An upper critical solution temperature responsive mesoporous silica, and a preparation method and application thereof

Abstract

The application discloses an upper critical solution temperature responsive mesoporous silicon and a preparation method and application thereof, and is characterized in that: mesoporous silicon is coated on the outside of copper sulfide nanoparticles (CuS NPs), and then the coated CuS NPs are subjected to amination by using an APTES solution, and then a temperature-sensitive material PEG-p(AAm-co-AN) is grafted to form the upper critical solution temperature responsive mesoporous silicon. The application has the advantages of simple method, easy operation and mild reaction condition; the final product has good stability, can be used for loading a chemotherapeutic drug doxorubicin hydrochloride, and can realize drug controlled release; has temperature response drug release performance, and the phase transition temperature of the carrier is 43 DEG C, which is suitable for the microenvironment of tumor tissues; can be subjected to photothermal therapy under the irradiation of a 980nm laser, and realizes the synergistic effect of photothermal therapy and chemotherapy.

Description

Technical Field

[0001] This invention relates to the preparation and application of nanomaterials, and particularly to an upper critical dissolution temperature responsive mesoporous silicon, its preparation method, and its application. Background Technology

[0002] Cancer is one of the most destructive diseases, and despite some progress in cancer treatment in recent years, it still threatens human health. Chemotherapy is currently one of the most commonly used methods for cancer treatment, but chemotherapy drugs are limited by factors such as multidrug resistance. To better treat cancer, nanomedicine delivery systems have been developed. Among them, mesoporous silica nanoparticles are widely used to deliver chemotherapy drugs for cancer treatment due to their good biocompatibility, high specific surface area, and porous structure.

[0003] In recent years, temperature-responsive materials have been developed, among which upper critical solution temperature (LCT) responsive polymers have attracted widespread attention. These polymers are hydrophobic below their critical solution temperature, but transform into hydrophilic substances when the temperature reaches above it. The critical solution temperature of LCT polymers is generally around 43°C, which does not cause irreversible damage to the human body. Furthermore, because cancer cells are susceptible to high temperatures, they hold great potential for cancer treatment. In addition, the phase transition behavior of these polymers during heating can be combined with photothermal therapy. When a photothermal agent is introduced into the system and driven by near-infrared or ultraviolet light, the photothermal agent converts light energy into heat energy, thereby triggering a phase transition in the temperature-responsive polymer. Utilizing these properties can provide new strategies for drug delivery.

[0004] When using nanomaterials to deliver chemotherapy drugs for cancer treatment, preventing premature leakage of the drugs during delivery and reducing severe toxic side effects on normal cells are crucial aspects. However, ordinary mesoporous materials can only encapsulate and deliver hydrophobic drugs, failing to meet the need for controlled drug release. Summary of the Invention

[0005] The first objective of this invention is to address the shortcomings of existing technologies by providing a method for preparing upper critical dissolution temperature-responsive mesoporous silica, thereby obtaining a nano-drug delivery system with drug-carrying and photothermal therapy functions. This overcomes the drawbacks of premature drug leakage and significant side effects during delivery, while also leveraging the synergistic effect of chemotherapy and photothermal therapy to achieve enhanced anti-tumor efficacy.

[0006] This invention first synthesizes CTAB-terminated CuS NPs, then prepares amino-modified CuS@MSN-NH2 using the sol-gel method, and prepares polyethylene glycol-poly(acrylamide-acrylonitrile) via conventional free radical polymerization. Finally, the polymer is grafted onto CuS@MSN-NH2 to obtain nanoparticles. The specific method of this invention is as follows:

[0007] Step (1), Preparation of CuS NPs:

[0008] Hexadecyltrimethylammonium bromide (CTAB) and copper chloride were dissolved in water and stirred. Sodium sulfide was then added and reacted in a water bath. After cooling, an aqueous solution of CuS NPs was obtained.

