Infrared light release hydrogen sulfide nano-reactor, preparation method thereof and application of infrared light release hydrogen sulfide nano-reactor in spinal cord injury repair
Patent Information
- Application Number
- CN202311832386.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-28
- Publication Date
- 2025-07-01
Smart Images
Figure CN120227457A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of material synthesis, and particularly relates to an infrared light-releasing hydrogen sulfide nanoreactor, a preparation method thereof, and an application thereof in spinal cord injury repair. Background Art
[0002] The spinal cord is an important part of the central nervous system and serves as a connection between the brain and peripheral nerve tissues by transmitting important information and reflexes (Lancet 2022, 24(13), 417–425). Spinal cord injury is a temporary or permanent impairment of spinal cord function under the action of external forces. It is estimated that there are 250,000 to 500,000 new cases of spinal cord injury worldwide each year, with the majority being young people. The mortality rate of patients with acute spinal cord injury ranges from 4% to 17% (Neurosurg. 2020, 43(2), 425–441). Spinal cord injuries are divided into traumatic and non-traumatic. After traumatic spinal cord injury occurs, although the primary mechanical compression can be relieved surgically, the secondary injury after spinal cord injury, whose molecular and cellular level mechanisms are very complex.
[0003] In addition, complex physiology and uncontrollable pathological processes may lead to unsatisfactory recovery results. The high incidence, high disability rate of the disease, and high economic consumption of treatment result in a reduced quality of life of patients after surgery. Traditional decompression surgery to reconstruct spinal stability and the use of high-dose intravenous methylprednisolone (MPSS) in the acute phase are the most common clinical treatment methods. Although surgical treatment can reconstruct spinal stability and relieve the compressed nerves, complete repair of the central nerve is still difficult to achieve. The use of high-dose MPSS to reduce early inflammatory responses also has problems such as low bioavailability and low aggregation efficiency (Nanoscale 2013, 5(19), 8821-36). It is worth noting that none of these treatments have shown the ability to promote axon and nerve regeneration, and the treatment effect is limited. Therefore, it is necessary to seek more effective treatment methods to solve these problems.
[0004] Medical gas therapy has been widely applied in clinical treatment and scientific research, providing solutions for various medical needs. Pharmaceutical molecules range from traditional gases (oxygen and nitrous oxide) to gases such as nitric oxide, carbon monoxide, and hydrogen sulfide, all of which have recently been shown to act as bioactive messenger molecules. Messenger molecules play a role in regulating the body's basic physiological activities after binding to multivalent metal elements in the body. In potential treatment methods for neurological diseases, clinical studies have found that medical gases have unique advantages over more traditional pharmacological strategies, such as the easy diffusibility, high permeability, and low drug resistance of gases. Among the gases commonly used in the current field of neurological diseases, nitric oxide and carbon monoxide have disadvantages such as inducing inflammation in the late stage of treatment and extremely low safety thresholds. Hydrogen sulfide has been verified to have the effects of vasoregulation, reducing oxidative stress response, and reducing inflammatory response, so it is more suitable for repairing spinal cord injury.
[0005] However, there are also significant controversies regarding current treatment methods. The unfamiliarity with gas therapy will, to a certain extent, lead to the resistance of patients, and the research and development of related products also face many difficulties. The property of gas as a natural substance makes it impossible to directly obtain high commercial value as a product. Therefore, some current studies have focused on the concept of a nano gas generator. Bu et al. loaded a photothermal-excitable nitric oxide donor onto an upconversion material-modified metal-organic framework, and through near-infrared triggering, used nitric oxide as a bioactive signal molecule to inhibit glial cell regeneration (Sci. Adv. 2020, 6(39), eabc3513). However, due to the inherent properties of nitric oxide, there are still defects in inducing inflammation in the later stage. Nevertheless, the idea of constructing a nano gas generator, as well as the advantages of controlled release, integration, and tunability of the nano gas generator, will contribute to the transformation and application of traditional gas therapy. Summary of the Invention
[0006] In view of the above problems, the present invention provides an infrared light-releasing hydrogen sulfide nano-reactor, innovatively proposing to in-situ control the release of hydrogen sulfide therapeutic gas through a light field to protect spinal cord neurons from secondary injury. And due to the good lipid solubility of hydrogen sulfide, hydrogen sulfide can easily pass through the blood-brain-spinal cord barrier and cell membrane barrier and enter the intracellular structure for the treatment of spinal cord injury.
[0007] In a first aspect, the present invention provides an infrared light-releasing hydrogen sulfide nano-reactor. The infrared light-releasing hydrogen sulfide nano-reactor includes an injectable PVA / PVP transparent conductive hydrogel and an SNSs / PEI-DTC complex filled inside the injectable PVA / PVP transparent conductive hydrogel; the SNSs / PEI-DTC complex includes a donor PEI-DTC and two-dimensional silicene nanosheets loaded on the surface of the donor PEI-DTC.
