Efficient sound-sensitive agent based on zinc oxide nanoparticles and preparation method of efficient sound-sensitive agent

Abstract

The invention relates to the field of functional nanometer materials and sound-sensitive agent preparations, in particular to a high-efficiency sound-sensitive agent ZnO (at) Ce6 (at) PEG based on zinc oxide nanoparticles and a preparation method thereof, and belongs to the technical field of functional inorganic nanometer materials. The preparation method comprises the following steps: weighing ZnO-OH nano powder and m PEG-NH2, mixing, and magnetically stirring for 12 hours; ce6, EDC and NHS are weighed at the same time and added into a DMSO solution to be magnetically stirred for 12 h, then the Ce6, the EDC and the NHS are mixed and magnetically stirred for 24 h, dialysate outside a dialysis bag is continuously replaced with ultrapure water and the dialysis bag for 3 days, centrifugation is conducted after dialysis is finished, supernate is removed, and sediment is dried; after the reaction is completed, a powdery ZnO (at) Ce6 (at) PEG sound-sensitive agent is obtained; the efficient sound-sensitive agent ZnO (at) Ce6 (at) PEG based on the zinc oxide nanoparticles, provided by the invention, has relatively good sound-sensitive performance and a good sonodynamic development prospect.

Description

Technical Field

[0001] The present invention relates to the field of functional nanomaterials and sonosensitizer preparations, in particular to a high-efficiency sonosensitizer ZnO@Ce6@PEG based on zinc oxide nanoparticles and a preparation method thereof, belonging to the technical field of functional inorganic nanomaterials. Background Art

[0002] As an inorganic antibacterial agent, ZnO nanomaterials have a wide band gap (3.37 eV) and large exciton binding energy, good near-ultraviolet scattering, good biocompatibility, piezoelectric properties of near-dimensional nanostructures and catalytic functions, and are one of the safest inorganic antibacterial agents. ZnO nanomaterials can work together with organic matter and ultrasound to achieve a synergistic effect, and can be further used as an inorganic piezoelectric nanocarrier with an attached sonosensitizer for in-depth antibacterial research.

[0003] After hydroxylation, -OH groups are formed on the outer surface of ZnO nanomaterials. The potential application is that they can combine with other groups through chemical reactions (dehydration condensation to form ester bonds) to form new substances, and further optimize the functions and characteristics, providing some prerequisites for the design of new materials.

[0004] Sonodynamic therapy (SDT) can use ultrasound to irradiate the tumor site, activate the sonosensitizer that selectively accumulates in the tumor tissue, and trigger a sonodynamic reaction to destroy the tumor tissue. The ideal sonosensitizer needs to have efficient sonodynamic response characteristics, excellent biocompatibility and tumor targeting, and be relatively stable and easy to store. Compared with organic small molecules, inorganic nanomaterials have the advantages of high stability, good hydrophilicity, strong circulation ability, and high tumor targeting.

[0005] At present, the application of inorganic nanomaterials as sonosensitizers is mainly reflected in: 1) using inorganic mesoporous materials with tumor microenvironment responsiveness as carriers of organic sonosensitizers, so that small molecules remain stable under normal physiological conditions, but are rapidly degraded and released in tumor tissues, thereby improving the tumor targeting of small molecules and preventing nonspecific damage; 2) using nanomaterials with their own sonodynamic effects as sonosensitizers, and modifying biocompatible polymers and targeting molecules on the surface to effectively improve the tumor aggregation of nanomaterials. However, if heavy metal elements are added to piezoelectric nanocomposites, a comprehensive evaluation is required to ensure biosafety. The uncertainty and cell damage caused by ultrasonic irradiation require strict control of ultrasonic (US) conditions (frequency, power, duty cycle, duration, etc.). These challenges restrict its development. Efficient piezoelectric sonosensitizers limit the further application of SDT. Researchers are also constantly seeking better solutions. Therefore, the development of inorganic SDT urgently needs to design new piezoelectric sonosensitizers that can effectively separate and migrate electron-hole pairs to improve the generation of reactive oxygen species (ROS). Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the existing inorganic piezoelectric nanosonosensitizer materials, and to provide a high-efficiency sonosensitizer ZnO@Ce6@PEG based on zinc oxide nanoparticles and a preparation method thereof, which has good sonodynamic therapy and development prospects.

