, etc. Another important advantage of MIPs is that, in contrast to other nanoparticle therapies, they do not require modification of targeting ligands, thus avoiding any additional grafting steps, making them greener, safer, more practical, economical, and environmentally friendly. In addition, with SS-DMONs as the core, its special dendritic structure can also provide a large specific surface area for the MIPs layer, exposing more degradation sites and imprinting sites on the surface.

[0005] At present, most of the targeted drug-loaded molecular imprinted polymers use a single tumor marker as a template molecule, so that the drug-loaded molecular imprinted polymer can not only target tumor cells but also carry anticancer drugs. However, a single template molecule inevitably causes a waste of the nanoparticle surface. Summary of the invention

[0006] The present invention aims to solve the problems of poor biocompatibility of molecular imprinted polymers, difficulty in degradation, low drug loading and encapsulation rate, few types of functional monomers, unclear degradation mechanism or difficulty in degradation, and insufficient interaction with target molecules; poor biocompatibility of MIPs; difficulty in releasing drugs loaded on nanoparticles or easy premature leakage of drugs, and limited recognition ability. A method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecular imprinted polymers is provided.

[0007] The preparation method of the pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecular imprinting polymers of the present invention is specifically carried out according to the following steps:

[0008] 1. Preparation of SS-DMONs:

[0009] The hexadecyltrimethylammonium chloride and triethanolamine were mixed and dissolved in water, stirred and reacted at 90-100°C for 5-10 minutes, tetraethyl orthosilicate A was added to the reaction solution and the stirring reaction was continued for 0.8-1.2 hours, the silica product was collected by centrifugation, and washed repeatedly with ethanol and water for 3-5 times; the obtained precipitate was condensed and refluxed at 75-80°C for 34-38 hours with ethanol to remove the hexadecyltrimethylammonium chloride, tetraethyl orthosilicate B and BTEPTS were added and the stirring reaction was continued at 90-100°C for 3-5 hours, and SS-D was obtained by centrifugation. MONs; the mass ratio of the hexadecyl trimethyl ammonium chloride to triethanolamine is 1:(0.03-0.05); the mass ratio of the hexadecyl trimethyl ammonium chloride to water is 1 g:(8-12) mL; the mass ratio of the hexadecyl trimethyl ammonium chloride to tetraethyl orthosilicate A is 1:(0.4-0.6); the mass ratio of the hexadecyl trimethyl ammonium chloride to tetraethyl orthosilicate B is 1:(0.4-0.6); the mass ratio of the hexadecyl trimethyl ammonium chloride to BTEPTS is 1:(0.8-1.2);

[0010] 2. Preparation of SS-DMONs / DOX-Ce6:

[0011] DOX and Ce6 were mixed and dissolved in a PBS solution, SS-DMONs were added thereto, and the mixture was subjected to ultrasonic treatment for 0.5-1.5 h, followed by stirring for reaction for 20-26 h, and then centrifuged to obtain SS-DMONs / DOX-Ce6; the pH of the PBS solution was 7.3-7.5; the mass ratio of DOX to Ce6 was 1:(0.8-1.2); the mass ratio of DOX to the volume of the PBS solution was 1 g:(1500-1700) mL; the mass ratio of DOX to SS-DMONs was 1:(1.6-2.4);

[0012] 3. Preparation of Cys-DA:

[0013] DA, L-Cys, dicyclohexylcarbodiimide and dimethylaminopyridine were mixed and dissolved in dichloromethane, and stirred in an ice-water bath for 2-4 hours; the mixed solution was filtered and evaporated under reduced pressure, then dissolved in an ice-cold acetone solution and evaporated under reduced pressure, and then recrystallized with cyclohexane and ethanol aqueous solution in turn to obtain a recrystallized product; the recrystallized product and BBr 3 Mix and dissolve in CH 2 Cl 2The reaction was stirred for 1-3 hours, methanol was added and the reaction was continued to be stirred for 1-3 hours, and the mixture was evaporated under reduced pressure; methanol was added to the mixture, and the solution was evaporated under reduced pressure to remove the formed trimethylborate; the residue was heated to remove other impurities; the solid product was washed with ethyl acetate, and when the thin layer chromatography showed only one chromatographic point, the reaction was terminated to obtain Gys-DA; the mass ratio of the DA to the volume of dichloromethane was 1g:(8-12)mL; the mass ratio of the L-Cys to the volume of dichloromethane was 1g:(6-8)mL; the mass ratio of the dicyclohexylcarbodiimide to the volume of dichloromethane was 1g:(7-9)mL; the mass ratio of the dimethylaminopyridine to the volume of dichloromethane was 1g:(5-7)mL; the mass ratio of the recrystallized product to CH 2 Cl 2 The volume ratio is 1g: (1800~2200) mL; the mass of the recrystallized product is about the same as that of BBr 3 The volume ratio is 1g: (160~200)mL;

[0014] 4. Preparation of dual template imprinted nanopolymers:

[0015] Gys-DA, dopamine, 4-aminophenylboronic acid, TrF, and SA were mixed and dispersed in deionized water, SS-DMONs / DOX-Ce6 was added thereto, and after ultrasonic dissolution, ammonium sulfate was added to initiate polymerization. After reacting for 22-26 hours under electromagnetic stirring, the mixture was washed with PBS for 3-5 times, separated and dried, and eluted with methanol solution to obtain SS-DMONs / DOX-Ce6@MIPs. The mass ratio of Gys-DA to the volume of deionized water was 1 g: (4800-5200) mL; the mass ratio of dopamine to the volume of deionized water was 1g:(4800~5200)mL; the mass ratio of the 4-aminophenylboronic acid to the volume ratio of deionized water is 1g:(4800~5200)mL; the mass ratio of the TrF to the volume ratio of deionized water is 1g:(4800~5200)mL; the mass ratio of the SA to the volume ratio of deionized water is 1g:(4800~5200)mL; the mass ratio of the SS-DMONs / DOX-Ce6 to the volume ratio of deionized water is 1g:(2400~2600)mL; the volume ratio of ammonium sulfate to deionized water is 1:(45~55).

[0016] Advantages of the present invention:

[0017] 1. The present invention uses transferrin and tumor markers together as template molecules, which improves the targeting ability of pH / GSH-responsive drug-loaded molecular imprinted polymers to tumors, allowing the imprinted polymers to carry more anticancer drugs and target the tumor environment.

[0018] 2. The nanoparticles prepared by the present invention combine multiple targeting forces (EPR effect, antigen-antibody interaction, molecular imprinting polymer-template molecule interaction) to jointly target tumor cells, which improves the therapeutic effect on tumor cells.

[0019] 3. The present invention uses biomaterials dopamine and L-cysteine ​​to prepare a degradable functional monomer named Cys-DA, which is pH / GSH sensitive in the slightly acidic environment of tumors and has the ability to specifically break and degrade chemical bonds. It is the first time that it is used as a functional monomer to prepare molecularly imprinted polymers.