[0008] Preferably, the mass ratio of the injectable PVA / PVP transparent conductive hydrogel to the SNSs / PEI-DTC complex is 1000:1 - 10000:1.
[0009] Preferably, the ratio of two-dimensional silicene nanosheets to PEI-DTC is 20 - 30 μg: 0.15 - 0.2 mg.
[0010] In a second aspect, the present invention provides a method for preparing an infrared light-responsive hydrogen sulfide nanoreactor. The method for preparing the infrared light-responsive hydrogen sulfide nanoreactor includes: Synthesizing and exfoliating two-dimensional silicene nanosheets by a wet chemical method; Modifying the surface of the two-dimensional silicene nanosheets with a hydrogen sulfide-producing donor PEI-DTC by thermal decomposition to obtain SNSs / PEI-DTC; Entirely wrapping SNSs / PEI-DTC in an injectable PVA / PVP transparent conductive hydrogel to form an infrared light-responsive hydrogen sulfide nanoreactor.
[0011] Preferably, the method for preparing two-dimensional silicene nanosheets includes: adding precursors CaSi2 and I2 to anhydrous acetonitrile, with a molar ratio of CaSi2 to I2 of 1:1, maintaining stirring under N2 atmosphere protection, and continuously reacting at room temperature for 2 - 3 weeks; centrifuging the reaction solution, taking the solid product, dispersing it in an organic solvent, and ultrasonically crushing it to obtain a dispersion containing two-dimensional silicene nanosheets; preferably, the ratio of CaSi2: I2: anhydrous acetonitrile is 570 - 575 mg: 1520 - 1530 mg: 160 - 170 mL; more preferably, repeatedly eluting and drying with absolute ethanol and deionized water to obtain two-dimensional silicene nanosheets.
[0012] Preferably, the method for preparing PEI-DTC includes: dissolving PEI and KOH in methanol, stirring until KOH is completely dissolved; adding CS2 to the obtained solution, stirring for 10 - 20 min, and drying to obtain PEI-DTC; preferably, the ratio of PEI: KOH: methanol is 500 - 505 mg: 650 - 655 mg: 50 - 55 mL; more preferably, the ratio of PEI: CS2 is 500 - 505 mg: 695 - 700 μL.
[0013] Preferably, PEI-DTC is loaded on the surface of two-dimensional silicene nanosheets by electrostatic adsorption. Preferably, at room temperature, two-dimensional silicene nanosheets are dispersed in water to prepare solution A; PEI-DTC is dissolved in deionized water to form solution B; solution A is centrifuged, the supernatant is poured off, and solution A is mixed with the same volume of solution B. After centrifugation, the supernatant is discarded to obtain the SNSs / PEI-DTC composite. More preferably, the concentration of two-dimensional silicene nanosheets in solution A is 0.02 - 0.03 mg / mL; the concentration of PEI-DTC in solution B is 0.15 - 0.2 mg / mL.
[0014] Preferably, the PVA / PVP hydrogel is prepared by the cyclic freezing and thawing method. Preferably, an aqueous PVA / PVP solution with a PVP concentration of 0.5 - 5 wt% is prepared; under stirring, the PVA / PVP solution is dissolved at a constant temperature of 90 - 95 °C for 4 - 4.5 hours; stirring is stopped, and the solution is left standing at 95 - 100 °C for 1.5 - 2 hours; it is frozen at -18 to -20 °C for 18 - 18.5 hours and thawed at room temperature for 6 - 6.5 hours. Preferably, the freezing and thawing are repeated 5 - 6 times.
[0015] Preferably, the SNSs / PEI-DTC composite is uniformly mixed with the PVA / PVP hydrogel to wrap the SNSs / PEI-DTC composite entirely within the injectable PVA / PVP transparent conductive hydrogel.
[0016] In a third aspect, the present invention provides the use of the infrared light-releasing hydrogen sulfide nanoreactor described in any one of the above in the preparation of a spinal cord injury repair product.
[0017] Preferably, the infrared light-releasing hydrogen sulfide nanoreactor is injected into the spinal cord injury site, and the activation effect of irradiation with an 808 nm infrared laser causes the nanogenerator to release hydrogen sulfide gas.
[0018] Beneficial effects
[0019] 1) The hydrogen sulfide nanogenerator provided by the present invention has stable hydrogen sulfide controlled release, anti-inflammatory, and spinal cord injury repair effects, and is applicable to the biomedical field including the treatment of the acute phase of spinal cord injury inflammation.
[0020] 2) The preparation process of the nanogenerator provided by the present invention is simple, the operation is convenient, it does not require complex and expensive equipment, and it is easy to realize industrial production.