[0009] Step (2), Preparation of CuS@MSN-NH2:

[0010] Triethanolamine (TEA) and hexadecyltrimethylammonium bromide (CTAB) were added to the CuS NPs aqueous solution obtained in step (1) and stirred. Then, tetraethyl orthosilicate (TEOS) was added for a water bath reaction. The silane coupling agent (APTES) was added to continue the reaction. After the reaction was completed, CuS@MSN-NH2 was obtained.

[0011] Step (3), Preparation of PEG-P(AAm-co-AN):

[0012] Acrylamide, acrylonitrile, and azobiscyanopentanoic acid were dissolved in dimethyl sulfoxide, mixed, and inert gas was introduced. The mixture was reacted at 50–60 °C. After the reaction was completed, the mixture was placed in an ice bath, and then placed in an excess solvent to stand. After centrifugation, washing, and vacuum drying, poly(acrylamide-acrylonitrile), i.e., P(AAm-co-AN), was obtained. Poly(acrylamide-acrylonitrile) and methoxy polyethylene glycol succinimide carbonate were then dissolved together in dimethyl sulfoxide and reacted to obtain polyethylene glycol-poly(acrylamide-acrylonitrile), i.e., PEG-P(AAm-co-AN).

[0013] Step (4): Preparation of upper critical solution temperature responsive mesoporous silicon C@M@P:

[0014] The polyethylene glycol-poly(acrylamide-acrylonitrile), 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC, and N-hydroxysuccinimide NHS obtained in step (3) were dissolved in dimethyl sulfoxide. After the reaction, CuS@MSN-NH2 obtained in step (2) was added to continue the reaction to obtain the upper critical dissolution temperature responsive mesoporous silica C@M@P.

[0015] Preferably, the molar ratio of CTAB, copper chloride and sodium sulfide in step (1) is 1:10:3 to 10, and more preferably 1:10:3.

[0016] Preferably, the molar ratio of TEA, CTAB, TEOS and APTES in step (2) is 0.1:1.6~5.1:0.5~1:0.5~1, and more preferably 0.1:1.6~5.1:0.5:0.5.

[0017] Preferably, the molar ratio of acrylonitrile, acrylamide, and azodicyanovalerate in step (3) is 1:3 to 10:0.03, and more preferably 1:3:0.03.

[0018] Preferably, in step (3), the molar ratio of poly(acrylamide-acrylonitrile) to methoxy polyethylene glycol succinimide carbonate is 1:5 to 10, more preferably 1:5.

[0019] Preferably, in step (4), the mass ratio of polyethylene glycol-poly(acrylamide-acrylonitrile), EDC, NHS, and CuS@MSN-NH2 is 5-5.5:1-5:1-2:2, and more preferably 5:1:1:2.

[0020] The second objective of this invention is to provide an upper critical dissolution temperature responsive mesoporous silica, prepared by the above method; this nanodelivery system can change the temperature by controlling the irradiation of external near-infrared light, thereby causing a phase transition in the polymer, opening the drug release channel, and at the same time, the increase in local temperature can also work in conjunction with chemotherapy to achieve better cancer treatment results.

[0021] A third objective of this invention is to provide the application of the above-described upper critical dissolution temperature responsive mesoporous silica in loading chemotherapeutic drugs, wherein the chemotherapeutic drugs include one or both of doxorubicin hydrochloride and elemol.

[0022] Preferably, the mass ratio of upper critical dissolution temperature responsive mesoporous silica to chemotherapeutic drugs is 2.5 to 20:1.

[0023] A fourth objective of this invention is to provide the application of the above-mentioned upper critical dissolution temperature responsive mesoporous silica in the preparation of tumor photothermal therapy synergistic chemotherapy drugs.

[0024] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0025] 1. This invention prepares core-shell CuS@MSN-NH2 nanoparticles by grafting critical dissolution temperature-responsive polymers onto the outer layer of mesoporous silica nanoparticles. Drugs are loaded into the mesopores, and the outermost layer is grafted with a polymer of polyethylene glycol-poly(acrylamide-acrylonitrile) for pore sealing, which also acts as a temperature-responsive switch, enabling controllable drug release under photothermal response. This invention uses copper sulfide nanoparticles as a photothermal agent, converting light energy into heat energy under near-infrared light irradiation. When the system temperature reaches the upper critical dissolution temperature of the polymer, the outer temperature-sensitive polymer expands, thereby controlling drug release. Furthermore, the increase in local temperature can synergistically work with chemotherapy to achieve better tumor treatment effects. This invention cleverly combines temperature response with photothermal therapy, which can be used in the biomedical field for the treatment of malignant tumors.