[0020] 4. The molecularly imprinted polymer prepared by the present invention has excellent biocompatibility, and the raw materials used are Cys-DA, dopamine, and 4-mercaptophenylboronic acid, all of which are biomaterials or non-toxic and harmless substances.

[0021] 5. The present invention uses multifunctional monomers to prepare molecularly imprinted polymers. Cys-DA and 4-mercaptophenylboronic acid are connected to dopamine, and the self-polymerization of dopamine is used to form a polymer. This avoids the use of additional cross-linking agents and initiators, and improves the biocompatibility of the polymer.

[0022] 6. The use of multifunctional monomers also increases the interaction between molecular imprinting polymers and template molecules, improving the adsorption capacity and imprinting effect.

[0023] 7. This new self-made degradable functional monomer (Cys-DA) can be used to prepare other molecular imprinting polymers or other medical polymer materials that are also pH / GSH sensitive and have good biocompatibility and responsive to degradation.

[0024] 8. The SS-DMONs / DOX-Ce6@MIPs prepared by the present invention are loaded with two drugs at the same time, namely, photosensitizer Ce6 and anticancer drug DOX. Photosensitizer Ce6 can produce toxic reactive oxygen species under 655nm laser irradiation, and together with anticancer drug DOX, it can play a chemical / photodynamic synergistic anticancer role.

[0025] 9. The SS-DMONs / DOX-Ce6@MIPs nanoparticles prepared by the present invention have specific recognition, good biocompatibility and pH / GSH sensitive degradation.

[0026] 10. Molecular imprinting polymer uses disulfide cross-linked dendritic organic mesoporous silica as a carrier to make the imprinted complex have a larger specific surface area, more recognition sites and degradation sites, and better selective adsorption performance.

[0027] 11. The SS-DMONs / DOX-Ce6@MIPs prepared by the present invention can selectively capture TrF in the blood, thereby resisting macrophage phagocytosis and immune stress, prolonging blood circulation time, and improving the internalization of nanomaterials by tumor cells through receptor-mediated endocytosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the ultra-high performance liquid chromatogram of Cys-DA obtained in step 3;

[0029] Figure 2 is the mass spectrum of Cys-DA obtained in step 3;

[0030] Figure 3 Transmission electron microscopy images of SS-DMONs;

[0031] Figure 4 Transmission electron microscopy image of SS-DMONs / DOX-Ce6@MIPs;

[0032] Figure 5 Infrared spectra of SS-DMONs, SS-DMONs / DOX-Ce6 and SS-DMONs / DOX-Ce6@MIPs; 1 represents SS-DMONs, 2 represents SS-DMONs / DOX-Ce6, and 3 represents SS-DMONs / DOX-Ce6@MIPs;

[0033] Figure 6 The bar graphs are the encapsulation efficiency and drug loading of SS-DMONs / DOX-Ce6@MIPs and MIPs for DOX and Ce6; 1 represents MIPs, and 2 represents SS-DMONs / DOX-Ce6@MIPs;

[0034] Figure 7 Comparison diagram of the selective adsorption of SA and its interfering substances by SS-DMONs / DOX-Ce6@NIPs and SS-DMONs / DOX-Ce6@MIPs in monodisperse solution or mixed solution; 1 represents the monodisperse solution group and 2 represents the mixed solution group;

[0035] Figure 8 Thermodynamic adsorption bar graphs of SA and TrF by SS-DMONs / DOX-Ce6@MIPs, MIPs and SS-DMONs / DOX-Ce6@NIPs; 1 represents SS-DMONs / DOX-Ce6@MIPs, 2 represents MIPs, and 3 represents SS-DMONs / DOX-Ce6@NIPs;

[0036] Fig. 9The gel electrophoresis analysis results of TrF captured from mouse blood samples; wherein lane 1 represents the eluate after SS-DMONs / DOX-Ce6@MIPs adsorbed mouse blood, lane 2 represents the supernatant of mouse blood adsorbed by SS-DMONs / DOX-Ce6@MIPs, lane 3 represents 100-fold diluted mouse blood, lane 4 represents 0.5 mg / mL TrF solution, and lane 5 represents the supernatant of mouse blood adsorbed by SS-DMONs / DOX-Ce6@NIPs;

[0037] Fig.10 The drug release of SS-DMONs / DOX-Ce6@MIPs under different environments, where 1 is pH = 5.5, [GSH] = 10mmol / L, 2 is pH = 7.4, [GSH] = 10mmol / L, 3 is pH = 5.5, [GSH] = 5mmol / L, and 4 is pH = 7.4, [GSH] = 5mmol / L;

[0038] Fig.11 The biodegradation transmission electron microscopy image of SS-DMONs / DOX-Ce6@MIPs nanoparticles in a microenvironment of pH=5.5 and [GSH]=10mmol / L for 5 hours;

[0039] Fig.12 The biodegradation transmission electron microscopy image of SS-DMONs / DOX-Ce6@MIPs nanoparticles in the microenvironment of pH=5.5 and [GSH]=10mmol / L for 20h.

[0040] Fig.13 Transmission electron microscopy image of biodegradation of SS-DMONs / DOX-Ce6@MIPs nanoparticles in a microenvironment of pH=5.5 and [GSH]=10mmol / L for 50h;

[0041] Fig.14 The biodegradation transmission electron microscopy image of SS-DMONs / DOX-Ce6@MIPs nanoparticles in a microenvironment of pH=5.5 and [GSH]=10mmol / L for 150h.

[0042] Fig.15CLSM images of active oxygen determination in MCF-7 cells after DCFH staining; 1 represents SS-DMONs / DOX-Ce6@MIPs + 655nm laser irradiation, 2 represents SS-DMONs / DOX-Ce6@MIPs, 3 represents SS-DMONs / DOX-Ce6@NIPs+ 655nm laser irradiation, 4 represents SS-DMONs / DOX-Ce6@NIPs, 5 represents Ce6 + 655nm laser irradiation, 6 represents SS-DMONs / DOX-Ce6 + 655nm laser irradiation, 7 represents SS-DMONs / DOX-Ce6, and 8 represents SS-DMONs +655nm laser irradiation;

[0043] Fig.16 SS-DMONs / DOX-Ce6@MIPs / TrF in MCF-7 cells FITC The fluorescence value of

[0044] Fig.17 The growth curve of tumor size in experimental mice; 1 represents the PBS group, 2 represents the SS-DMONs / DOX-Ce6 group, and 3 represents the SS-DMONs / DOX-Ce6@MIPs group;

[0045] Fig.18 The H&E staining comparison of the main organs of the tumor sections of each treatment group on the 14th day; 1 represents the PBS group, 2 represents the SS-DMONs / DOX-Ce6 group, and 3 represents the SS-DMONs / DOX-Ce6@MIPs group. DETAILED DESCRIPTION

[0046] Specific implementation method 1: In this implementation method, a preparation method of pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecular imprinting polymers is specifically carried out according to the following steps:

[0047] 1. Preparation of SS-DMONs:

[0048] The hexadecyltrimethylammonium chloride and triethanolamine were mixed and dissolved in water, stirred and reacted at 90-100°C for 5-10 minutes, tetraethyl orthosilicate A was added to the reaction solution and the stirring reaction was continued for 0.8-1.2 hours, the silica product was collected by centrifugation, and washed repeatedly with ethanol and water for 3-5 times; the obtained precipitate was condensed and refluxed at 75-80°C for 34-38 hours with ethanol to remove the hexadecyltrimethylammonium chloride, tetraethyl orthosilicate B and BTEPTS were added and the stirring reaction was continued at 90-100°C for 3-5 hours, and SS-D was obtained by centrifugation. MONs; the mass ratio of the hexadecyl trimethyl ammonium chloride to triethanolamine is 1:(0.03-0.05); the mass ratio of the hexadecyl trimethyl ammonium chloride to water is 1 g:(8-12) mL; the mass ratio of the hexadecyl trimethyl ammonium chloride to tetraethyl orthosilicate A is 1:(0.4-0.6); the mass ratio of the hexadecyl trimethyl ammonium chloride to tetraethyl orthosilicate B is 1:(0.4-0.6); the mass ratio of the hexadecyl trimethyl ammonium chloride to BTEPTS is 1:(0.8-1.2);

[0049] 2. Preparation of SS-DMONs / DOX-Ce6:

[0050] DOX and Ce6 were mixed and dissolved in a PBS solution, SS-DMONs were added thereto, and the mixture was subjected to ultrasonic treatment for 0.5-1.5 h, followed by stirring for reaction for 20-26 h, and then centrifuged to obtain SS-DMONs / DOX-Ce6; the pH of the PBS solution was 7.3-7.5; the mass ratio of DOX to Ce6 was 1:(0.8-1.2); the mass ratio of DOX to the volume of the PBS solution was 1 g:(1500-1700) mL; the mass ratio of DOX to SS-DMONs was 1:(1.6-2.4);

[0051] 3. Preparation of Cys-DA:

[0052] DA, L-Cys, dicyclohexylcarbodiimide and dimethylaminopyridine were mixed and dissolved in dichloromethane, and stirred in an ice-water bath for 2-4 hours; the mixed solution was filtered and evaporated under reduced pressure, then dissolved in an ice-cold acetone solution and evaporated under reduced pressure, and then recrystallized with cyclohexane and ethanol aqueous solution in turn to obtain a recrystallized product; the recrystallized product and BBr 3 Mix and dissolve in CH 2 Cl 2The reaction was stirred for 1-3 hours, methanol was added and the reaction was continued to be stirred for 1-3 hours, and the mixture was evaporated under reduced pressure; methanol was added to the mixture, and the solution was evaporated under reduced pressure to remove the formed trimethylborate; the residue was heated to remove other impurities; the solid product was washed with ethyl acetate, and when the thin layer chromatography showed only one chromatographic point, the reaction was terminated to obtain Gys-DA; the mass ratio of the DA to the volume of dichloromethane was 1g:(8-12)mL; the mass ratio of the L-Cys to the volume of dichloromethane was 1g:(6-8)mL; the mass ratio of the dicyclohexylcarbodiimide to the volume of dichloromethane was 1g:(7-9)mL; the mass ratio of the dimethylaminopyridine to the volume of dichloromethane was 1g:(5-7)mL; the mass ratio of the recrystallized product to CH 2 Cl 2 The volume ratio is 1g: (1800~2200) mL; the mass of the recrystallized product is about the same as that of BBr 3 The volume ratio is 1g: (160~200)mL;

[0053] 4. Preparation of dual template imprinted nanopolymers:

[0054] Gys-DA, dopamine, 4-aminophenylboronic acid, TrF, and SA were mixed and dispersed in deionized water, SS-DMONs / DOX-Ce6 was added thereto, and after ultrasonic dissolution, ammonium sulfate was added to initiate polymerization. After reacting for 22-26 hours under electromagnetic stirring, the mixture was washed with PBS for 3-5 times, separated and dried, and eluted with methanol solution to obtain SS-DMONs / DOX-Ce6@MIPs. The mass ratio of Gys-DA to the volume of deionized water was 1 g: (4800-5200) mL; the mass ratio of dopamine to the volume of deionized water was 1g:(4800~5200)mL; the mass ratio of the 4-aminophenylboronic acid to the volume ratio of deionized water is 1g:(4800~5200)mL; the mass ratio of the TrF to the volume ratio of deionized water is 1g:(4800~5200)mL; the mass ratio of the SA to the volume ratio of deionized water is 1g:(4800~5200)mL; the mass ratio of the SS-DMONs / DOX-Ce6 to the volume ratio of deionized water is 1g:(2400~2600)mL; the volume ratio of ammonium sulfate to deionized water is 1:(45~55).

[0055] The other impurities described in this embodiment are mainly methyl bromide and hydrobromic acid.

[0056] In this embodiment, two biomaterials, L-cysteine ​​and dopamine, are polymerized to prepare the functional monomer of molecular imprinting polymer, and the molecular imprinting polymer is wrapped on the surface of disulfide bridged dendritic mesoporousorganosilicas; the prepared disulfide bridged dendritic mesoporousorganosilicas nanoparticles (SS-DMONs) are used as a reservoir for drug molecules, have a large specific surface area and high drug loading efficiency, and have good biocompatibility and easy surface functionalization. Compared with traditional MSNs, SS-DMONs will have greater application potential in the field of biology.

[0057] This embodiment of the present invention proposes for the first time a new functional monomer of molecular imprinting polymer prepared from biomaterials, and uses it as the main functional monomer, and uses 4-mercaptophenylboronic acid as the auxiliary functional monomer. At the same time, for the first time, transferrin and tumor marker (SA) are used as dual template molecules to prepare a molecular imprinting polymer with clear interaction between the functional monomer and the template molecule and a clear degradation mechanism. The use of dual template molecules can not only improve the targeting ability of molecular imprinting polymers for tumor cells, but also make full use of non-imprinting sites on the surface of the polymer. It is coated on the surface of drug-loaded SS-DMONs, which not only greatly improves the biocompatibility of the drug carrier, the circulation time in the body, the targeting effect, and the aggregation effect of the carrier in the tumor area, but also can avoid the premature leakage of drugs in the carrier to harm normal tissue cells. In addition, two drugs are loaded in SS-DMONs, namely, anticancer drug DOX and photosensitizer Ce6. Both will be internalized into tumor cells through TrF and TfR-mediated endocytosis on the surface of tumor cells, and then released into the cancer cell cytoplasm as the MIPs layer is degraded in the TME. Under laser irradiation, the embedded Ce6 will produce a large amount of toxic reactive oxygen species, which will achieve chemical / photodynamic synergistic therapy with DOX.