[0021] 3) The nanogenerator provided by the present invention can achieve a rapid recovery in the acute phase of inflammation within 2 weeks, and the effect is obvious. And it has an obvious behavioral recovery for 28 days. The nanogenerator material of the present invention will have good application prospects as a biomedical material. Description of the drawings
[0022] Figure 1 It is the TEM image of the silicene nanosheets prepared in Example 1.
[0023] Figure 2 It is the ultraviolet spectrum (UV) image of PEI (bottom) and PEI-DTC prepared in Example 1 (top).
[0024] Figure 3 It is the SEM image of the PVA / PVP transparent conductive hydrogel prepared in Example 1.
[0025] Figure 4 It is the AC impedance image of the conductive hydrogel. The PVP concentrations of PVA / PVP-1, PVA / PVP-2, and PVA / PVP-3 are 0.5 wt%, 1 wt%, and 5 wt% respectively.
[0026] Figure 5 It is the effect image of the SNSs / PEI-DTC complex scavenging ROS.
[0027] Figure 6 It is the construction and surgical schematic diagram of the nanoreactor.
[0028] Figure 7 It is the tissue section images of HE, Nissl, and LFB staining of the nanoreactor and the SCI group. Detailed implementation manners
[0029] The present invention is further illustrated by the following implementation manners. It should be understood that the following implementation manners are only used to illustrate the present invention, rather than limiting the present invention.
[0030] The infrared light-releasing hydrogen sulfide nanoreactor (which can also be called "hydrogen sulfide nano gas generator" or "near-infrared light in-situ hydrogen sulfide-releasing nanoreactor") described in the present invention includes an injectable PVA / PVP transparent conductive hydrogel and an SNSs / PEI-DTC complex filled inside the injectable PVA / PVP transparent conductive hydrogel; the SNSs / PEI-DTC complex includes a donor PEI-DTC and two-dimensional silicene nanosheets loaded on the surface of the donor PEI-DTC. The infrared light-releasing hydrogen sulfide nanoreactor described in the present invention combines two-dimensional silicene with good biosecurity, a PEI-DTC donor, and an injectable PVA / PVP transparent conductive hydrogel. Two-dimensional silicene has excellent photothermal conversion efficiency. The PEI-DTC donor is different from common hydrolyzable donors on the market and can achieve in-vivo slow release by thermal decomposition. The PVA / PVP hydrogel has good light transmittance and conductivity, can be used as a good carrier for the nano gas generator, and can replace the injured spinal cord to transmit electrical signals. Two-dimensional silicene can fully decompose PEI-DTC to generate H2S gas, which can pass through the blood-brain-spinal cord barrier and cell membrane barrier, enabling the treatment to enter the cells.
[0031] Preferably, the mass ratio of the injectable PVA / PVP transparent conductive hydrogel to the SNSs / PEI-DTC complex is 1000:1 - 10000:1; the ratio of two-dimensional silicene nanosheets to PEI-DTC is 20 - 30 μg:0.15 - 0.2 mg. At the above ratios, when the SNSs / PEI-DTC is irradiated with 808 nm infrared laser, the released hydrogen sulfide concentration is within the biosafety range. And at the ratios of the hydrogel and the complex, the maximum temperature of the nanogenerator under infrared laser irradiation is about 37 °C, which meets the human safety temperature. Preferably, the mass ratio of the injectable PVA / PVP transparent conductive hydrogel to the SNSs / PEI-DTC complex is 2000:1 - 8000:1. More preferably, the mass ratio of the injectable PVA / PVP transparent conductive hydrogel to the SNSs / PEI-DTC complex is 2000:1 - 6000:1. Additionally optionally, the proportion of PVP in the PVA / PVP transparent conductive hydrogel is 0.5 - 5 wt%.
[0032] The preparation method of the infrared light-released hydrogen sulfide nanoreactor of the present invention may include: synthesizing and exfoliating two-dimensional silicene nanosheets by a wet chemical method; modifying the surface of the two-dimensional silicene nanosheets with a donor PEI-DTC that thermally decomposes to produce hydrogen sulfide to obtain Si / PEI-DTC; wrapping the Si / PEI-DTC as a whole in an injectable PVA / PVP transparent conductive hydrogel to form an infrared light-released hydrogen sulfide nanoreactor. Specific descriptions will be given below.
