Antioxidant nano double-response drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, and discloses an antioxidant nano double-response drug delivery system and a preparation method and application thereof.The preparation method comprises the steps that mesoporous bioactive glass nano-particles with the particle size being 100-400 nm, the aperture being 2-10 nm and the specific surface area being 200-800 m < 2 > / g are adopted, amino functionalization is conducted on the surfaces, and the mesoporous bioactive glass nano-particles are prepared; the preparation method comprises the following steps: adsorbing a micromolecular antioxidant drug to mesoporous channels and surfaces of nanoparticles by using a rotary evaporation method, and finally grafting a pH-sensitive and ROS-responsive gated polymer by adopting an amide reaction to construct a nano drug delivery system capable of releasing the antioxidant drug in a pH-ROS dual-response manner, and the nano drug delivery system has good biocompatibility, and can be used for preparing a drug delivery system for the antioxidant drug in a pH-ROS dual-response manner. Antioxidant drugs can be released by intelligently responding to low pH and high ROS; by releasing small-molecule antioxidant drugs, free radicals are eliminated, oxidative stress is relieved, inflammatory response is inhibited, and the hydrogel has good application prospects in the fields of diabetes mellitus chronic wound healing, tissue engineering drug release, cancer treatment, tumor resistance and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to an antioxidant nano dual-responsive drug delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetes is a common chronic metabolic disease clinically, and diabetic wounds are one of the most common diabetic complications. Currently, the number of adult diabetic patients globally exceeds 537 million, and the incidence rate of diabetic chronic wounds exceeds 25%. The treatment of diabetic chronic wounds has become a major challenge facing global health. The wounds of normal skin restore normal physiological functions after experiencing four processes: hemostasis, inflammation, proliferation, and remodeling. However, due to the excessive inflammatory response, high oxidative stress level, high blood glucose, insufficient angiogenesis, and extremely high risk of bacterial infection in diabetic wounds, the healing process is chronically stalled at the inflammation stage and gradually develops into chronic wounds, bringing a heavy economic burden and pain to diabetic patients, and even increasing the risk of amputation and death.

[0003] Traditional wound dressings for treating diabetic chronic wounds have a single function and are difficult to adapt to the complex microenvironment of the wounds, and it is difficult to meet the treatment requirements during the healing process. Therefore, wound dressings combined with a stimulus-responsive nano drug delivery system can specifically respond to the special pathological microenvironment (such as pH, glucose, reactive oxygen species, etc.) at the wound site, and achieve on-demand responsive release by loading drugs, bioactive substances, etc., and can effectively promote the healing of diabetic chronic wounds. Currently, intelligent drug delivery systems that can synergistically respond to multiple pathological characteristics (such as pH / enzyme / reactive oxygen species, etc.) are still rarely reported.

[0004] Mesoporous bioactive glass nanoparticles (MBGNs) have characteristics such as good biocompatibility, large specific surface area, uniform particle size and pore size structure, and easy surface modification, and are good carriers for drugs or bioactive substances, etc. Since there are a large number of silanol groups on the surface of MBGNs, according to specific requirements, a nano drug delivery system with intelligent responsive release of drugs in one or two pathological environments can be custom-designed by combining different molecules or drugs. Small molecule antioxidant drugs can rapidly scavenge free radicals and can well cope with the pathological environment of excessive oxidative stress in diabetic chronic wounds. The diabetic chronic wound site has the characteristics of low pH and high ROS. Based on the characteristics of this pathological environment, a nano drug delivery system with responsive intelligent release of small molecule antioxidant drugs can be designed to reduce the oxidative stress at the wound site, slow down the inflammatory reaction, and achieve antioxidant and anti-inflammatory effects to promote the healing of diabetic chronic wounds. Therefore, a nano drug delivery system loaded with small molecule antioxidant drugs and having dual responsiveness to pH and ROS has good application prospects, and in addition to the treatment of diabetic wounds, it also has great application potential in tissue engineering drug release, cancer targeted therapy, and anti-tumor. Summary of the Invention

[0005] The primary object of the present invention is to provide an antioxidant nano dual-responsive drug delivery system, its preparation method and application. This preparation method combines surface modification, rotary evaporation for physical adsorption of antioxidant drugs, and amidation reaction to obtain a nano drug delivery system that can intelligently respond to pH and ROS to release antioxidant drugs. At the same time, the present invention also provides the biological research of the above materials in vitro, and the influence of the above materials on the expression of inflammatory factors secreted by macrophages under simulated hyperglycemic and high oxidative stress conditions.