[0058] In the present embodiment, DOX represents doxorubicin, BTEPTS represents bis[3-(triethoxysilyl)propyl]tetrasulfide, Ce6 represents photosensitizer chlorin e6, DA represents dopamine, L-Cys represents L-cysteine, TrF represents transferrin, TfR represents transferrin receptor, and SA represents sialic acid.

[0059] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that: the mass ratio of hexadecyl trimethyl ammonium chloride to triethanolamine in step 1 is 1:0.04; the mass ratio of the hexadecyl trimethyl ammonium chloride to water is 1g:10mL; the mass ratio of the hexadecyl trimethyl ammonium chloride to tetraethyl orthosilicate A is 1:0.5; the mass ratio of the hexadecyl trimethyl ammonium chloride to tetraethyl orthosilicate B is 1:0.5; the mass ratio of the hexadecyl trimethyl ammonium chloride to BTEPTS is 1:1. The rest is the same as specific embodiment 1.

[0060] Specific implementation method 3: This implementation method is different from specific implementation methods 1 to 2 in that: the mass ratio of DOX to Ce6 in step 2 is 1:1; the mass ratio of DOX to PBS solution is 1g:1600mL; the mass ratio of DOX to SS-DMONs is 1:2. Others are the same as specific implementation methods 1 to 2.

[0061] Specific embodiment 4: This embodiment is different from specific embodiments 1 to 3 in that: in step 3, the mass ratio of DA to dichloromethane is 0.92 g:10 mL; the mass ratio of L-Cys to dichloromethane is 1.33 g:10 mL; the mass ratio of dicyclohexylcarbodiimide to dichloromethane is 1.25 g:10 mL; the mass ratio of dimethylaminopyridine to dichloromethane is 1.5 g:10 mL. Others are the same as specific embodiments 1 to 3.

[0062] Specific embodiment 5: This embodiment is different from the specific embodiments 1 to 4 in that the mass of the recrystallized product in step 3 is the same as that of CH 2 Cl 2 The volume ratio is 1g:2000mL; the mass of the recrystallized product is the same as that of BBr 3 The volume ratio is 1g:180mL. Others are the same as those in the first to fourth embodiments.

[0063] Specific implementation method 6: This implementation method is different from the specific implementation methods 1 to 5 in that: the CH 2 Cl 2 With BBr 3 The volume ratio is 1: 1. The rest is the same as that of the first to fifth embodiments.

[0064] Specific embodiment 7: This embodiment differs from specific embodiments 1 to 6 in that the volume ratio of ethanol to water in the ethanol aqueous solution in step 3 is 1:5. The rest is the same as specific embodiments 1 to 6.

[0065] Specific embodiment eight: This embodiment differs from specific embodiments one to six in that: in step three, the reaction is monitored by thin-layer chromatography of an eluent; the eluent is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol in the mixture is 4:1. The rest is the same as specific embodiments one to six.

[0066] Specific embodiment 9: This embodiment is different from specific embodiments 1 to 6 in that: in step 4, the mass ratio of Gys-DA to deionized water is 1g:5000mL; the mass ratio of dopamine to deionized water is 1g:5000mL; the mass ratio of 4-aminophenylboronic acid to deionized water is 1g:5000mL; the mass ratio of TrF to deionized water is 1g:5000mL; the mass ratio of SA to deionized water is 1g:5000mL. Others are the same as specific embodiments 1 to 6.

[0067] Specific embodiment 10: This embodiment differs from specific embodiments 1 to 6 in that: in step 4, the mass ratio of SS-DMONs / DOX-Ce6 to deionized water is 1 g:2500 mL; the concentration of ammonium sulfate is 7 mg / mL; the volume ratio of ammonium sulfate to deionized water is 1:50. Others are the same as specific embodiments 1 to 6.

[0068] The beneficial effects of the present invention are verified by the following examples:

[0069] Example 1: A method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecular imprinting polymers is specifically carried out according to the following steps:

[0070] 1. Preparation of SS-DMONs:

[0071] 2 g of hexadecyltrimethylammonium chloride and 0.08 g of triethanolamine were mixed and dissolved in 20 mL of water, stirred and reacted at 95°C for 5 min, 1 g of tetraethyl orthosilicate A was added to the reaction solution and continued to stir and react for 1 h, the silica product was collected by centrifugation, and washed repeatedly with ethanol and water for 3 times; the obtained precipitate was condensed and refluxed at 78°C for 36 h with ethanol to remove the unreacted hexadecyltrimethylammonium chloride, 1 g of tetraethyl orthosilicate B and 2 g of BTEPTS were added and continued to stir and react at 95°C for 4 h, and SS-DMONs were obtained by centrifugation;

[0072] 2. Preparation of SS-DMONs / DOX-Ce6:

[0073] 25 mg DOX and 25 mg Ce6 were mixed and dissolved in 40 mL PBS solution with a pH of 7.4, 50 mg SS-DMONs was added thereto, and after ultrasonic treatment for 1 h, the mixture was stirred and reacted for 24 h, and SS-DMONs / DOX-Ce6 was obtained by centrifugation;

[0074] 3. Preparation of Cys-DA:

[0075] 0.92 g DA, 1.33 g L-Cys, 1.25 g dicyclohexylcarbodiimide and 1.5 g dimethylaminopyridine were mixed and dissolved in 10 mL dichloromethane, and stirred in an ice-water bath for 3 h. The mixed solution was filtered and evaporated under reduced pressure, then dissolved in an ice-cold acetone solution and evaporated under reduced pressure, and then recrystallized using cyclohexane and ethanol aqueous solution in turn to obtain a recrystallized product. 0.01 g of the recrystallized product and 1.8 mL BBr 3 Mix and dissolve in 20 mL CH 2 Cl 2 The mixture was stirred for 2 hours, 20 mL of methanol was added and the stirring reaction was continued for 2 hours, and the mixture was evaporated under reduced pressure; excess methanol was added to the mixture, and the solution was evaporated under reduced pressure to remove the formed trimethylborate; the residue was heated to remove other impurities; the solid product was washed with 10 mL of ethyl acetate, and when the thin layer chromatography showed only one chromatographic point, the reaction was terminated to obtain Gys-DA; the volume ratio of ethanol to water in the ethanol-water solution was 1:5; the reaction was monitored by thin layer chromatography of the eluent during the reaction; the eluent was a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol in the mixture was 4:1;

[0076] 4. Preparation of dual template imprinted nanopolymers:

[0077] 10 mg Gys-DA, 10 mg dopamine, 10 mg 4-aminophenylboronic acid, 10 mg TrF, and 10 mg SA were mixed and dispersed in 50 mL deionized water, and 20 mg SS-DMONs / DOX-Ce6 was added thereto. After ultrasonic dissolution, 1 mL of 7 mg / mL ammonium sulfate was added to initiate polymerization. After reacting for 24 h under electromagnetic stirring, the mixture was washed three times with PBS, separated and dried, and eluted with methanol solution to obtain SS-DMONs / DOX-Ce6@MIPs.