[0033] Preparation of two-dimensional silicene nanosheets. The precursors CaSi2 and I2 were added to anhydrous acetonitrile, and the molar ratio of CaSi2 to I2 was 1:1. Under stirring and N2 atmosphere protection, the reaction was continuously carried out at room temperature for 2 - 3 weeks; the reaction solution was centrifuged, and the solid product was taken and dispersed in an organic solvent for ultrasonic fragmentation to obtain a dispersion containing two-dimensional silicene nanosheets. When in use, the organic solvent in the dispersion was removed, washed with water and absolute ethanol, and then dried to obtain two-dimensional silicene nanosheets. In some technical solutions, 570 - 575 mg of CaSi2 and 1520 - 1530 mg of I2 powder (molar ratio = 1:1) were added to 160 - 170 mL of anhydrous acetonitrile and stirred. Under N2 atmosphere protection, the reaction was continuously carried out at room temperature for 2 - 3 weeks. Centrifuged and washed with anhydrous acetonitrile, stored in an NMP solution, and ultrasonically fragmented at an ultrasonic power of 150 - 200 W for 5 - 8 h to obtain a dispersion containing two-dimensional silicene nanosheets. As an example, 575 mg of CaSi2 and 1524 mg of I2 powder (molar ratio = 1:1) were added to 160 mL of anhydrous acetonitrile and stirred. Under N2 atmosphere protection, the reaction was continuously carried out at room temperature for 2 weeks; centrifuged and washed with anhydrous acetonitrile, stored in an NMP solution, and ultrasonically fragmented at an ultrasonic power of 150 - 200 W for 5 - 8 h to obtain a dispersion containing two-dimensional silicene nanosheets. As an example, the thickness of the two-dimensional silicene nanosheets is 1 - 5 nm.
[0034] Preparation of PEI-DTC (polyethyleneimine copolymerized dithiocarbamate). PEI and KOH were dissolved in methanol and stirred until KOH was completely dissolved; CS2 was added to the resulting solution and stirred for 10 - 20 min, and then dried to obtain PEI-DT. In some technical solutions, 500 - 505 mg of PEI (0.02 - 0.0202 mmol) and 650 - 655 mg of KOH (11.6 - 11.7 mmol) were dissolved in 50 - 55 mL of methanol and stirred until KOH was completely dissolved. The solution was purged with N2 for 5 - 10 min to completely expel oxygen, and 695 - 700 μL of CS2 (11.6 - 11.69 mmol) was slowly added to the mixed solution. Stir for 10 - 20 min. The color of the solution turned light yellow. After rotary evaporation, the solid sample PEI-DTC was obtained.
[0035] Load PEI-DTC on the surface of two-dimensional silicene nanosheets by electrostatic adsorption. At room temperature, disperse two-dimensional silicene nanosheets in water to prepare solution A; dissolve PEI-DTC in deionized water to form solution B; centrifuge solution A, pour off the supernatant, and mix it with the same volume of solution B as solution A. After centrifugation, discard the supernatant to obtain the SNSs / PEI-DTC composite. In some technical solutions, at room temperature, disperse 20-30 mg of solid silicene nanosheets in 1000 mL of deionized water to prepare solution A with a concentration of 20-30 ppm. Dissolve 15-20 mg of PEI-DTC in 100 mL of deionized water to form solution B. Centrifuge 1 mL of solution A, pour off the supernatant, and mix it with 1 mL of solution B. After centrifugation, discard the supernatant to obtain SNSs / PEI-DTC (two-dimensional silicene nanosheets with PEI-DTC loaded on the surface). As an example, at room temperature, disperse 30 mg of two-dimensional silicene nanosheets in 1000 mL of water to prepare solution A with a concentration of 30 ppm; dissolve 20 mg of PEI-DTC in 100 mL of deionized water to form solution B; centrifuge 1 mL of solution A, pour off the supernatant, and mix it with 1 mL of solution B. After centrifugation, discard the supernatant to obtain SNSs / PEI-DTC.
[0036] Prepare the PVA / PVP hydrogel using the cyclic freeze-thaw method. During freezing, the PVA chains form hydrogen bonds through hydroxyl groups, and the polymer chains are locally ordered to form polymer crystallization. Adding PVP and through cyclic freeze-thaw, the crystallinity of PVA is continuously enhanced, thereby forming a hydrogel with crystal regions as physical cross-linking points. As an example, prepare an aqueous solution of PVA / PVP with 0.5-5 wt% PVP. This ratio refers to the proportion of PVP in PVA / PVP. As an example but not limited to this, the amount of water used can be 2-20 times the total mass of PVA / PVP. Under magnetic stirring, dissolve the solution at a constant temperature of 90-95 °C for 4-4.5 hours. Stop stirring and let it stand at 95-100 °C for 1.5-2 hours. Place it in a freezer at -18--20 °C for 18-18.5 hours and thaw at room temperature for 6-6.5 hours. Preferably, repeat freezing and thawing 5-6 times. As an example, use the cyclic freeze-thaw method to prepare the PVA / PVP hydrogel; preferably, prepare a PVA / PVP solution with 1 wt% PVP; under stirring, dissolve the PVA / PVP solution at a constant temperature of 90 °C for 4 hours; stop stirring and let it stand at 95 °C for 2 hours; place it in a freezer at -20 °C for 18 hours and thaw at room temperature for 6 hours.