[0007] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0008] In the first aspect, the present invention provides a high-efficiency sonosensitizer ZnO@Ce6@PEG based on zinc oxide nanoparticles, wherein the ZnO@Ce6@PEG sonosensitizer is composed of a composite of a piezoelectric nanomaterial ZnO-OH, a sonosensitizer and an organic substance, and the ZnO-OH material is a powdered nanostructured ZnO-OH obtained by hydroxylation of ZnO NPs.

[0009] In a second aspect, the present invention provides a method for preparing a ZnO@Ce6@PEG sonosensitizer, the method being as follows:

[0010] Step S1: Preparation of hydroxylated ZnO Nps: slowly add zinc oxide nanomaterials into a 250 ml round-bottom flask preheated in an oil bath, then slowly add 100 ml of 30% hydrogen peroxide solution into the round-bottom flask containing zinc oxide nanomaterials, stir uniformly in the oil bath to condense and reflux for 12 h, then cool to room temperature, then transfer the product in the round-bottom flask into a 50 ml centrifuge tube, centrifuge at 9000 rpm for 5 min, remove the supernatant, and then wash with deionized water and centrifuge for more than 3 times; dry the precipitate at 80 degrees Celsius for 24 h; after the reaction, obtain powdered ZnO-OH Nps;

[0011] Step S2: Take 100 ml of deionized water, add the ZnO-OH nanopowder obtained in step S1, add an appropriate amount of mPEG-NH2 powder, and then stir the mixture at 500 rpm for 12 hours. At the same time, take 1 mg Ce6, 0.64 mg EDC, and 0.42 mg NHS into 10 ml DMSO solution, and stir at 500 rpm for 12 hours to fully activate the carboxyl groups therein; mix the modified ZnO-OH solution with the activated Ce6 solution and stir at 1000 rpm for 24 hours, and replace the dialysate outside the bag with ultrapure water and dialysis bag (MW: 5000) for 3 days without interruption. After the dialysis is completed, the obtained solution is centrifuged at 9500 rpm for 10 minutes. After removing the supernatant, the precipitate is dried at 80 degrees Celsius for 24 hours; after the reaction is complete, a powdered ZnO@Ce6@PEG sonosensitizer is obtained.

[0012] Preferably, the specific steps for preparing the hydroxylation of ZnO Nps in step S1 are as follows: 3 g of zinc oxide nanomaterial is slowly added to a 250 ml round-bottom flask preheated in an oil bath, and then 100 ml of 30% hydrogen peroxide solution is slowly added to the round-bottom flask containing the zinc oxide nanomaterial, and the mixture is stirred at a constant speed of 30 rpm in the oil bath for 12 hours and then cooled to room temperature, and then the product in the round-bottom flask is transferred to a 50 ml centrifuge tube, centrifuged at 9000 rpm for 5 minutes, and after removing the supernatant, it is washed with deionized water and centrifuged 5 times, and centrifuged at 9500 rpm for 10 minutes per cycle until the supernatant gradually becomes clear; the supernatant is removed, and the precipitate is dried at 80 degrees Celsius for 24 hours; after the reaction is completed, a powdered ZnO-OH nanomaterial is obtained.

[0013] Preferably, in step S2, 1 g of ZnO-OH nanopowder and 0.1 g of mPEG-NH2 are used.

[0014] Preferably, in step S2, 0.64 mg EDC and 0.42 mg NHS are added to 10 ml DMSO solution, and after sufficient stirring, 1 mg Ce6 powder is slowly added thereto.

[0015] Preferably, in step S2, the dialysate outside the bag is replaced every 4-6 hours during the dialysis process, and the dialysate temperature is controlled at 4 degrees Celsius as much as possible.

[0016] Preferably, in step S2, the product obtained after drying is ground with an agate mortar and stored in a refrigerator at 4 degrees Celsius.

[0017] In a third aspect, the present invention provides the application of the ZnO@Ce6@PEG sonosensitizer in sonodynamic therapy.

[0018] The present invention has the following beneficial effects: (1) The ZnO@Ce6@PEG sonosensitizer prepared by the present invention has a tetrahedral hole structure.