[0078] For comparison, the polymerization method of non-imprinted polymers (SS-DMONs / DOX-Ce6@NIPs) was the same as that of SS-DMONs / DOX-Ce6@MIPs, but without the addition of SA and TrF. The preparation and treatment of MIPs were the same as those of SS-DMONs / DOX-Ce6@MIPs, but without the addition of SS-DMONs.

[0079] 5. Calculation of drug loading capacity

[0080] The drug encapsulation efficiency (EE) and drug loading capacity (LC) of SS-DMONs / DOX-Ce6@MIPs and MIPs were calculated according to formula (1) (2).

[0081] (1)

[0082] (2)

[0083] Where W is the mass, and the concentrations of DOX and Ce6 were determined by HPLC.

[0084] 6. Selective adsorption

[0085] 5 mg SS-DMONs / DOX-Ce6@NIPs were added into 10 mL bovine hemoglobin, 10 mL CA125, 10 mL IgA, 10 mL L-tryptophan, 10 mL N-acetyl-D-galactosamine, 10 mL N-acetyl-D-glucosamine, 10 mL folic acid, and 10 mL Ca2+, respectively. 2+ Solution, 10mL Fe 3+ Solution, 10mL Cl - The monodisperse solutions of SS-DMONs / DOX-Ce6@NIPs were prepared in several groups; 5 mg of SS-DMONs / DOX-Ce6@MIPs were added to 10 mL of bovine hemoglobin, 10 mL of CA125, 10 mL of IgA, 10 mL of L-tryptophan, 10 mL of N-acetyl-D-galactosamine, 10 mL of N-acetyl-D-glucosamine, 10 mL of folic acid, and 10 mL of Ca2+, with a concentration of 50 mg / L. 2+ Solution, 10mLFe 3+ Solution, 10mLCl - In the solution, several groups of SS-DMONs / DOX-Ce6@MIPs monodisperse solutions were prepared;

[0086] Then, 50 mg / L of bovine hemoglobin, CA125, IgA, L-tryptophan, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, folic acid, Ca 2+ Solution, Fe 3+ Solution, Cl -The solution was configured into a mixed solution; 5 mg SS-DMONs / DOX-Ce6@MIPs and 5 mg SS-DMONs / DOX-Ce6@NIPs were added to the mixed solutions of two groups of substances with more than 10 mL, respectively; ultrasonic treatment was performed for 5 min, and the mixture was allowed to stand for 25 min. The adsorption amount of each substance by SS-DMONs / DOX-Ce6@MIPs and SS-DMONs / DOX-Ce6@NIPs was determined by UPLC and ICP-OES.

[0087] VII. Analysis of Actual Selective Capture Capability

[0088] 50 mg of SS-DMONs / DOX-Ce6@MIPs and 50 mg of SS-DMONs / DOX-Ce6@NIPs were dispersed in 10 mL of mouse blood sample diluted 100 times with Tris-HCl buffer (pH=7.0, 10 mmol / L). The two mixtures were centrifuged and the proteins adsorbed on the polymers were eluted with 2% acetic acid-2% SDS (V / V) solution. Finally, the initial solution, adsorption solution and elution solution were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detection using 12% polyacrylamide separation gel (Bio-Rad).

[0089] 8. Calculation of adsorption amount

[0090] 10mg SS-DMONs / DOX-Ce6@MIPs were added to 10mL SA and 10mL TrF (1~100mg / L) solutions, respectively, and soaked for 60 min; 10mg SS-DMONs / DOX-Ce6@NIPs were added to 10mL SA and 10mL TrF (1~100mg / L) solutions, respectively, and soaked for 60min; then the static adsorption capacity of SA or TrF was determined by UV-Vis method. The adsorption amount can be calculated by the following formula (3), and the imprinting factor can be calculated by (4).

[0091] (3)

[0092] In the formula, C 0 (mg / mL) is the initial concentration of SA or TrF, C eq (mg / mL) is the concentration of SA or TrF after adsorption by SS-DMONs / DOX-Ce6@MIPs or SS-DMONs / DOX-Ce6@NIPs, V (mL) is the volume of the added solution, and M (mg) is the mass of SS-DMONs / DOX-Ce6@MIPs or SS-DMONs / DOX-Ce6@NIPs.

[0093] (4)

[0094] In the formula, Q MIPs and Q NIPs (mg / g) are the maximum adsorption amounts of SA or TrF by SS-DMONs / DOX-Ce6@MIPs and SS-DMONs / DOX-Ce6@NIPs, respectively.

[0095] IX. Drug Release Experiment

[0096] 5 mg of different nanoparticles were dispersed in 40 mL PBS buffer with pH = 7.4, 5.5 and [GSH] = 5, 10 mM and then placed in a thermostatic oscillator at 37 °C and 120 rpm. The samples were centrifuged at specified time intervals for 10 min and the drug concentration released from the materials was determined by liquid chromatography. The drug release rate was calculated according to (7).

[0097] (7)

[0098] Where M t is the drug release amount at time t, M ∞ For drug loading.

[0099] 10. Degradation Experiment

[0100] SS-DMONs / DOX-Ce6@MIPs were immersed in PBS solutions with different pH values ​​(7.4 or 5.5) and different concentrations of GSH (5 or 10 mM). Then, the solutions were placed in a magnetic stirring water bath at 37 °C. The samples obtained at different times were centrifuged.

[0101] XI. Synergistic Anti-tumor Assay

[0102] The ROS generation of SS-DMONs / DOX-Ce6@MIPs, SS-DMONs / DOX-Ce6@NIPs, SS-DMONs / DOX-Ce6, and Ce6 was characterized using 2',7'-dichlorodihydrofluorescein diethyl ester (DCFH-DA). Briefly, 2 mL of 20 μg / mL SS-DMONs / DOX-Ce6@NIPs, SS-DMONs / DOX-Ce6, SS-DMONs / DOX-Ce6@MIPs, and Ce6 were mixed with 10 μL of DCFH-DA and illuminated at 655 nm (100 mW / cm 2 ) CLSM characterization was performed after laser irradiation for 10 minutes. Non-fluorescent DCFH reacted with ROS to generate fluorescent DCFH-DA.

[0103] 12. Anti-tumor therapy in mice

[0104] Female Balb / c nude mice (5-6 weeks old, 20-25 g) were used to establish the tumor model. 4T1 cells were propagated to 1×10 6 After reaching the density, the tumor model was prepared. 4T1 cells were collected by centrifugation and dispersed into PBS solution, and injected subcutaneously into the right front flank of healthy nude mice. When the average tumor size reached 80-100 mm 3 At about 14 days, the tumor-bearing mouse model was successfully established. The 4T1 tumor-bearing mice were divided into PBS, SS-DMONs / DOX-Ce6, SS-DMONs / DOX-Ce6@NIPs and SS-DMONs / DOX-Ce6@MIPs groups. Eight hours after drug injection, the tumor sites of 4T1 tumor-bearing mice were irradiated with 655nm laser for 10 minutes. Irradiation was performed every other day. The tumor size was recorded every other day. After 14 days of feeding, the main organs were stained with hematoxylin and eosin (H&E).