[0006] To achieve the above object and other related objects, the first aspect of the present invention provides a preparation method of a nano drug delivery system capable of dual-responsive release of antioxidant drugs in response to pH-ROS, including the following steps:

[0007] (1) Surface amino-functionalization of mesoporous bioactive glass nanoparticles: Mesoporous bioactive glass nanoparticles with a particle size of 100 - 400 nm, mainly composed of Si, Ca, and O, prepared by using CTAB as a template agent and the sol-gel method, are ultrasonically dispersed in ethanol. A silane coupling agent is added, and the reaction is carried out at 80 °C in a water bath for 2 h. After centrifugation, the supernatant is discarded, and it is dried at 50 °C for 24 h.

[0008] (2) Loading of small molecule antioxidant drugs: The amino-functionalized mesoporous bioactive glass nanoparticles obtained in step (1) are ultrasonically dispersed in methanol. A small molecule antioxidant drug is added, and after ultrasonic dispersion and dissolution until uniform, it is rotary evaporated until the solvent is completely evaporated, and then dried at 50 °C for 24 h to obtain drug-loaded nanoparticles.

[0009] (3) Grafting of gated polymers: The drug-loaded nanoparticles obtained in step (2) are ultrasonically dispersed in dichloromethane. A condensing agent PyBOP, HOBt, DIPEA are added, and a ROS-responsive polymer is added. After reacting at room temperature for 24 h, centrifugation is carried out, and the supernatant is discarded. After drying at 50 °C for 3 - 5 h, it is redispersed in deionized water. A pH-sensitive polymer is added, and the reaction is carried out at room temperature for 24 h. After centrifugation, the supernatant is discarded, washed 3 times with deionized water, and then freeze-dried to obtain a nano drug delivery system capable of dual-responsive release of antioxidant drugs in response to pH-ROS.

[0010] Preferably, the concentration of the ethanol is 50 - 100%.

[0011] The silane coupling agent is aminopropyltriethoxysilane, aminopropyltrimethoxysilane, 2-aminoethyl-aminopropyl-trimethoxysilane, or aminoethylaminopropylmethyldimethoxysilane.

[0012] Preferably, the reaction mass percentage of the silane coupling agent to ethanol is 0.1 - 0.5%.

[0013] Preferably, the small molecule antioxidant drug is gallic acid, quercetin, salidroside, resveratrol or baicalin.

[0014] The drug loading concentration of the small molecule antioxidant drug is 20 - 60 mg / mL, and the drug loading rate is 20 - 60%.

[0015] Preferably, the molar ratio of the condensing agents PyBOP, HOBt, and DIPEA is 1:1:1.2 - 1:1:2.

[0016] Preferably, the ROS-responsive polymer is one of DSPE-PEG-S-S-COOH, DSPE-PEG-Se-Se-COOH, PLGA-S-S-COOH, PEG-Se-Se-COOH, PEG-TK-COOH.

[0017] The molecular weight of the ROS-responsive polymer is 3000 - 10000 Da.

[0018] Preferably, the pH-sensitive polymer is polyacrylic acid or polymethacrylic acid.

[0019] The molecular weight of the polyacrylic acid or polymethacrylic acid is 10000 - 30000 Da.

[0020] Preferably, the mass ratio of the ROS-responsive polymer to the pH-sensitive polymer is 1:1 - 1:5.

[0021] The second aspect of the present invention lies in providing a nano-drug delivery system capable of dual pH-ROS responsive release of antioxidant drugs, which is prepared by the above preparation method.

[0022] The third aspect of the present invention lies in providing an in vitro antioxidant performance study of the above nano-drug delivery system.

[0023] Preferably, one of the in vitro antioxidant performance studies of the above nano-drug delivery system is the DPPH free radical scavenging experiment.

[0024] Preferably, one of the in vitro antioxidant performance studies is to detect the scavenging ability of intracellular ROS by the DCFH-DA reactive oxygen fluorescence probe after co-culturing the leaching solution containing the above nano-drug delivery system with L929s cells.

[0025] The fourth aspect of the present invention lies in providing an in vitro biological study of the above nano-drug delivery system.

[0026] Preferably, one of the in vitro biological studies of the above nano-drug delivery system is the detection of cell proliferation and toxicity experiments.

[0027] One of the in vitro biological studies of the above-mentioned nano drug delivery system is to study the effect of the above-mentioned material extract on the expression of inflammatory factors secreted by macrophages through real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) experiments.

[0028] A nano drug delivery system is prepared by the above-mentioned preparation method.

[0029] Application of a nano drug delivery system in drug preparation.