[0037] The two-dimensional silicene nanosheets with surface-loaded PEI-DTC were uniformly mixed with PVA / PVP hydrogel to obtain an infrared light-released hydrogen sulfide nanoreactor. As a classic photothermal material, silicene has good photothermal conversion efficiency under infrared light irradiation, which is beneficial to stimulate PEI-DTC to generate hydrogen sulfide. In order to avoid the inflammatory reaction caused by high temperature, PVA / PVP transparent conductive hydrogel was synthesized by the cyclic freeze-thaw method. It can not only protect biological tissues by using the high specific heat capacity of water, but also the conductivity of the hydrogel can be used as a functional device to replace damaged nerves to transmit electrical signals.
[0038] The present invention first proposes a nano gas generator for thermally controlled release of hydrogen sulfide gas through an optical field in situ to protect spinal cord neurons from secondary injury. The above nano gas generator was injected into the spinal cord injury site, and the activation effect of irradiation with an 808 nm infrared laser was used to make the nano gas generator release hydrogen sulfide gas. The injectable hydrogen sulfide nano generator can be used for early inflammation relief and promotion of injury repair in spinal cord injury, and has good biosafety. The present invention has important scientific significance and application value for expanding the preparation method and application scope of injectable hydrogen sulfide gas generator materials for spinal cord injury repair.
[0039] The following further lists examples to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the protection scope of the present invention. The specific process parameters and the like in the following examples are only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description herein, rather than being limited to the specific values in the following examples.
[0040] Example 1
[0041] 160 mL of anhydrous acetonitrile (CH3CN) was added to a 1000 mL round-bottom flask, and 575 mg of CaSi2 and 1524 mg of I2 powder (molar ratio = 1:1) were added in sequence. The resulting mixture was magnetically stirred at room temperature for 2 weeks. The reaction system was always under nitrogen protection throughout the reaction process. After the reaction, the product was washed 3 times with anhydrous acetonitrile and dispersed in 50 mL of NMP, and ultrasonically broken at an ultrasonic power of 200 W for 6 h. When taking samples, the solid product was collected by centrifugation, repeatedly eluted with anhydrous ethanol and deionized water, and dried to obtain few-layer silicene nanosheets with uniform size.
[0042] At room temperature, 500 mg of PEI (0.02 mmol) and 650 mg of KOH (11.6 mmol) were dissolved in 50 mL of methanol, and stirred until KOH was completely dissolved. N2 was introduced into the resulting mixed solution and maintained for 15 s to completely expel the oxygen in the solution. 695 μL of CS2 (11.6 mmol) was slowly added to the solution, and stirred for 10 min. The solution color changed to light yellow. Rotary evaporation was carried out to obtain PEI-DTC.
[0043] 9.9 g (99 wt%) of polyvinyl alcohol (PVA) and 0.1 g (1 wt%) of polyvinylpyrrolidone (PVP) were dissolved in 100 mL of deionized water. The mixed solution was placed in a round-bottom flask and magnetically stirred at a constant temperature of 90 °C for 4 h, and then left to stand at 95 °C for 2 h. It was frozen at -20 °C for 18 h and thawed at room temperature for 6 h. Taking this as a cycle, the freezing and thawing steps were repeated 5 times to obtain an injectable PVA / PVP transparent conductive hydrogel.
[0044] At room temperature, solid silicene nanosheets were dispersed in deionized water to prepare solution A with a concentration of 30 ppm. 2 mg of PEI-DTC was dissolved in 10 mL of deionized water to form solution B. 1 mL of solution A was centrifuged, the supernatant was poured off, and it was mixed with 1 mL of solution B. After centrifugation, the supernatant was discarded and dried to obtain the SNSs / PEI-DTC complex (solid, about 2 mg). The SNSs / PEI-DTC complex was vortexed into 5 g of PVA / PVP hydrogel to obtain a hydrogen sulfide nano gas generator.
[0045] Figure 1 It is the TEM image of the silicene nanosheets prepared in Example 1. It can be seen that the few-layer silicene has good dispersibility.
[0046] Figure 2 It is the ultraviolet spectrum (UV) image of PEI and PEI-DTC prepared in Example 1. It can be seen that PEI-DTC has two new absorption peaks at 260 nm and 290 nm, and these two peaks respectively correspond to the dithiocarbamate salts formed by the reaction of primary and secondary amine groups in the polymer PEI with carbon disulfide. The above results confirm the successful synthesis of the polymer PEI-DTC.
[0047] Figure 3 It is the SEM image of the PVA / PVP transparent conductive hydrogel prepared in Example 1. It can be seen that the hydrogel has an obvious porous channel structure, which helps the gas to escape.