[0019] (2) The ZnO@Ce6@PEG sonosensitizer prepared by the present invention has a wider band gap than that of ZnO nanomaterials, so that ZnO@Ce6@PEG has a larger specific surface area and more active centers, which prevents the rapid recombination of electron-hole pairs, thereby generating more electron holes, increasing the yield of ROS, and achieving better sonodynamic therapy.

[0020] (3) The ZnO@Ce6@PEG sonosensitizer obtained by the present invention has better sonosensitivity and higher ROS yield under ultrasound, and has good prospects for the development of SDT. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is a physical picture of the ZnO@Ce6@PEG sonosensitizer prepared in Example 1;

[0022] Figure 2 is a transmission electron microscopy image of the ZnO@Ce6@PEG sonosensitizer prepared in Example 2;

[0023] Figure 3 It is the Fourier transform infrared spectroscopy image of the ZnO-OH nanomaterial, the ZnO@Ce6@PEG sonosensitizer and the ZnO nanomaterial prepared in Example 2;

[0024] Figure 4 It is the calculated band gap image of the ultraviolet-visible spectrum of the ZnO@Ce6@PEG sonosensitizer and ZnO nanomaterial prepared in Example 2;

[0025] Figure 5 This is the image of the decrease in absorbance of the ZnO@Ce6@PEG sonosensitizer prepared in Example 3 under different ultrasound action times;

[0026] Figure 6 It is the absorption spectra of the ZnO@Ce6@PEG sonosensitizer and sonosensitizer Ce6 prepared in Example 3 under simultaneous ultrasound action for different time periods; Implementation

[0027] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention. Example 1: A highly efficient sonosensitizer based on zinc oxide nanoparticles ZnO@Ce6@PEG

[0028] Step S1 Preparation of hydroxylated ZnO Nps: 3 g of zinc oxide nanomaterial is slowly added to a 250 ml round-bottom flask preheated in an oil bath, and then 100 ml of 30% hydrogen peroxide solution is slowly added to the round-bottom flask containing the zinc oxide nanomaterial. The mixture is stirred at 30 rpm in the oil bath for 12 h and then cooled to room temperature. The product in the round-bottom flask is then transferred to a 50 ml centrifuge tube and centrifuged at 9000 rpm for 5 min. After removing the supernatant, the mixture is washed with deionized water and centrifuged for 5 times. The mixture is centrifuged at 9500 rpm for 10 min per cycle until the supernatant gradually becomes clear. The supernatant is removed and the precipitate is dried at 80 degrees Celsius for 24 h. After the reaction is completed, a powdered ZnO-OH nanomaterial is obtained.

[0029] Step S2: Take 100 ml of deionized water, add the ZnO-OH nanopowder obtained in step S1, add an appropriate amount of mPEG-NH2 powder, and then stir the mixture at 500 rpm for 12 hours. At the same time, take 1 mg Ce6, 0.64 mg EDC, and 0.42 mg NHS into 10 ml DMSO solution, and stir at 500 rpm for 12 hours to fully activate the carboxyl groups therein; mix the modified ZnO-OH solution with the activated Ce6 solution and stir at 1000 rpm for 24 hours, and replace the dialysate outside the bag with ultrapure water and dialysis bag (MW: 5000) for 3 days without interruption. After the dialysis is completed, the obtained solution is centrifuged at 9500 rpm for 10 minutes. After removing the supernatant, the precipitate is dried at 80 degrees Celsius for 24 hours; after the reaction is complete, a powdered ZnO@Ce6@PEG sonosensitizer is obtained.

[0030] Example 2: Synthesis and characterization of the microstructure and morphology of the prepared ZnO@Ce6@PEG sonosensitizer.

[0031] After the ZnO nanomaterial is hydroxylated, ZnO-OH is obtained. The steps of loading the sonosensitizer Ce6 and the organic substance PEG through chemical reaction to finally synthesize the ZnO@Ce6@PEG sonosensitizer are as follows:

[0032] Step S1, take 100ml of deionized water, add 1g of ZnO-OH nanopowder thereto, then add 0.1g of mPEG-NH2 powder, and then stir the mixture at 500rpm for 12h. At the same time, take 1mgCe6, 0.64mgEDC, and 0.42mgNHS into 10mlDMSO solution, and stir at 500rpm for 12h to fully activate the carboxyl groups therein; after the modified ZnO-OH solution and the activated Ce6 solution are mixed, stir at 1000rpm for 24h, and replace the dialysate outside the bag with ultrapure water and dialysis bag (MW: 5000) for 3 days without interruption. After the dialysis is completed, the obtained solution is centrifuged at 9500rpm for 10min, and after removing the supernatant, the precipitate is dried at 80 degrees Celsius for 24h; the obtained product after drying is ground with an agate mortar to finally obtain a powdered ZnO@Ce6@PEG sonosensitizer (such as Figure 1 shown).