[0030] By loading small molecule antioxidants, a nano drug delivery system can effectively scavenge free radicals, and the DPPH free radical scavenging rate is 80-98%.

[0031] Preferably, the inflammatory factors secreted by the macrophages are IL-4, IL-10, TGF-β and IL-6.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] (1) The nano drug delivery system prepared by the present invention, which can release antioxidant drugs in a pH-ROS dual-responsive manner, has a high drug loading capacity, can respond to the low pH of the microenvironment and high ROS to control the release of small molecule antioxidants, is suitable for the drug delivery treatment of diabetic chronic wounds, drug release in tissue engineering, cancer targeted therapy and controlled release of anti-tumor drugs, has stable performance and is convenient for storage, and has good application prospects.

[0034] (3) The nano drug delivery system prepared by the present invention has good biocompatibility with a variety of cells and has good antioxidant performance.

[0035] (4) The nano drug delivery system prepared by the present invention has a down-regulating effect on the inflammatory factor IL-6 secreted by macrophages and has a significant up-regulating effect on the anti-inflammatory factors IL-4, IL-10, TGF-β. Description of the Drawings

[0036] Figure 1 It is the high-magnification transmission electron microscope image and EDS energy spectrum element analysis chart of Example 1 of the present invention.

[0037] Figure 2 It is the particle size distribution statistical chart of Example 3 of the present invention.

[0038] Figure 3 It is the response release drug curve diagram of Example 1 of the present invention under different conditions.

[0039] Figure 4 It is the cell proliferation experiment result chart of Examples 1, 4, and 5 of the present invention.

[0040] Figure 5DPPH scavenging rate graphs of Examples 1, 2, and 4 of the present invention

[0041] Figure 6 Fluorescence quantitative graphs of intracellular ROS scavenging experiments of Examples 1, 2, and 4 of the present invention.

[0042] Figure 7 RT-qPCR gene expression graphs of Examples 1 and 5 of the present invention.

[0043] Figure 8 Flow chart of the preparation method of the nano-drug delivery system of the present invention. Detailed implementation manners

[0044] The following combines specific examples to further illustrate the technical solutions described in the present invention, but the implementation manners of the present invention are not limited thereto.

[0045] Example 1

[0046] Weigh 1.0 g of MBGNs and ultrasonically disperse them in 100 mL of 50% ethanol. Pipette 0.2 mL of aminopropyltrimethoxysilane and add it dropwise to the above solution. React at 80 °C in a water bath for 2 h, centrifuge, discard the supernatant, wash with deionized water 3 times, and dry the precipitate at 50 °C for 24 h to obtain surface-aminated mesoporous bioactive glass nanoparticles M-NH2. Weigh 0.5 g of M-NH2 and ultrasonically disperse it in 10 mL of methanol. Add 200 mg (20 mg / mL) of salidroside, ultrasonically disperse and dissolve evenly, use a rotary evaporator to evaporate until the solvent is completely evaporated, and dry at 50 °C for 24 h to obtain drug-loaded nanoparticles M-NH2-Sal. Weigh 0.5 g of M-NH2-Sal and ultrasonically disperse it in 10 mL of dichloromethane. Add 21.56 mg of PyBOP, 5.60 mg of HOBt, and 13.78 μL of DIPEA (molar ratio 0.04 mmol:0.04 mmol:0.08 mmol = 1:1:2), add 200 mg of PEG-TK-COOH, react at room temperature for 24 h, then centrifuge, discard the supernatant, dry at 50 °C for 3 - 5 h and then redisperse in deionized water. Add 300 mg of polymethacrylic acid, react at room temperature for 24 h, centrifuge, discard the supernatant, wash with deionized water 3 times and then freeze-dry for 24 h to obtain the pH / ROS dual-responsive nano-drug delivery system.

[0047] Example 2

[0048] Weigh 1.0 g of MBGNs and ultrasonically disperse them in 100 mL of 50% ethanol. Pipette 0.2 mL of aminopropyltrimethoxysilane and add it dropwise to the above solution. React at 80 °C in a water bath for 2 h, then centrifuge, discard the supernatant, wash 3 times with deionized water, and dry the precipitate at 50 °C for 24 h to obtain surface - amino - functionalized mesoporous bioactive glass nanoparticles M - NH₂. Weigh 0.5 g of M - NH₂ and ultrasonically disperse it in 10 mL of methanol. Add 200 mg (20 mg / mL) of gallic acid, ultrasonically disperse and dissolve evenly, use a rotary evaporator to evaporate the solvent until it dries up, and dry at 50 °C for 24 h to obtain drug - loaded nanoparticles M - NH₂ - GA. Weigh 0.5 g of M - NH₂ - GA and ultrasonically disperse it in 10 mL of dichloromethane. Add 21.56 mg of PyBOP, 5.60 mg of HOBt, 13.78 μL of DIPEA (molar ratio 0.04 mmol:0.04 mmol:0.08 mmol = 1:1:2), add 200 mg of PEG - TK - COOH, react at room temperature for 24 h, then centrifuge, discard the supernatant, dry at 50 °C for 3 - 5 h, redisperse in deionized water, add 300 mg of polymethacrylic acid, react at room temperature for 24 h, centrifuge, discard the supernatant, wash 3 times with deionized water, and then freeze - dry for 24 h to obtain the pH / ROS dual - responsive nano - drug delivery system.