[0048] Figure 4It is the AC impedance diagram of the conductive hydrogel. PVA / PVP-1 has PVP = 0.5 wt%. PVA / PVP-2 has PVP = 1 wt%. PVA / PVP-3 has PVP = 5 wt%. It can be seen that with the increase of PVA, the conductivity improves.
[0049] The cell efficacy test was evaluated by ROS scavenging experiment. The samples for the experiment were divided into a control group (Control), a control + SNSs / PEI-DTC group (Control+SNSs / PEI-DTC), an H2O2 group, and an H2O2 + SNSs / PEI-DTC group. H2O2 + SNSs / PEI-DTC group: 200 μM H2O2 and 1 mL SNSs / PEI-DTC were added to PC12 cells for co-incubation. The specific operation was as follows: PC12 cells (Cell Bank of the Chinese Academy of Sciences) were seeded into a 96-well plate at a density of 1×10 4 / well, and then cultured in a CO2 incubator at 37 °C with humid air containing 5% CO2 for 24 h to allow the cells to adhere. Then the PC12 cells were treated with 200 μM H2O2 incomplete medium for 2 h to induce oxidative stress and overexpression of reactive oxygen species in PC12 cells. Subsequently, the medium was aspirated, and fresh serum-free culture medium containing silicene nanosheets loaded with PEI-DTC (200 μg / mL) was added and incubated for another 8 h. After the incubation was completed, the culture medium was removed, and the cells were washed 3 times with fresh PBS. Then, CCK-8 solution diluted tenfold with the medium was added to each well, and the plate was placed in a CO2 incubator at 37 °C with humid air containing 5% CO2 and co-incubated for another 2 h. Finally, the absorbance was measured on a microplate reader (λ = 450 nm).
[0050] Control group (Control): PC12 cells were not treated and grew normally. The specific operation was as follows: PC12 cells (Cell Bank of the Chinese Academy of Sciences) were seeded into a 96-well plate at a density of 1×10 4 / well, and then cultured in a CO2 incubator at 37 °C with humid air containing 5% CO2 for 24 h to allow the cells to adhere.
[0051] Control + SNSs / PEI-DTC group (Control+SNSs / PEI-DTC): 1 mL SNSs / PEI-DTC was added to PC12 cells for co-incubation. The specific operation was as follows: PC12 cells (Cell Bank of the Chinese Academy of Sciences) were seeded into a 96-well plate at a density of 1×10 4 / well, and then cultured in a CO2 incubator at 37 °C with humid air containing 5% CO2 for 24 h to allow the cells to adhere. Subsequently, the medium was aspirated, and fresh serum-free culture medium containing silicene nanosheets loaded with PEI-DTC (200 μg / mL) was added and incubated for another 8 h.
[0052] H2O2 group: PC12 cells were co-incubated with 200 μM H2O2. The specific operation was as follows: PC12 cells (Cell Bank of the Chinese Academy of Sciences) were seeded into a 96-well plate at a density of 1×10 4 / well, and then cultured in a CO2 incubator at 37 °C with humid air containing 5% CO2 for 24 h to allow the cells to adhere. Then, the PC12 cells were treated with 200 μM H2O2 incomplete medium for 2 h to induce oxidative stress in PC12 cells.
[0053] Figure 5 It is the effect diagram of ROS scavenging by SNSs / PEI-DTC. It can be seen that after adding the SNSs / PEI-DTC complex, ROS was effectively scavenged.
[0054] The cytotoxicity index was expressed as the percentage of the cell viability after sample treatment relative to the cell viability of the untreated blank control group. The results showed that the nano gas generator could effectively scavenge reactive oxygen species in oxidative stress cells, thereby playing a role in protecting oxidative stress cells and anti-apoptosis, and had good potential for biological applications.
[0055] Figure 6 It is the construction and surgical schematic diagram of the nano gas generator. Using the liquid-phase exfoliation method, the precursor calcium silicide crystal was exfoliated into multi-layer and few-layer silicene. After the hydrogen sulfide donor PEI-DTC and silicene SNSs were combined by electrostatic adsorption, the whole was vortexed into the PVA / PVP transparent conductive hydrogel to prepare the hydrogen sulfide nano gas generator. The generator was placed at the spinal cord injury site with a 1 mL syringe, and the wound was sutured and then irradiated with infrared light for treatment.
[0056] In the animal experiment, the hydrogen sulfide nano gas generator was placed in a 1 mL syringe and injected into the spinal cord injury site of C57BL / 6 mice. Starting from the seventh day after the operation, NIR (808 nm infrared light) was irradiated for 10 min on days 0, 3, and 5.
[0057] Figure 7 It is the tissue section diagrams of HE, Nissl, and LFB staining of the nano gas generator and the SCI (spinal cord injury) group. Light micrographs of HE (hematoxylin-eosin staining), Nissl (Nissl Staining), and LFB (FAST BLUE) staining after repair and sectioning from the spinal cord injury site. More Nissl bodies and longer myelin sheaths appeared in the tissues around the injury in the experimental group, and the repair effect was obvious.