[0026] 2. The CuS NPs prepared by this invention have significant absorption in the near-infrared region of around 980 nm, which can convert light energy into heat energy with high photothermal conversion efficiency. CuS NPs are simple to prepare, low in cost, and have no long-term toxicity as photothermal agents.

[0027] 3. The drug-loaded nanoparticles prepared by the method of the present invention have a simple process. In the drug loading experiment, the nanocomposite material with photothermal response and controllable drug release has a large drug loading capacity and a significant drug control release effect.

[0028] 4. The thermosensitive polymer selected in this invention has the characteristic of responding to the upper critical dissolution temperature. As the temperature increases, it can achieve a hydrophobic to hydrophilic transition. Furthermore, the polymer exhibits stable and repeatable characteristics during heating and cooling cycles, making it suitable for sealing mesoporous silica and releasing drugs. Attached Figure Description

[0029] Figure 1 The images and performance test results of CuS NPs are shown below, including: A) Transmission electron microscopy (TEM) image, B) Particle size distribution map, C) UV-Vis-NIR absorption curve, and D) Photothermal conversion curve (980 nm, 2 W / cm²). 2 );

[0030] Figure 2 Transmission electron microscopy image of CuS@MSN-NH2;

[0031] Figure 3 Transmission electron microscopy image of C@M@P;

[0032] Figure 4 Zeta potential diagrams for CuS NPs, CuS@MSN-NH2, PEG-p(AAm-co-AN), and C@M@P;

[0033] Figure 5 The permeability of PEG-p(AAm-co-AN) aqueous solution varies with temperature;

[0034] Figure 6 The drug release curve for C@M@P-Dox;

[0035] Figure 7 Cell viability after co-incubation of 4T1 cells with different concentrations of C@M@P;

[0036] Figure 8 The cell survival rate after co-incubation with 4T1 cells in each treatment group is shown. Detailed Implementation

[0037] To make the technical problems, technical solutions, and advantages of the present invention clearer, a detailed description will be provided below in conjunction with the accompanying drawings and specific embodiments. The technical solutions of the present invention will be further illustrated below with reference to specific embodiments.

[0038] Example 1

[0039] Add 85 mg CuCl2 and 100 mg hexadecyltrimethylammonium bromide (CTAB) to 400 ml of water and stir magnetically at room temperature for 30 minutes. Add 121 mg Na2S to the reaction solution and stir magnetically at room temperature for 5 minutes. Then transfer the solution to a 90°C water bath and react for 15 minutes to obtain a green CuS-CTAB solution, which can be stored for later use.

[0040] Example 2

[0041] Add 85 mg CuCl2 and 100 mg cetyltrimethylammonium bromide (CTAB) to 400 ml of water and stir magnetically at room temperature for 30 minutes. Add 106 mg Na2S to the reaction solution and stir magnetically at room temperature for 5 minutes. Then transfer to a 90°C water bath and react for 15 minutes to obtain a green CuS-CTAB solution, which can be stored for later use.

[0042] Example 3

[0043] Weigh 500 mg of CTAB and dissolve it in 20 ml of the CuS NPs aqueous solution prepared in Example 1. Add 18 μl of triethanolamine and magnetically stir at room temperature for 1 h. Then, slowly add 200 μl of TEOS to the above reaction solution and magnetically stir the resulting mixture in a 90 °C water bath for 1 h. Next, add 200 μl of APTES and continue magnetically stirring at 90 °C for 1 h. After the reaction is complete, allow it to cool and stand, centrifuge at 10,000 rpm for 5 min, and wash three times with distilled water. Redisperse the collected sample in 12.5 ml of methanol, add 100 mg of sodium chloride, and magnetically stir at room temperature for 24 h. After the reaction is complete, centrifuge at 10,000 rpm for 5 min and wash three times with methanol. Repeat this operation three times. After the final reaction, centrifuge at 10,000 rpm for 5 min, wash three times with methanol, and then wash twice with pure water. Collect the product to obtain CuS@MSN-NH2.