[0105] Analysis, characterization and application of dual-templated molecularly imprinted polymer nanoparticles with biodegradable functional monomers

[0106] 1. LC-MS characterization of Cys-DA:

[0107] The present invention prepares Cys-DA and uses UPLC-MS for qualitative analysis. When the liquid chromatography acquisition time is 9.66 min ( Figure 1 ) at the mass spectrum ( Figure 2 The mass-to-charge ratios of the two peaks in the graph are 256 and 279, respectively, and the difference between the two is 23. Therefore, it can be determined that 256 is the molecular ion peak of the substance ([M] + ), 279 is the [M+Na] + Peak. Molecular formula matching was performed on the peak with a mass-to-charge ratio of 256 (the element composition is C, H, O, N, S). Taking into account factors such as the exact mass deviation, isotope distribution, and unsaturation, it was inferred that the most likely molecular formula of the substance is C 11 H 16 N 2 O 3 S, compared with the theoretical molecular weight and isotope distribution, the results were in good agreement.

[0108] 2. Characterization of the morphology of SS-DMONs and SS-DMONs / DOX-Ce6@MIPs using transmission electron microscopy

[0109] The surface morphology of the synthesized SS-DMONs and SS-DMONs / DOX-Ce6@MIPs was characterized by transmission electron microscopy. Figure 3, 4, it can be seen that SS-DMONs have a special dendritic spherical structure. A uniform film with a thickness of about 10 nm is formed on the surface of SS-DMONs, indicating that MIPs are successfully coated on the surface of SS-DMONs. This thin and uniform film is conducive to the rapid mass transfer and recognition of SA and TrF by SS-DMONs / DOX-Ce6@MIPs in vivo. In addition, the diameter of SS-DMONs prepared by the present invention is about 100 nm, and the diameter of SS-DMONs / DOX-Ce6@MIPs is about 113 nm. SS-DMONs / DOX-Ce6@MIPs uses SS-DMONs with a special dendritic spherical structure as a matrix, which is easy to load drugs and provides a large specific surface area for the MIPs layer, so that more imprinting sites and a large number of protonated degradation sites in the MIPs layer are exposed on the surface.

[0110] 3. FTIR characterization of SS-DMONs, SS-DMONs / DOX-Ce6 and SS-DMONs / DOX-Ce6@MIPs

[0111] SS-DMONs, SS-DMONs / DOX-Ce6, and SS-DMONs / DOX-Ce6@MIPs nanomaterials were characterized by infrared spectroscopy ( Figure 5 ). In curve a, 467cm -1 The absorption peak at 1093cm is the infrared absorption peak of SS. -1 The absorption peak at 2926cm is the symmetrical stretching vibration peak of Si-O. -1 The absorption peaks at 1664cm are CH stretching vibration peaks, which come from BTEPTS or tetraethyl orthosilicate used to prepare SS-DMONs. Compared with curve a, curve b still has SS, Si-O and CC stretching vibration peaks. At the same time, the absorption peak at 1664cm -1 and 1529cm -1 The benzene ring skeleton vibration peaks at 1319 cm-1 are derived from the loaded DOX and Ce6. Compared with curves a and b, curve c has an additional peak at 1319 cm-1. -1 The stretching vibration peak of the CS bond in the functional monomer Cys-DA is 1356 cm -1 The stretching vibration peak of BO bond in the functional monomer 4-mercaptophenylboronic acid is shown at 1400 nm, indicating that an imprinting layer containing Cys-DA and 4-mercaptophenylboronic acid as functional monomers was prepared on the surface of SS-DMONs.

[0112] 4. Encapsulation efficiency and drug loading of MIPs and SS-DMONs / DOX-Ce6@MIPs

[0113] like Figure 6As shown in the figure, the drug loading capacity (LC) and encapsulation effect (EE) of MIPs and SS-DMONs / DOX-Ce6@MIPs for DOX and Ce6 were further investigated. The MIPs prepared by the traditional method had poor loading capacity for Ce6 and DOX. Due to the special structure of SS-DMONs and the π-π stacking and electrostatic force between SS-DMONs and drugs, the LC and EE values ​​of SS-DMONs / DOX-Ce6@MIPs were higher than those of MIPs. SS-DMONs / DOX-Ce6@MIPs had high LC (DOX: 47.63%±0.95%, Ce6: 47.25%±0.95%) and EE (DOX: 90.96%±1.82%, Ce6: 89.58%±1.79%).

[0114] 5. Selective adsorption of SA and its interfering substances by SS-DMONs / DOX-Ce6@MIPs and SS-DMONs / DOX-Ce6@NIPs

[0115] Depend on Figure 7 It can be seen that there are interfering substances and structural analogs (bovine hemoglobin, CA125, IgA, L-serine, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, folic acid, Na + , Fe 3+ ,Cl - ), SS-DMONs / DOX-Ce6@MIPs has the largest adsorption capacity for SA and TrF. This is because after eluting SA and TrF from SS-DMONs / DOX-Ce6@MIPs, imprinting sites that bind to SA and TrF through covalent and non-covalent forces such as borate bonds, disulfide bonds, and hydrogen bonds are left in its cavities. However, SS-DMONs / DOX-Ce6@NIPs do not have forces and imprinting cavities that match SA and TrF in structure, size, and functional groups, so the adsorption capacity of template molecules is low. The above results verify that SS-DMONs / DOX-Ce6@MIPs can specifically bind to SA and TrF through specific chemical bonds in complex systems without being affected by interfering substances.

[0116] 6. Thermodynamic adsorption of SS-DMONs / DOX-Ce6@MIPs and SS-DMONs / DOX-Ce6@NIPs

[0117] Depend on Figure 8It can be seen that with the increase of the initial concentration of SA and TrF, the adsorption amount of SA and TrF by SS-DMONs / DOX-Ce6@MIPs increased. The maximum adsorption amount of SA by SS-DMONs / DOX-Ce6@MIPs was 29.16 mg / g, and the maximum adsorption amount of TrF by SS-DMONs / DOX-Ce6@MIPs was 87.67 mg / g, which was much larger than that of MIPs and SS-DMONs / DOX-Ce6@NIPs prepared by traditional methods. This can be attributed to the fact that SS-DMONs / DOX-Ce6@MIPs contains a large number of three-dimensional imprinted cavities that match SA and TrF in shape, size and functional groups. This cavity contains imprinted sites that bind to SA and TrF through covalent and non-covalent forces such as boronate bonds, disulfide bonds, and hydrogen bonds, which increases the binding stability between the polymer and the template molecule. On the other hand, it can be attributed to the use of SS-DMONs as the matrix. The unique dendritic mesoporous structure of SS-DMONs provides a larger specific surface area for the MIPs layer, which can expose more imprinted cavities and increase the adsorption possibility between the polymer and the template molecules.

