Preparation of functionalized nanosulfur and application thereof in wound repair

Abstract

The application discloses a kind of functionalized nano sulfur preparation and its application in wound repair.The application uses PEG200 and elemental sulfur as reaction raw materials, does not introduce other organic solvents, is prepared by muffle furnace gradient heating mode at 30-70 DEG C safe reaction temperature, and obtains the spherical nano sulfur of 50-100nm particle size, has certain repair effect to normal mouse wound and diabetic refractory wound, and does not cause toxicity in vivo, with higher biological safety.Therefore, it can be used for wound repair, especially refractory wound repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of wound repair, in particular to a functionalized nano-sulfur preparation and its application in wound repair. BACKGROUND

[0002] Sulfur element as a key element of oxygen family has a variety of biological functions, but free sulfur element is unstable and has low biological activity, and currently nano-sulfur is considered as a high-activity and high-stability form. In recent years, there are many reports on the preparation process of nano-sulfur, which mainly includes the following three kinds: 1) using organic acid or inorganic acid to catalyze sodium thiosulfate, and then further modifying by various surfactants to obtain nano-sulfur; 2) using water-in-oil microemulsion system, using organic solvent as oil phase and sulfur-containing solution as water phase to prepare sulfur nanoparticles; 3) using electrochemical oxidation-reduction reaction to catalyze the reaction to obtain nano-sulfur.

[0003] Although there are many synthesis processes of nano-sulfur at present, these synthesis processes have certain defects: 1) a large amount of organic solvent is used, so that the biological activity of the prepared nano-sulfur is affected, and it is difficult to apply to in vivo research; 2) the preparation reaction system is relatively complex, the cost is high and the process amplification is unstable. Therefore, it is a key problem to be solved to develop a simple and green nano-sulfur synthesis process. SUMMARY

[0004] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a functionalized nano-sulfur preparation method.

[0005] Another purpose of the present application is to provide functionalized nano-sulfur prepared by the method.

[0006] Still another purpose of the present application is to provide the application of the functionalized nano-sulfur.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] A functionalized nano-sulfur preparation method, comprising the following steps:

[0009] Sublimed sulfur is added to PEG200, stirred and mixed at a temperature of 20-40℃ (preferably 25-30℃), then transferred to a high-temperature sealed reaction kettle, first raised from 20-40℃ to 60-80℃ within 20-40 minutes, maintained at 60-80℃ for 30-60 minutes, then lowered from 60-80℃ to 25-35℃ within 15-30 minutes, then water is added after taking out, then dialyzed to obtain PEG atomized nano-sulfur particles, namely the functionalized nano-sulfur.

[0010] The amount of the sublimed sulfur is 1-5 mL of PEG200 per mg of the sublimed sulfur; preferably 1 mL of PEG200 per mg of the sublimed sulfur.

[0011] The reaction condition is preferably as follows: first, the temperature is raised from 30 DEG C to 70 DEG C in 30 minutes, then maintained at 70 DEG C for 30 minutes, and then the temperature is lowered from 70 DEG C to 30 DEG C in 30 minutes.

[0012] The water is added in an equal volume.

[0013] The water is preferably distilled water.

[0014] The dialysis is performed by using a dialysis bag with a molecular weight cut-off of 1000 kDa; preferably, the dialysis is performed by using a dialysis bag with a molecular weight cut-off of 1000 kDa for 72-120 hours; more preferably, the dialysis is performed by using a dialysis bag with a molecular weight cut-off of 1000 kDa for 72 hours.

[0015] The dialysis liquid used for the dialysis is preferably a PBS buffer solution.

[0016] A functionalized nano-sulfur is prepared by the method of any one of the above.

[0017] The functionalized nano-sulfur is a spherical nano-sulfur with a particle size of 50-100 nm.

[0018] The functionalized nano-sulfur is used for preparing a product for wound repair.