[0033] Step S2, characterize the ZnO@Ce6@PEG sonosensitizer. First, the ZnO@Ce6@PEG nanomaterial has a unique tetrahedral structure (such as Figure 2 As shown in the figure, it can be clearly seen from the transmission electron microscope that the ZnO@Ce6@PEG nanomaterial has a regular structure, the layers are closely arranged, the particle size is larger than before coating, the average diameter is about 15nm, and the particle size distribution is uniform.

[0034] Fourier transform infrared spectroscopy was used to compare and analyze ZnO NPs, ZnO-OH and ZnO@Ce6@PEG (e.g. Figure 3 As shown in Figure 2, ZnO-OH is at 3430 cm-1 compared with ZnO. -1 There is an obvious decrease in the absorption peak at , and the hydroxyl stretching vibration peak appears, proving that the hydroxyl group is successfully loaded;

[0035] Compared with ZnO-OH and ZnO@Ce6@PEG, we can see that at 2930 cm -1 There is an obvious decrease in the stretching peak at the position, which belongs to the stretching vibration peak caused by C=O. The infrared structure analysis shows that the carboxyl group has been successfully loaded, and the hydroxylated ZnONPs has successfully combined with the free Ce6, which also leads to an increase in the peak value at the original 3430cm-1 hydroxyl stretching vibration peak, because the hydroxyl group is consumed in chemical reactions, which also lays the foundation for the subsequent ultrasound-excited piezoelectric sonodynamic therapy.

[0036] The band gap was calculated by UV-visible spectroscopy (e.g. Figure 4 As shown in the figure, the band gap of ZnO NPs is measured to be 3.25ev, and the band gap of ZnO@Ce6@PEG is measured to be 4.10ev, which increases from 3.25ev to 4.10ev. Due to the addition of Ce6, ZnO@Ce6@PEG has a larger specific surface area and more active centers, which prevents the rapid recombination of electron-hole pairs, thereby generating more electron holes, enhancing the yield of ROS, and achieving a better SDT effect.

[0037] Example 3: Evaluation of sonosensitivity of ZnO@Ce6@PEG sonosensitizer

[0038] In order to further evaluate the sonosensitivity of ZnO@Ce6@PEG nanomaterials, the mixture was ultrasonically treated with DPBF (concentration of 20ug / ml) for 0s, 10s, 20s, 30s, 60s, and 90s, and the generation of reactive oxygen species was determined by measuring the decrease in absorbance (the concentration of ZnO@Ce6@PEG was 0.2mg / ml) (e.g. Figure 5 As shown in the figure, the absorbance decreases with the increase of ultrasound action time within a certain range, and can decrease by 25.38% at 90s, indicating that the ZnO@Ce6@PEG nanomaterial acts quickly, has good sonosensitivity, has a high active oxygen production rate under ultrasound, and has good prospects for the development of sonodynamics.

[0039] The ZnO@Ce6@PEG sonosensitizer was compared with the original sonosensitizer Ce6, and ultrasound was applied for 0s, 10s, 20s, 30s, 60s, and 90s respectively. At each time point, the absorbance decrease percentage of the ZnO@Ce6@PEG sonosensitizer was higher than that of Ce6, indicating that the ZnO@Ce6@PEG sonosensitizer can produce more ROS and has a better SDT effect.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A highly efficient sonosensitizer ZnO@Ce6@PEG based on zinc oxide nanoparticles, characterized in that: The ZnO@Ce6@PEG sonosensitizer is composed of a ZnO nanomaterial loaded with a sonosensitizer dihydrochlorin (Ce6) and a hydrophilic organic polyethylene glycol (PEG). The ZnO@Ce6@PEG sonosensitizer needs to be prepared in two steps: first, zinc oxide nanoparticles (ZnO Nps) are hydroxylated to obtain ZnO-OH Nps, and then a ZnO@Ce6@PEG sonosensitizer is prepared, which is compounded by chemical reaction with the sonosensitizer Ce6, and finally the ZnO@Ce6@PEG sonosensitizer is obtained after an outer layer of PEG is wrapped.