[0118] 7. Analysis of the actual selective capture ability of SS-DMONs / DOX-Ce6@MIPs and SS-DMONs / DOX-Ce6@NIPs

[0119] Using 100-fold diluted mouse blood as the real sample, the actual selective capture ability of SS-DMONs / DOX-Ce6@MIPs for TrF was determined ( Fig. 9 ). Lane 3 and lane 4 are 100-fold diluted mouse blood samples and TrF (0.5 mg / mL) samples, respectively. After adsorption by SS-DMONs / DOX-Ce6@MIPs, TrF was almost completely removed from lane 2, while only TrF was present in lane 1, indicating that SS-DMONs / DOX-Ce6@MIPs has a high specific capture ability for TrF in mouse blood. Lane 5 is similar to lane 3, indicating that SS-DMONs / DOX-Ce6@NIPs has non-specific recognition of TrF in mouse blood. The selective capture of TrF by SS-DMONs / DOX-Ce6@MIPs can be attributed to the MIPs layer on its surface. Therefore, the SS-DMONs / DOX-Ce6@MIPs prepared by the present invention is expected to selectively capture TrF in the blood circulation of organisms without being affected by interfering substances.

[0120] 8. Drug release

[0121] The ideal drug delivery system is that nanoparticles deliver drugs to specific locations, slowly release a certain dose of drugs, and maintain an appropriate drug concentration for a certain period of time to produce a therapeutic effect. On the one hand, drug release that is too fast will damage normal cells, and on the other hand, drug release that is too slow will not achieve a therapeutic effect. Based on this, the present invention designed a variety of simulation environments to observe the drug release of SS-DMONs / DOX-Ce6@MIPs. Fig.10 It can be seen that as SS-DMONs / DOX-Ce6@MIPs degrade in different simulated environments, their drug release amounts continue to increase. SS-DMONs / DOX-Ce6@MIPs has a sustained release effect on drugs, and as the H + As the concentration of GSH increases, the amount of drug released increases, and the amount of drug in the environment gradually increases. This is because the outer MIPs membrane protects the internal drug, and the -SS- and -CO-NH- in the MIPs layer can be specifically broken and degraded under the slightly acidic / highly reducing conditions of TME. As the outer MIPs protective membrane gradually breaks, the inner core SS-DMONs / DOX-Ce6 is exposed, and the H in the environment + The π-π stacking and electrostatic interaction between SS-DMONs and DOX / Ce6 can be destroyed. The above results show that the SS-DMONs / DOX-Ce6@MIPs prepared by the present invention has a good sustained release effect on drugs, avoiding the premature leakage of DOX and Ce6, thereby reducing the harm to normal cells, and can maintain an appropriate drug concentration in tumor cells to produce a therapeutic effect.

[0122] 9. Characterization of the degradation morphology of SS-DMONs / DOX-Ce6@MIPs using transmission electron microscopy

[0123] Through transmission electron microscopy observation, as SS-DMONs / DOX-Ce6@MIPs and H + and GSH (pH = 5.5, [GSH] = 10mmol / L), Fig.11 In the figure, the outline of SS-DMONs / DOX-Ce6@MIPs became blurred, and it is speculated that the outer polymer film and the dendritic structure of SS-DMONs were destroyed first. Fig.12 In the case of SS-DMONs, after 20 h, due to the gradual opening of -SS- and -Si-O-, only a small number of -Si-O- bonds remained in the corroded hollow structure. Fig.13 , 14, as the degradation reaction proceeds, SS-DMONs / DOX-Ce6@MIPs gradually turns into small fragments. Therefore, it is shown that the imprinted polymer nanoparticles prepared by using biomaterials in the present invention have good biodegradability.

[0124] 10. Synergistic anti-tumor assay

[0125] DCFH was selected as the ROS indicator for fluorescence imaging of MCF-7 cells. Fig.15 As shown in the figure, compared with other experimental groups, the MCF-7 cells incubated with SS-DMONs / DOX-Ce6@MIPs after TrF adsorption produced the strongest green fluorescence after 655nm laser irradiation. This shows that compared with SS-DMONs / DOX-Ce6@NIPs, SS-DMONs / DOX-Ce6, and Ce6, SS-DMONs / DOX-Ce6@MIPs can carry the most DOX and Ce6 into tumor cells, increase the uptake rate of tumor cells, and thus enhance the therapeutic effect of chemo / photodynamic synergistic therapy.

[0126] 11. Endocytosis assay

[0127] To facilitate quantitative evaluation, we used the fluorescent dye FITC to label TrF (TrF FITC ), and then prepare the adsorbed TrF FITC SS-DMONs / DOX-Ce6@MIPs (SS-DMONs / DOX-Ce6@MIPs / TrF FITC ). Fig.16 It shows that SS-DMONs / DOX-Ce6@MIPs / TrF in MCF-7 cells blocked by TrF FITC The fluorescence of SS-DMONs / DOX-Ce6@MIPs / TrF FITC In summary, SS-DMONs / DOX-Ce6@MIPs can internalize TrF into cells through the specific affinity between TrF and TfR, thereby increasing the specific uptake of molecularly imprinted nanoparticles by tumor cells.

[0128] 12. Anti-tumor therapy in mice

[0129] exist Fig.17 In the experiment, compared with the control group (PBS and SS-DMONs / DOX-Ce6 group), the tumor growth in the SS-DMONs / DOX-Ce6@MIPs group was significantly inhibited. The first reason is that the outer polymer of SS-DMONs / DOX-Ce6@MIPs has a protective effect on the anti-tumor drugs loaded in the internal SS-DMONs, thereby carrying more anti-cancer drugs to the tumor cells. The second reason is that after SS-DMONs / DOX-Ce6@MIPs targets tumor cells through multiple forces, Ce6 and DOX jointly play a chemical / photodynamic synergistic anti-cancer role under laser irradiation.

[0130] The mouse tumors and major organs (heart, liver, spleen, lungs, and kidneys) were removed and subjected to H&E staining. Fig.18 As shown in the figure, no obvious physiological abnormalities such as inflammatory necrosis and organ damage were found in the tissue sections of the main organs of mice. For mouse tumor tissue, the cells in the control groups (PBA group and SS-DMONs / DOX-Ce6 group) were dense, with only a small number of scattered necrotic areas. However, large areas of necrosis were clearly visible in the tumor tissue of the SS-DMONs / DOX-Ce6@MIPs group, and the therapeutic effect was better than that of the control group. The results show that the SS-DMONs / DOX-Ce6@MIPs designed in this study have good biocompatibility, good biosafety and minimal adverse reactions.