[0049] Example 3

[0050] Weigh 1.0 g of MBGNs and ultrasonically disperse them in 100 mL of 50% ethanol. Pipette 0.2 mL of aminopropyltriethoxysilane and add it dropwise to the above solution. React at 80 °C in a water bath for 2 h, then centrifuge, discard the supernatant, wash 3 times with deionized water, and dry the precipitate at 50 °C for 24 h to obtain surface - amino - functionalized mesoporous bioactive glass nanoparticles M - NH₂. Weigh 0.5 g of M - NH₂ and ultrasonically disperse it in 10 mL of methanol. Add 200 mg (20 mg / mL) of salidroside, ultrasonically disperse and dissolve evenly, use a rotary evaporator to evaporate the solvent until it dries up, and dry at 50 °C for 24 h to obtain drug - loaded nanoparticles M - NH₂ - Sal. Weigh 0.5 g of M - NH₂ - Sal and ultrasonically disperse it in 10 mL of dichloromethane. Add 21.56 mg of PyBOP, 5.60 mg of HOBt, 13.78 μL of DIPEA (molar ratio 0.04 mmol:0.04 mmol:0.08 mmol = 1:1:2), add 200 mg of PEG - TK - COOH, react at room temperature for 24 h, then centrifuge, discard the supernatant, dry at 50 °C for 3 - 5 h, redisperse in deionized water, add 300 mg of polymethacrylic acid, react at room temperature for 24 h, centrifuge, discard the supernatant, wash 3 times with deionized water, and then freeze - dry for 24 h to obtain the pH / ROS dual - responsive nano - drug delivery system.

[0051] Example 4

[0052] Weigh 1.0 g of MBGNs and ultrasonically disperse them in 100 mL of 50% ethanol. Pipette 0.2 mL of aminopropyltrimethoxysilane and add it dropwise to the above solution. React at 80 °C in a water bath for 2 h, centrifuge, discard the supernatant, wash 3 times with deionized water, and dry the precipitate at 50 °C for 24 h to obtain surface-aminated mesoporous bioactive glass nanoparticles M-NH₂. Weigh 0.5 g of M-NH₂ and ultrasonically disperse it in 10 mL of methanol. Add 600 mg (60 mg / mL) of salidroside, ultrasonically disperse and dissolve evenly, use a rotary evaporator to evaporate until the solvent is completely evaporated, and dry at 50 °C for 24 h to obtain drug-loaded nanoparticles M-NH₂-Sal. Weigh 0.5 g of M-NH₂-Sal and ultrasonically disperse it in 10 mL of dichloromethane. Add 21.56 mg of PyBOP, 5.60 mg of HOBt, and 13.78 μL of DIPEA (molar ratio 0.04 mmol:0.04 mmol:0.08 mmol = 1:1:2), add 200 mg of PEG-TK-COOH, react at room temperature for 24 h, then centrifuge, discard the supernatant, dry at 50 °C for 3 - 5 h, redisperse in deionized water, add 300 mg of polymethacrylic acid, react at room temperature for 24 h, centrifuge, discard the supernatant, wash 3 times with deionized water, and then freeze-dry for 24 h to obtain the pH / ROS dual-responsive nanodrug delivery system.