2. The method for preparing ZnO-OH Nps according to claim 1, characterized in that: The method is as follows: Step S1: Preparation of hydroxylated ZnO Nps: Slowly add zinc oxide nanomaterials into a 250 ml round-bottom flask preheated in an oil bath, then slowly add 100 ml of 30% hydrogen peroxide solution into the round-bottom flask containing the zinc oxide nanomaterials, stir uniformly in the oil bath, condense and reflux for 12 h, and then cool to room temperature, then transfer the product in the round-bottom flask to a 50 ml centrifuge tube, centrifuge at 9000 rpm for 5 min, remove the supernatant, and then wash with deionized water and centrifuge for more than 3 times; dry the precipitate at 80 degrees Celsius for 24 h; after the reaction, obtain powdered ZnO-OH Nps.

3. The preparation method of the ZnO@Ce6@PEG sonosensitizer according to claim 1, characterized in that: The method is as follows: Step S2: Preparation of ZnO@Ce6@PEG sonosensitizer: Take 100 ml of deionized water, add the ZnO-OH nanopowder obtained in step S1, add an appropriate amount of methoxypolyethylene glycolamine (mPEG-NH2), and then stir the mixture magnetically at 500 rpm for 12 h. At the same time, take 1 mg of Ce6, 0.64 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and 0.42 mg N-hydroxysuccinimide (NHS) was added to 10 ml of dimethyl sulfoxide (DMSO) solution and magnetically stirred at 500 rpm for 12 hours to fully activate the carboxyl groups therein; the modified ZnO-OH solution was mixed with the activated Ce6 solution and magnetically stirred at 1000 rpm for 24 hours, and the dialysate outside the bag was replaced with ultrapure water and a dialysis bag (MW: 5000) for 3 days without interruption. After the dialysis, the obtained solution was centrifuged at 9500 rpm for 10 minutes, and after removing the supernatant, the precipitate was dried at 80 degrees Celsius for 24 hours; after the reaction was complete, a powdered ZnO@Ce6@PEG sonosensitizer was obtained.

4. The method for preparing ZnO-OH Nps according to claim 2, characterized in that: Step S1 The specific steps for the preparation of hydroxylated ZnO Nps are as follows: 3 g of zinc oxide nanomaterial is slowly added to a 250 ml round-bottom flask preheated in an oil bath, and then 100 ml of 30% hydrogen peroxide solution is slowly added to the round-bottom flask containing the zinc oxide nanomaterial. The mixture is stirred at 30 rpm in the oil bath for 12 hours and then cooled to room temperature. The product in the round-bottom flask is then transferred to a 50 ml centrifuge tube and centrifuged at 9000 rpm for 5 minutes. After removing the supernatant, the mixture is washed with deionized water and centrifuged for 5 times, and centrifuged at 9500 rpm for 10 minutes per cycle until the supernatant gradually becomes clear. The supernatant is removed and the precipitate is dried at 80 degrees Celsius for 24 hours. After the reaction is completed, a powdered ZnO-OH nanomaterial is obtained.

5. The method for preparing the ZnO@Ce6@PEG sonosensitizer according to claim 3, characterized in that: In step S2, 1 g of ZnO-OH nanopowder and 0.1 g of mPEG-NH2 were used.

6. The method for preparing the ZnO@Ce6@PEG sonosensitizer according to claim 3, characterized in that: In step S2, 0.64 mg EDC and 0.42 mg NHS were added to 10 ml DMSO solution, and after sufficient stirring, 1 mg Ce6 powder was slowly added thereto.

7. The method for preparing the ZnO@Ce6@PEG sonosensitizer according to claim 3, characterized in that: In step S2, the dialysate outside the bag is replaced every 4-6 hours during the dialysis process, and the dialysate temperature needs to be controlled at 4 degrees Celsius.

8. The method for preparing the ZnO@Ce6@PEG sonosensitizer according to claim 3, characterized in that: In step S2, the product obtained after drying is ground with an agate mortar and stored in a refrigerator at 4 degrees Celsius.

Images