[0019] The wound repair includes normal human or animal wound repair, or diabetic human or animal wound repair (repair of diabetic refractory wounds).

[0020] The product includes a medicine, etc.

[0021] The present application has the following advantages and effects relative to the prior art:

[0022] 1. The present application provides a new nano-sulfur preparation process: PEG200 and elemental sulfur are used as reaction raw materials, no other organic solvent is introduced, and a simple gradient heating recrystallization method of a muffle furnace is used to prepare spherical nano-sulfur with a particle size of 50-100 nm, which has a certain repair effect on normal mouse wounds and diabetic refractory wounds.

[0023] 2、Prior art documents have reported that nanosulfur is prepared by using PEG and sublimed sulfur. Compared with the preparation process of the present application, the reaction temperature is above 110℃ (about 110℃-150℃), the prepared nanosulfur is rod-shaped structure, and the biological activity of the prepared nanosulfur is not studied. The preparation process proposed in the present application is carried out at a safe reaction temperature of 30-70℃, spherical structure is obtained, and the nanosulfur is further applied to wound repair. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a functionalized nanosulfur preparation process optimization result graph; wherein, A is a transmission electron microscope graph of the functionalized nanosulfur prepared under different PEG molecular weight conditions; B is a transmission electron microscope graph of the functionalized nanosulfur prepared at different temperatures; C is a graph of the influence of the functionalized nanosulfur on the proliferation of vascular endothelial cells (HUVEC) and fibroblasts (HSF).

[0025] Figure 2 is a functionalized nanosulfur morphology characterization result graph; wherein, A is a functionalized nanosulfur solution; B is a functionalized nanosulfur particle size distribution graph; C is a transmission electron microscope graph of the functionalized nanosulfur; D is an atomic force microscope graph of the functionalized nanosulfur and its quantitative analysis result graph.

[0026] Figure 3 is a functionalized nanosulfur on normal mouse skin wound repair condition graph; wherein, A is an experimental process schematic diagram; B is a normal mouse skin wound repair healing condition; C is a normal mouse skin wound healing area; D is a normal mouse wound tissue HE section staining result.

[0027] Figure 4 is a functionalized nanosulfur on diabetic mouse skin wound repair condition graph; wherein, A is an experimental process schematic diagram; B is a diabetic mouse skin wound repair healing condition; C is a diabetic mouse skin wound healing area; D is a diabetic mouse wound tissue HE section staining result.

[0028] Figure 5 is a functionalized nanosulfur in vivo biological safety evaluation result graph; wherein, A is the influence of the functionalized nanosulfur on the development of zebrafish embryos and larvae; B is the survival of zebrafish larvae and the body length and heart rate during the development process; C is the influence of the functionalized nanosulfur on the organs in the mouse body (HE section staining result); D is a mouse body weight change graph; E is a mouse blood index. DETAILED DESCRIPTION

[0029] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0030] The sublimed sulfur (purity 99.99%) and polyethylene glycol (PEG200, PEG400, PEG800, PEG1200) involved in the embodiments of the present invention were all purchased from Sigma-Aldrich.

[0031] Example 1

[0032] 1. Optimization of preparation conditions for novel functionalized nano-sulfur

[0033] 1.1 To obtain novel nano-sulfur with stable nanostructure and pro-repair activity, we optimized the preparation process of nano-sulfur. Different nano-sulfurs were obtained by changing the molecular weight of the PEG used and the reaction temperature during the preparation process. The specific steps are as follows:

[0034] (1) PEGs of different molecular weights

[0035] Accurately weigh 10 mg of sublimed sulfur, and separately measure 10 mL of PEG200, PEG400, PEG800, and PEG1200. Dissolve the weighed sublimed sulfur in the PEG solutions and magnetically stir for 10 minutes at 25–30 °C. Then transfer the solution to a high-temperature sealed reactor. Set the muffle furnace to program the temperature increase (from 30 °C to 70 °C within 30 minutes), maintain 70 °C for 30 minutes, and then program the temperature decrease (from 70 °C to 30 °C within 30 minutes). Remove the high-temperature sealed reactor and transfer the reaction solution to a beaker, immediately adding an equal volume of distilled water. Dialyze the solution using a dialysis bag with a molecular weight cutoff of 1000 kDa for 72 hours (using PBS buffer as the dialysate) to obtain PEG-atomized sulfur nanoparticles.