[0058] Example 2
[0059] Add 160 mL of anhydrous acetonitrile (CH3CN) to a 1000 mL round-bottom flask, and successively add 575 mg of CaSi2 and 1524 mg of I2 powder (molar ratio = 1:1). The resulting mixture is magnetically stirred at room temperature for 2 weeks. The reaction system is always under nitrogen protection throughout the reaction process. After the reaction is completed, the product is washed 3 times with anhydrous acetonitrile and dispersed in 50 mL of NMP, and ultrasonically crushed at an ultrasonic power of 200 W for 6 h. When taking samples, the solid product is collected by centrifugation, repeatedly eluted with absolute ethanol and deionized water, and dried to obtain few-layer silicene nanosheets with uniform size.
[0060] At room temperature, dissolve 500 mg of PEI (0.02 mmol) and 650 mg of KOH (11.6 mmol) in 50 mL of methanol, and stir until KOH is completely dissolved. Pass N2 into the resulting mixed solution and keep it for 15 s to completely expel the oxygen in the solution. Slowly add 695 μL of CS2 (11.6 mmol) to the solution and stir for 10 min. The color of the solution turns light yellow. Rotary evaporation is carried out to obtain PEI-DTC.
[0061] Dissolve 9.95 g (99.5 wt%) of polyvinyl alcohol (PVA) and 0.05 g (0.5 wt%) of polyvinylpyrrolidone (PVP) in 100 mL of deionized water. Place the mixed solution in a round-bottom flask, magnetically stir it at a constant temperature of 90 °C for 4 h, and then let it stand at 95 °C for 2 h. Freeze it at -20 °C for 18 h and thaw it at room temperature for 6 h. Take this as a cycle and repeat the freezing and thawing steps 5 times to obtain an injectable PVA / PVP transparent conductive hydrogel.
[0062] At room temperature, disperse solid silicene nanosheets in deionized water to prepare solution A with a concentration of 30 ppm. Dissolve 2 mg of PEI-DTC in 10 mL of deionized water to form solution B. Centrifuge 1 mL of solution A, pour off the supernatant, and mix it with 1 mL of solution B. After centrifugation, discard the supernatant and dry it to obtain the SNSs / PEI-DTC complex (solid, about 2 mg). Vortex the SNSs / PEI-DTC complex into 5 g of PVA / PVP hydrogel to obtain a hydrogen sulfide nano gas generator.
[0063] Example 3
[0064] Add 160 mL of anhydrous acetonitrile (CH3CN) to a 1000 mL round-bottom flask, and successively add 575 mg of CaSi2 and 1524 mg of I2 powder (molar ratio = 1:1). The resulting mixture is magnetically stirred at room temperature for 2 weeks. The reaction system is always under nitrogen protection throughout the reaction process. After the reaction is completed, the product is washed 3 times with anhydrous acetonitrile and dispersed in 50 mL of NMP, and ultrasonically fragmented at an ultrasonic power of 200 W for 6 h. When taking samples, the solid product is collected by centrifugation, repeatedly eluted with anhydrous ethanol and deionized water, and dried to obtain few-layer silicene nanosheets with uniform size.
[0065] At room temperature, dissolve 500 mg of PEI (0.02 mmol) and 650 mg of KOH (11.6 mmol) in 50 mL of methanol, and stir until KOH is completely dissolved. Pass N2 into the resulting mixed solution and keep it for 15 s to completely expel the oxygen in the solution. Slowly add 695 μL of CS2 (11.6 mmol) to the solution and stir for 10 min. The color of the solution turns light yellow. Rotate evaporate to obtain PEI-DTC.
[0066] Dissolve 9.5 g (95 wt%) of polyvinyl alcohol (PVA) and 0.5 g (5 wt%) of polyvinylpyrrolidone (PVP) in 100 mL of deionized water. Place the mixed solution in a round-bottom flask, magnetically stir it at a constant temperature of 90 °C for 4 h, and then let it stand at 95 °C for 2 h. Freeze it at -20 °C for 18 h and thaw it at room temperature for 6 h. Take this as a cycle and repeat the freezing and thawing steps 5 times to obtain an injectable PVA / PVP transparent conductive hydrogel.
[0067] At room temperature, disperse solid silicene nanosheets in deionized water to prepare solution A with a concentration of 30 ppm. Dissolve 2 mg of PEI-DTC in 10 mL of deionized water to form solution B. Centrifuge 1 mL of solution A, pour off the supernatant, and mix it with 1 mL of solution B. After centrifugation, discard the supernatant to obtain the SNSs / PEI-DTC complex (solid, about 2 mg). Vortex the SNSs / PEI-DTC complex into 5 g of PVA / PVP hydrogel to obtain a hydrogen sulfide nano gas generator.