Claims

1. A method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecular imprinting polymers, characterized in that The method is specifically carried out according to the following steps:

1. Preparation of SS-DMONs: The hexadecyltrimethylammonium chloride and triethanolamine were mixed and dissolved in water, and stirred at 90-100°C for 5-10 minutes. Tetraethyl orthosilicate A was added to the reaction solution and the stirring reaction was continued for 0.8-1.2 hours. The silica product was collected by centrifugation and washed repeatedly with ethanol and water for 3-5 times. The obtained precipitate was condensed and refluxed at 75-80°C for 34-38 hours with ethanol to remove the hexadecyltrimethylammonium chloride, and tetraethyl orthosilicate B and bis[3-(triethoxysilyl)propyl]tetrasulfide BTEPTS were added and the reaction was continued at 90-100°C for 3-5 minutes. h, centrifuged to obtain SS-DMONs; the mass ratio of hexadecyltrimethylammonium chloride to triethanolamine is 1:(0.03-0.05); the mass ratio of hexadecyltrimethylammonium chloride to water is 1g:(8-12)mL; the mass ratio of hexadecyltrimethylammonium chloride to tetraethyl orthosilicate A is 1:(0.4-0.6); the mass ratio of hexadecyltrimethylammonium chloride to tetraethyl orthosilicate B is 1:(0.4-0.6); the mass ratio of hexadecyltrimethylammonium chloride to BTEPTS is 1:(0.8-1.2); 2. Preparation of SS-DMONs / DOX-Ce6: Doxorubicin DOX and dihydrochlorin Ce6 were mixed and dissolved in a PBS solution, SS-DMONs were added thereto, and the mixture was subjected to ultrasonic treatment for 0.5-1.5 h, followed by stirring for reaction for 20-26 h, and then centrifuged to obtain SS-DMONs / DOX-Ce6; the pH of the PBS solution was 7.3-7.5; the mass ratio of DOX to Ce6 was 1:(0.8-1.2); the mass ratio of DOX to the volume of the PBS solution was 1 g:(1500-1700) mL; the mass ratio of DOX to SS-DMONs was 1:(1.6-2.4); 3. Preparation of Cys-DA: Dopamine DA, L-cysteine ​​L-Cys, dicyclohexylcarbodiimide and dimethylaminopyridine are mixed and dissolved in dichloromethane, and stirred in an ice-water bath for 2-4 hours; the mixed solution is filtered and evaporated under reduced pressure, then dissolved in an ice acetone solution and evaporated under reduced pressure, and then recrystallized using cyclohexane and ethanol aqueous solution in turn to obtain a recrystallized product; the recrystallized product and BBr3 are mixed and dissolved in CH2Cl2, stirred for 1-3 hours, methanol is added and the stirring reaction is continued for 1-3 hours, and the mixture is evaporated under reduced pressure; methanol is added to the mixture, and the solution is evaporated under reduced pressure to remove the formed trimethylborate; the residue is heated to remove other impurities; the solid product is washed with ethyl acetate When the thin layer chromatography shows only one chromatographic point, the reaction is terminated to obtain Gys-DA; the mass ratio of the DA to the volume of dichloromethane is 1g: (8-12) mL; the mass ratio of the L-Cys to the volume of dichloromethane is 1g: (6-8) mL; the mass ratio of the dicyclohexylcarbodiimide to the volume of dichloromethane is 1g: (7-9) mL; the mass ratio of the dimethylaminopyridine to the volume of dichloromethane is 1g: (5-7) mL; the mass ratio of the recrystallized product to the volume of CH2Cl2 is 1g: (1800-2200) mL; the mass ratio of the recrystallized product to the volume of BBr3 is 1g: (160-200) mL; 4. Preparation of dual template imprinted nanopolymers: Gys-DA, dopamine, 4-aminophenylboronic acid, transferrin TrF, and sialic acid SA were mixed and dispersed in deionized water, SS-DMONs / DOX-Ce6 was added thereto, and after ultrasonic dissolution, ammonium sulfate was added to initiate polymerization. After reacting for 22-26 hours under electromagnetic stirring, the mixture was washed with PBS for 3-5 times, separated and dried, and eluted with methanol solution to obtain SS-DMONs / DOX-Ce6@MIPs. The mass ratio of Gys-DA to deionized water was 1 g: (4800-5200) mL. The mass ratio of dopamine to deionized water was The volume ratio is 1g: (4800~5200) mL; the volume ratio of the mass of the 4-aminophenylboronic acid to the deionized water is 1g: (4800~5200) mL; the volume ratio of the mass of the TrF to the deionized water is 1g: (4800~5200) mL; the volume ratio of the mass of the SA to the deionized water is 1g: (4800~5200) mL; the volume ratio of the mass of the SS-DMONs / DOX-Ce6 to the deionized water is 1g: (2400~2600) mL; the volume ratio of the ammonium sulfate to the deionized water is 1: (45~55).

2. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that The mass ratio of hexadecyltrimethylammonium chloride to triethanolamine in step 1 is 1:0.04; the mass ratio of the hexadecyltrimethylammonium chloride to water is 1g:10mL; the mass ratio of the hexadecyltrimethylammonium chloride to tetraethyl orthosilicate A is 1:0.5; the mass ratio of the hexadecyltrimethylammonium chloride to tetraethyl orthosilicate B is 1:0.5; the mass ratio of the hexadecyltrimethylammonium chloride to BTEPTS is 1:

1.

3. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that In step 2, the mass ratio of DOX to Ce6 is 1:1; the mass ratio of DOX to the volume of PBS solution is 1 g:1600 mL; and the mass ratio of DOX to SS-DMONs is 1:

2.

4. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that In step 3, the mass ratio of DA to dichloromethane is 0.92 g:10 mL; the mass ratio of L-Cys to dichloromethane is 1.33 g:10 mL; the mass ratio of dicyclohexylcarbodiimide to dichloromethane is 1.25 g:10 mL; the mass ratio of dimethylaminopyridine to dichloromethane is 1.5 g:10 mL.

5. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that The mass ratio of the recrystallized product in step 3 to the volume ratio of CH2Cl2 is 1g:2000mL; the mass ratio of the recrystallized product to the volume ratio of BBr3 is 1g:180mL.

6. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that The volume ratio of CH2Cl2 to BBr3 in step 3 is 1:

1.

7. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that In step 3, the volume ratio of ethanol to water in the ethanol-water solution is 1:

5.

8. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that During the reaction in step 3, the reaction is monitored by thin layer chromatography of the eluent; the eluent is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol in the mixture is 4:

1.

9. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that In step 4, the mass ratio of Gys-DA to deionized water is 1 g:5000 mL; the mass ratio of dopamine to deionized water is 1 g:5000 mL; the mass ratio of 4-aminophenylboronic acid to deionized water is 1 g:5000 mL; the mass ratio of TrF to deionized water is 1 g:5000 mL; the mass ratio of SA to deionized water is 1 g:5000 mL.

10. The method for preparing pH / GSH-responsive drug-loaded biodegradable targeted nanoparticles based on dual-template molecularly imprinted polymers according to claim 1, characterized in that In step 4, the mass ratio of SS-DMONs / DOX-Ce6 to deionized water is 1 g:2500 mL; the concentration of ammonium sulfate is 7 mg / mL; and the volume ratio of ammonium sulfate to deionized water is 1:50.

Images