[0044] Example 4

[0045] Weigh 500 mg of CTAB and dissolve it in 20 ml of the CuS NPs aqueous solution prepared in Example 1. Add 18 μl of triethanolamine and magnetically stir at room temperature for 1 h. Then, slowly add 100 μl of TEOS to the above reaction solution and magnetically stir the resulting mixture in a 90 °C water bath for 1 h. Subsequently, add 100 μl of APTES and continue magnetically stirring at 90 °C for 1 h. After the reaction is complete, allow it to stand and cool, centrifuge at 10,000 rpm for 5 min, and wash three times with distilled water. Redisperse the collected sample in 12.5 ml of methanol, add 100 mg of sodium chloride, and magnetically stir at room temperature for 24 h. After the reaction is complete, centrifuge at 10,000 rpm for 5 min and wash three times with methanol. Repeat this operation three times. After the last reaction is complete, centrifuge at 10,000 rpm for 5 min, wash three times with methanol, and then wash twice with pure water. Collect the product to obtain CuS@MSN-NH2.

[0046] Example 5

[0047] Accurately weigh 2g of acrylamide into a three-necked flask, add 30ml of dimethyl sulfoxide, and then inject 0.661ml of acrylonitrile, mixing thoroughly. Slowly add 0.6ml of a 3.7% (w / w) azodicyanovalerate solution to the above mixture, mixing thoroughly under magnetic stirring, and purging with nitrogen for 1 hour. Then transfer the entire reaction system to a 60℃ water bath and react with magnetic stirring for 6 hours. After the reaction is complete, transfer to an ice-water bath for rapid cooling. After cooling, slowly add the above solution to 300ml of methanol to precipitate the polymer. Let the methanol solution containing the precipitate stand overnight at -20℃. Discard part of the supernatant, centrifuge the remaining methanol solution containing the precipitate at 10000 rpm for 10 minutes, and wash the resulting precipitate three times with methanol. Finally, vacuum dry the product for 24 hours to obtain the white product poly(acrylamide-acrylonitrile), i.e., p(AAm-co-AN). Accurately weigh 5 mg of synthesized poly(acrylamide-acrylonitrile) and 6.58 mg of methoxy polyethylene glycol disuccinimide carbonate, add 2 ml of dimethyl sulfoxide, and magnetically stir at 50 °C for 48 h. Then transfer the solution to a dialysis bag, dialyze for 48 h, and freeze dry to obtain the white product polyethylene glycol-poly(acrylamide-acrylonitrile), i.e., PEG-p(AAm-co-AN).

[0048] Example 6

[0049] Weigh 5.5 mg of polyethylene glycol-poly(acrylamide-acrylonitrile) from Example 5, add 1 ml of dimethyl sulfoxide, 1.6 mg of EDC, and 1 mg of NHS, and magnetically stir at 50 °C for 1 h; then add 2 mg of CuS@MSN-NH2 from Example 3, and magnetically stir at 50 °C for 48 h. The solution is then transferred to a dialysis bag, dialyzed for 48 h, and then freeze-dried to obtain C@M@P.

[0050] Example 7

[0051] Disperse 5 mg of elemene in 5 ml of water and sonicate to obtain solution A. Add 5 mg of C@M@P from Example 6 to 400 μl of solution A and magnetically stir at room temperature in the dark for 24 h to fully load the nanomaterial with the drug. Centrifuge the drug-loaded nanoparticles at 10,000 rpm for 5 min, collect the drug-loaded nanoparticles, and wash them twice with water to remove the drug adhering to the surface of the material.