[0036] (2) Different reaction temperatures

[0037] The method is the same as step (1) above, except that the molecular weight of the PEG used is 200 (PEG200). The muffle furnace is programmed to heat up (from 30°C to 70°C, 110°C, and 150°C within 30 minutes), and then maintained at this temperature (70°C, 110°C, and 150°C) for 30 minutes. Then the muffle furnace is programmed to cool down (from this temperature to 30°C within 30 minutes), while maintaining the reaction temperature at 30°C is used as a control.

[0038] The morphology of the nano-sulfur prepared by different preparation processes was observed by transmission electron microscopy. The results showed that with the increase of the molecular weight of PEG, the prepared nano-sulfur was more prone to aggregation (A); and it was found that the nano-sulfur prepared under different reaction temperature conditions had different morphologies, in which the nano-structure with complete spherical shape was obtained under the condition of 70°C, while the nano-sulfur prepared under the condition of 150°C was rod-shaped structure (B). Figure 1 Figure 1

[0039] 1.2 Further, we used the nano-sulfur prepared by different processes to treat the blood vessel endothelial cells and fibroblasts related to wound repair, and by determining the promotion of cell proliferation, the process of preparing nano-sulfur was optimized. The specific steps are as follows:

[0040] The blood vessel endothelial cells (HUVEC) and fibroblasts (HSF) were purchased from ATCC, and the cells in logarithmic growth phase were digested and inoculated into 96-well plates at a density of 3500 cells per well, with 6 replicate wells in each group. After 24 hours, the DMEM complete culture medium in the wells was aspirated and replaced with 100 μL of DMEM medium containing 0.05% (v / v) fetal bovine serum (FBS) per well, and starved for 24 hours. Then 90 μL of drug (nano-sulfur prepared by different molecular weight of PEG or different reaction temperature, final concentration of 20 μM) dissolved in DMEM containing 0.05% (v / v) FBS was added, and the cells were treated for 48 hours. Among them, the DMEM medium without drug was used as a control, and 10 μL of CCK8 solution was added to the wells. Next, the cells were incubated in the cell incubator for another 2 hours. Finally, the absorbance at 450 nm was detected by a microplate reader, and the cell proliferation rate was calculated, and the average value was calculated by three times of repetition.

[0041] The results are shown in Figure 1 C, it was found that the nano-sulfur obtained by using PEG200 and 70°C reaction condition had the best cell proliferation activity.

[0042] In summary, by optimizing the molecular weight of PEG and the reaction temperature, through the observation of the morphology and the determination of the cell activity, the reaction condition of using PEG200 and 70°C was finally optimized as the best preparation process of nano-sulfur.

[0043] 2. Preparation and characterization of new functionalized nano-sulfur

[0044] 2.1 Preparation of new functionalized nano-sulfur

[0045] ​​Accurately weigh 10 mg of sublimed sulfur and measure 10 mL of PEG200. Dissolve the sublimed sulfur in PEG200 and magnetically stir for 10 minutes at 25–30 °C. Then transfer the solution to a high-temperature sealed reactor and program the muffle furnace temperature (increasing from 30 °C to 70 °C within 30 minutes), maintaining 70 °C for 30 minutes, followed by a programmable temperature decrease (reducing from 70 °C to 30 °C within 30 minutes). Remove the high-temperature sealed reactor and transfer the reaction solution to a beaker, immediately adding an equal volume of distilled water. Dialyze the solution using a dialysis bag with a molecular weight cutoff of 1000 kDa for 72 hours (using PBS buffer as the dialysate) to obtain PEG-atomized sulfur nanoparticles (Nano-S).