Claims
1. An infrared light-released hydrogen sulfide nano-reactor, characterized in that, The infrared light-releasing hydrogen sulfide nano-reactor includes an injectable PVA / PVP transparent conductive hydrogel and an SNSs / PEI-DTC complex filled inside the injectable PVA / PVP transparent conductive hydrogel; the SNSs / PEI-DTC complex includes a donor PEI-DTC and two-dimensional silicene nanosheets loaded on the surface of the donor PEI-DTC.
2. The infrared light-releasing hydrogen sulfide nano-reactor according to claim 1, characterized in that, The mass ratio of the injectable PVA / PVP transparent conductive hydrogel to the SNSs / PEI-DTC complex is 1000:1 - 10000:
1.
3. The infrared light-releasing hydrogen sulfide nano-reactor according to claim 1 or 2, characterized in that, The mass ratio of the two-dimensional silicene nanosheets to PEI-DTC is 20 - 30 μg:0.15 - 0.2 mg.
4. The preparation method of the infrared light-releasing hydrogen sulfide nano-reactor according to any one of claims 1 to 3, characterized in that, The preparation method includes: Synthesizing and exfoliating two-dimensional silicene nanosheets by a wet chemical method; Modifying the donor PEI-DTC that thermally decomposes to produce hydrogen sulfide on the surface of the two-dimensional silicene nanosheets to obtain an SNSs / PEI-DTC complex; Wholly wrapping the SNSs / PEI-DTC complex in the injectable PVA / PVP transparent conductive hydrogel to form an infrared light-releasing hydrogen sulfide nano-reactor.
5. The preparation method according to claim 4, characterized in that, The method for preparing two-dimensional silicene nanosheets includes: adding precursors CaSi2 and I2 to anhydrous acetonitrile, with the molar ratio of CaSi2 to I2 being 1:1, maintaining stirring under N2 atmosphere protection, and continuously reacting at room temperature for 2 - 3 weeks; centrifuging the reaction solution, taking the solid product, dispersing it in an organic solvent and ultrasonically crushing it to obtain a dispersion containing two-dimensional silicene nanosheets; preferably, the ratio of CaSi2:I2:anhydrous acetonitrile is 570 - 575 mg:1520 - 1530 mg:160 - 170 mL; more preferably, repeatedly eluting and drying with absolute ethanol and deionized water to obtain two-dimensional silicene nanosheets.
6. The preparation method according to claim 4 or 5, characterized in that, The method for preparing PEI-DTC includes: dissolving PEI and KOH in methanol, stirring until KOH is completely dissolved; adding CS2 to the obtained solution, stirring for 10 - 20 min, and drying to obtain PEI-DTC; preferably, the ratio of PEI:KOH:methanol is 500 - 505 mg:650 - 655 mg:50 - 55 mL; more preferably, the ratio of PEI:CS2 is 500 - 505 mg:695 - 700 μL.
7. The preparation method according to any one of claims 4 to 6, characterized in that, Loading PEI-DTC on the surface of two-dimensional silicene nanosheets by electrostatic adsorption; preferably, at room temperature, dispersing two-dimensional silicene nanosheets in water to prepare solution A; dissolving PEI-DTC in deionized water to form solution B; centrifuging solution A, pouring off the supernatant, and mixing it with solution B of the same volume as solution A, centrifuging and discarding the supernatant to obtain an SNSs / PEI-DTC complex; more preferably, the concentration of two-dimensional silicene nanosheets in solution A is 0.02 - 0.03 mg / mL; the concentration of PEI-DTC in solution B is 0.15 - 0.2 mg / mL.
8. The preparation method according to any one of claims 4 to 7, characterized in that, Prepare the PVA / PVP hydrogel using the cyclic freeze-thaw method; preferably, prepare an aqueous PVA / PVP solution with a PVP concentration of 0.5-5 wt%; under stirring, dissolve the PVA / PVP solution at a constant temperature of 90-95 °C for 4-4.5 hours; stop stirring, and let it stand at 95-100 °C for 1.5-2 hours; place it in a freezer at -18 to -20 °C for 18-18.5 hours, and thaw it at room temperature for 6-6.5 hours; preferably, repeat the freezing and thawing 5-6 times.
9. The preparation method according to any one of claims 4 to 8, characterized in that, Uniformly mix the SNSs / PEI-DTC complex with the PVA / PVP hydrogel to entirely encapsulate the SNSs / PEI-DTC complex within the injectable PVA / PVP transparent conductive hydrogel.
10. Use of the infrared light-releasing hydrogen sulfide nanoreactor according to any one of claims 1 to 3 in the preparation of a spinal cord injury repair product.