[0052] Example 8

[0053] Disperse 5 mg of doxorubicin hydrochloride (Dox) in 5 ml of water and sonicate to obtain solution A. Add 2.5 mg of C@M@P from Example 6 to 100 μl of solution A and stir magnetically at room temperature in the dark for 24 h to fully load the nanomaterials with the drug. Centrifuge the drug-loaded nanoparticles at 10,000 rpm for 5 min, collect the drug-loaded nanoparticles, and wash them twice with water to remove the drug adhering to the surface of the material.

[0054] The encapsulation efficiency and drug loading of doxorubicin hydrochloride in Examples 7 and 8 were tested, and the results are as follows: C@M@P:Dox = 2.5:1, encapsulation efficiency 13%, drug loading 6.1%; C@M@P:Dox = 2.5:1, encapsulation efficiency 32%, drug loading 7.2%.

[0055] Test Example 1

[0056] The material of Example 1 was characterized. Transmission electron microscopy (TEM) images and particle size distribution maps are shown below. Figure 1 As can be seen from AB, uniformly dispersed CuS NPs with a particle size of 10-18 nm were prepared.

[0057] The ultraviolet-visible-near-infrared absorption and photothermal conversion of Example 1 were tested. Figure 1 CD shows that CuS NPs aqueous solution has strong absorption at 980 nm and good photothermal conversion efficiency.

[0058] The materials of Examples 3 and 6 were characterized. The transmission electron microscope (TEM) image of CuS@MSN-NH2 obtained in Example 3 is shown below. Figure 2 The prepared CuS@MSN-NH2 particles were uniform in size, exhibiting a core-shell structure and spherical shape, with a particle size ranging from 48 to 56 nm. The transmission electron microscope image of C@M@P obtained in Example 6 is shown below. Figure 3 As can be seen, C@M@P with good morphology was prepared.

[0059] The materials of Examples 1, 3, 5 and 6 were characterized by potential. Figure 4The figures show the Zeta potentials of CuS-CTAB, CuS@MSN-NH2, PEG-p(AAm-co-AN), and C@M@P. The results indicate a potential reversal in the mesoporous silica potential before and after amino modification, confirming the successful modification. Positively charged CuS@MSN-NH2 and negatively charged PEG-p(AAm-co-AN) ultimately yielded C@M@P with a potential of 5.15 ± 0.72 mV.

[0060] The upper critical dissolution temperature of the material in Example 5 was tested. Figure 5 The figure shows the change in transmittance of polyethylene glycol-poly(acrylamide-acrylonitrile) aqueous solution with temperature. When the temperature rises above 43℃, the transmittance approaches 100% and remains stable, indicating that 43℃ is the upper critical dissolution temperature of polyethylene glycol-poly(acrylamide-acrylonitrile).

[0061] Test the in vitro release of the material in Example 8. Figure 6 The figure shows the in vitro cumulative release rate of C@M@P-Dox. At 37°C, Dox release is very slow, but at 45°C, the cumulative release rate after 48 hours can reach over 70%. Furthermore, the release under laser-induced temperature elevation is similar to that at 45°C, indicating that the release of C@M@P-Dox is temperature-responsive.

[0062] The cytotoxicity of cells tested in Examples 6 and 8 was evaluated using the MTT assay. 4T1 cells were seeded in 96-well plates and incubated. Different concentrations of C@M@P (0-200 μg / ml) were added to each well, and the cells were incubated for another 24 h. Then, 33 μl of MTT solution (5 mg / ml) was added to each well, and after 4 h of incubation, the liquid in the wells was removed, and 100 μl of dimethyl sulfoxide was added to each well. The plates were then shaken using a microplate reader, and the absorbance at 570 nm was measured. The cell viability of each group was obtained by comparing the absorbance values ​​of the experimental and control groups.

[0063] like Figure 7 As shown, the experimental results indicate that even at a C@M@P concentration of 200 μg / mL, cell viability remains greater than 80%, thus demonstrating that C@M@P has good biocompatibility.