[0046] 2.2 Morphological Characterization

[0047] Nano-sulfur was prepared using the PEG atomization method. Measurements of the functionalized nano-sulfur solution revealed the Tyndall effect, confirming its colloidal properties. Figure 2 A). Morphological observation using a Malvern particle size analyzer and transmission electron microscopy revealed that the PEG200-modified nano-sulfur nanoparticles formed uniformly dispersed nanospheres with a particle size of 50–100 nm. Figure 2 B and 2C). Further observation of the morphology of the prepared nano-sulfur nanoparticles using atomic force microscopy revealed that the atomic force data and transmission electron microscopy data were consistent. Figure 2 D). The above data proves that the present invention has successfully prepared novel functionalized nano-sulfur with a particle size of 50-100 nm.

[0048] 3. The effect of novel functionalized nano-sulfur on wound repair in normal mice

[0049] To verify the effect of the novel functionalized nano-sulfur on skin wound repair, we constructed a mouse model of full-thickness skin loss on the back. Every other day, we administered a 50 μL subcutaneous injection at two fixed points around the wound. On the eighth day, the mice were euthanized, and the repaired wound tissue was collected for HE section staining. The specific steps are as follows:

[0050] Balb / c mice (purchased from Guangdong Experimental Animal Center) of 6-8 weeks old and weighing 18-22 g were selected, 6 in each group. After anesthesia with sodium pentobarbital (purchased from Sigma-Aldrich), a full-thickness skin wound model was established on the back of the mice using a skin punch with a diameter of 6 mm. After establishing the dorsal skin wound model, two injection points were fixed around the wound, and 50 uL of drug molecules (concentration of 200 nM, functionalized nanosulfur prepared in 2.1 above) were subcutaneously injected, and the same volume of PBS was used as a control. Drug administration was performed every other day, and the corresponding wound healing data were collected. At the same time, wound tissue was collected on the eighth day, fixed with paraformaldehyde, prepared into paraffin blocks and made into tissue sections. The effect of nanosulfur on skin tissue wound repair was observed by HE staining.

[0051] The results are shown in Figure 3 It was found that the healing area of the dorsal wound of the mice treated with nanosulfur for six days was larger than that of the blank control group. On the eighth day, it was found that the wound of the nanosulfur treatment group had scabbed and fallen off, while the wound of the blank group had not healed. Further HE section of the wound tissue found that the structure of the epidermis and dermis of the newly generated skin tissue after nanosulfur treatment was more complete. In summary, the novel functionalized nanosulfur constructed in the present application has a certain repair effect on the wound of normal mice.

[0052] 4. Effect of novel functionalized nanosulfur on wound repair of diabetic mice

[0053] In addition to the normal skin wound repair effect, wound repair disorders caused by metabolic disorders in the body are also key scientific problems in wound repair, among which diabetic refractory wounds are a more difficult wound repair problem in clinical practice. Therefore, in this part, we used db / db mice to evaluate whether the functionalized nanosulfur constructed by us has a certain effect on the refractory wounds caused by diabetes. - / db - Diabetic mouse model was used to evaluate whether the functionalized nanosulfur constructed by us had a certain effect on the refractory wounds caused by diabetes. The specific steps are as follows:

[0054] Select 6-8 weeks old db- / db- diabetic mice (purchased from Guangdong Province Experimental Animal Center) weighing 30-35 g, 6 in each group. After sodium pentobarbital (purchased from Sigma-Aldrich) anesthesia, a full skin layer injury model was established on the back of the diabetic mouse using a skin puncher with a diameter of 6 mm. After establishing the diabetic refractory wound injury model, two injection points were fixed around the wound, and 50 uL of drug molecules (concentration of 200 nM, functionalized nanosulfur prepared in 2.1 above) were subcutaneously injected, and the same volume of PBS was used as a control. Drug administration was performed every other day, and the corresponding wound healing data were collected. At the same time, on the tenth day, wound tissue was taken, fixed with paraformaldehyde, prepared into paraffin blocks and made into tissue sections. The effect of nanosulfur on skin tissue wound tissue repair was observed by HE staining.