[0053] Example 5

[0054] Weigh 1.0 g of MBGNs and ultrasonically disperse them in 100 mL of 50% ethanol. Pipette 0.2 mL of aminopropyltrimethoxysilane and add it dropwise to the above solution. React at 80 °C in a water bath for 2 h, centrifuge, discard the supernatant, wash 3 times with deionized water, and dry the precipitate at 50 °C for 24 h to obtain surface-aminated mesoporous bioactive glass nanoparticles M-NH₂. Weigh 0.5 g of M-NH₂ and ultrasonically disperse it in 10 mL of methanol. Add 200 mg (20 mg / mL) of salidroside, ultrasonically disperse and dissolve evenly, use a rotary evaporator to evaporate until the solvent is completely evaporated, and dry at 50 °C for 24 h to obtain drug-loaded nanoparticles M-NH₂-Sal. Weigh 0.5 g of M-NH₂-Sal and ultrasonically disperse it in 1 mL of dichloromethane. Add 10.78 mg of PyBOP, 2.80 mg of HOBt, and 6.89 μL of DIPEA (molar ratio 0.02 mmol:0.02 mmol:0.04 mmol = 1:1:2), add 100 mg of PEG-TK-COOH, react at room temperature for 24 h, then centrifuge, discard the supernatant, dry at 50 °C for 3 - , redisperse in deionized water, add 400 mg of polymethacrylic acid, react at room temperature for 24 h, centrifuge, discard the supernatant, wash 3 times with deionized water, and then freeze-dry for 24 h to obtain the pH / ROS dual-responsive nanodrug delivery system.

Claims

1. A preparation method of an antioxidant nano dual-responsive drug delivery system, characterized in that, It includes the following steps: Perform amino-functionalization on the surface of mesoporous bioactive glass nanoparticles using a silane coupling agent. Adsorb small molecule antioxidant drugs into the mesoporous channels and on the surface through the rotary evaporation method. Then graft a pH-sensitive and ROS-responsive gating polymer onto the surface of the drug-loaded nanoparticles through an amidation reaction to construct a nano-drug delivery system capable of dual pH-ROS-responsive release of antioxidant drugs.

2. The preparation method according to claim 1, characterized in that, In the obtained nano-drug delivery system, the nanoparticles of mesoporous bioactive glass are prepared by the sol-gel method using CTAB as a template agent. Its main components are Si, Ca, and O, and the particle size is 100 - 400 nm.

3. The preparation method according to claim 1, characterized in that, Specifically, it includes the following steps: (1) Surface amino-functionalization of mesoporous bioactive glass nanoparticles: Use mesoporous bioactive glass nanoparticles, ultrasonically disperse them in ethanol, add a silane coupling agent, react under a water bath, centrifuge, discard the supernatant, and dry. (2) Loading of small molecule antioxidant drugs: Ultrasonically disperse the amino-functionalized mesoporous bioactive glass nanoparticles obtained in step (1) in methanol, add small molecule antioxidant drugs, ultrasonically disperse and dissolve them evenly, rotary evaporate until the solvent is completely evaporated, and dry to obtain drug-loaded nanoparticles. (3) Grafting of the gating polymer: Ultrasonically disperse the drug-loaded nanoparticles obtained in step (2) in dichloromethane, add condensing agents PyBOP, HOBt, DIPEA, add a ROS-responsive polymer, react at room temperature, centrifuge, discard the supernatant, redisperse in deionized water after drying, add a pH-sensitive polymer, react at room temperature, centrifuge, discard the supernatant, wash with deionized water, and then freeze-dry to obtain a nano-drug delivery system capable of dual pH-ROS response.

4. The preparation method according to claim 3, characterized in that, In step (1), the silane coupling agent is aminopropyltriethoxysilane, aminopropyltrimethoxysilane, 2-aminoethyl-aminopropyl-trimethoxysilane, or aminoethylaminopropylmethyldimethoxysilane, and the addition ratio is 0.1 - 0.5%.

5. The preparation method according to claim 3, characterized in that, In step (2), the small molecule antioxidant drug is gallic acid, quercetin, salidroside, resveratrol, or baicalin. The drug-loading concentration of the small molecule antioxidant drug is 20 - 60 mg / mL, and the drug-loading rate is 20 - 60%.

6. The preparation method according to claim 3, wherein In step (3), the molar ratio of the condensing agents PyBOP, HOBt, and DIPEA is 1:1:1.2 - 1:1:2; the ROS-responsive polymer is one of DSPE-PEG-S-S-COOH, DSPE-PEG-Se-Se-COOH, PLGA-S-S-COOH, PEG-Se-Se-COOH, PEG-TK-COOH, and the molecular weight is 3000 - 10000 Da.

7. The preparation method according to claim 3, characterized in that, In step (3), the pH-sensitive polymer is polymethacrylic acid, and the molecular weight is 10000 - 30000 Da; the mass ratio of the ROS-responsive polymer to the pH-sensitive polymer is 1:1 - 1:

5.

8. An antioxidant nano dual-responsive drug delivery system, characterized in that, It is prepared by the preparation method described in any one of claims 1 - 7.

9. Application of an antioxidant nano-dual-responsive drug delivery system in drug preparation.

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