[0064] The in vitro antitumor effects of Examples 6 and 8 were evaluated using the MTT assay. Seven cell experiments were set up. 4T1 cells were seeded in 96-well plates and incubated in an incubator. After the cells grew to a suitable density, the corresponding nanomaterials or drugs for each group were added for co-incubation. For the laser group, after 4 hours of co-incubation, the cells were irradiated with a laser for 5 minutes (980nm, 2w / cm²). 2The cells were incubated for another 20 hours. Then, MTT solution (33 μl, 5 mg / ml) was added to each well. After incubation for 4 hours, the liquid in the wells was aspirated, and 100 μl of dimethyl sulfoxide was added. The cells were shaken using a microplate reader, and the absorbance value at 570 nm was measured to obtain the cell viability of each group.

[0065] The results are as follows Figure 8 As shown, the C@M@P-Dox group after laser irradiation ( Figure 8 The cell survival rate was only 10% (C@M@P-Dox+L), indicating that the hyperthermia caused by increased temperature can synergistically reduce the cell survival rate and achieve a better anti-tumor effect.

Claims

1. A method for preparing upper critical dissolution temperature responsive mesoporous silicon, characterized in that, The preparation method includes the following steps: Step (1), Preparation of CuS NPs: Hexadecyltrimethylammonium bromide (CTAB) and copper chloride were dissolved in water and stirred. Sodium sulfide was then added and reacted in a water bath. After cooling, an aqueous solution of CuS NPs was obtained. Step (2), Preparation of CuS@MSN-NH2: Triethanolamine (TEA) and hexadecyltrimethylammonium bromide (CTAB) were added to the CuS NPs aqueous solution obtained in step (1) and stirred. Then, tetraethyl orthosilicate (TEOS) was added for a water bath reaction. The silane coupling agent (APTES) was added to continue the reaction. After the reaction was completed, CuS@MSN-NH2 was obtained. Step (3), Preparation of PEG-P(AAm-co-AN): Acrylamide, acrylonitrile, and azobiscyanopentanoic acid were dissolved in dimethyl sulfoxide, mixed, and inert gas was introduced. The mixture was reacted at 50–60 °C. After the reaction was completed, the mixture was placed in an ice bath, allowed to stand in an excess solvent, centrifuged, washed, and vacuum dried to obtain P(AAm-co-AN). P(AAm-co-AN) was then dissolved in dimethyl sulfoxide with methoxy polyethylene glycol succinimide carbonate to obtain PEG-P(AAm-co-AN). Step (4): Preparation of upper critical solution temperature responsive mesoporous silicon C@M@P: The PEG-P(AAm-co-AN), 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC, and N-hydroxysuccinimide NHS obtained in step (3) were dissolved in dimethyl sulfoxide. After the reaction, CuS@MSN-NH2 obtained in step (2) was added to continue the reaction to obtain the upper critical dissolution temperature responsive mesoporous silica C@M@P.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of CTAB, copper chloride and sodium sulfide is 1:10:3 to 10.

3. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of TEA, CTAB, TEOS and APTES is 0.1:1.6~5.1:0.5~1:0.5~1.

4. The preparation method according to claim 1, characterized in that, In step (3), the molar ratio of acrylonitrile, acrylamide, and azodicyanovalerate is 1:3 to 10:0.

03.

5. The preparation method according to claim 1, characterized in that, In step (3), the molar ratio of P(AAm-co-AN) and methoxy polyethylene glycol succinimide carbonate is 1:5 to 10.

6. The preparation method according to claim 1, characterized in that, In step (4), the mass ratio of PEG-P(AAm-co-AN), EDC, NHS, and CuS@MSN-NH2 is 5-5.5:1-5:1-2:

2.

7. An upper critical dissolution temperature responsive mesoporous silicon, prepared by the method described in any one of claims 1-6.

8. The application of the upper critical dissolution temperature responsive mesoporous silica according to claim 7 in loading chemotherapeutic drugs, characterized in that, The chemotherapy drugs include one or both of doxorubicin hydrochloride and elemol.

9. The application according to claim 8, characterized in that, The mass ratio of upper critical dissolution temperature responsive mesoporous silica to chemotherapeutic drugs is 2.5–20:

1.

10. The use of the upper critical dissolution temperature responsive mesoporous silica as described in claim 7 in the preparation of tumor photothermal therapy synergistic chemotherapy drugs.

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