[0055] The results are shown in Figure 4 As shown, we found that after every other day of drug administration, the blank group showed ulceration of the wound tissue on the tenth day, and the wound showed no healing trend. When treated with the new functionalized nanosulfur of the application, we found that the wounds of diabetic mice did not ulcerate and gradually healed. Further, we found that the dermal layer thickness and the integrity of the epidermis of the wound tissue were improved after drug treatment. In summary, the new functionalized nanosulfur we constructed can inhibit the formation of diabetic refractory wound ulceration and improve the repair of the wound.

[0056] 5 In vivo safety evaluation of new functionalized nanosulfur

[0057] In vivo biological safety is a key indicator for the application of biomaterials. In order to evaluate the in vivo biological safety of the new functionalized nanosulfur constructed, we used the zebrafish larva development model to evaluate its acute toxicity in vivo, and the specific steps are as follows:

[0058] (1) Zebrafish development toxicity evaluation:

[0059] The zebrafish is a conventional commercially available AB line wild type zebrafish, which is cultured to adulthood in the laboratory according to the conventional method, and then the adult female and male zebrafish are spawned, the fish eggs are collected, and the fish eggs are placed in a 24-well plate, 10 fish eggs per well. 1 mL of functionalized nanosulfur solution with a concentration of 200 nM prepared in 2.1 above was added to the well plate, 3 replicates were set, and PBS was used as a blank control. Photographs were taken at 0 hours, 24 hours, and 48 hours, and the survival rate of zebrafish larvae was counted. The body length and heart rate of zebrafish larvae were counted at 48 hours, and the average value was calculated three times to evaluate the development toxicity of nanosulfur to zebrafish.

[0060] (2) In vivo toxicity evaluation in mice:

[0061] Select 6-8 weeks old weight of 18-22 g balb / c mice (purchased from Guangdong Province Experimental Animal Center). After sodium pentobarbital anesthesia (purchased from Sigma-Aldrich), subcutaneous injection of drug three times, every other day, a single drug dose of 50 uL (200 nM, prepared in 2.1 above functionalized nanosulfur), with the same volume of PBS as control, statistics of its body weight change; after the eighth day, euthanize the mice, and detect the blood biochemical indicators, and take its viscera for HE section to observe whether there is structural lesions.

[0062] Results as shown in Figure 5 compared with the blank control group (PBS), nanosulfur has no toxicity to the development of zebrafish embryos and larvae, and has no teratogenicity; further by statistics of the body length and heart rate of zebrafish larvae during development, it is found that the drug has no significant effect on the development of zebrafish. In addition to using zebrafish to evaluate its toxicity, we also evaluated the in vivo toxicity of the new functionalized nanosulfur to mice by statistics of the body weight change of mice, blood indicators and HE section observation of the morphology of viscera, and found that the new functionalized nanosulfur constructed has no toxic effect on the viscera of mice in vivo. In summary, the two model organisms of zebrafish and mice verify that the new functionalized nanosulfur constructed does not cause toxicity in vivo and has high biological safety.

[0063] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for preparing functionalized nano-sulfur, characterized in that, Includes the following steps: Sublimed sulfur was added to PEG200 and mixed at 20–40 °C. The mixture was then transferred to a high-temperature sealed reactor. The temperature was first raised from 20–40 °C to 60–80 °C over 20–40 minutes, maintained at 60–80 °C for 30–60 minutes, and then lowered from 60–80 °C to 25–35 °C over 15–30 minutes. After removal, water was added, and the mixture was dialyzed to obtain PEG-atomic sulfur nanoparticles, i.e., the functionalized sulfur nanoparticles.

2. The method for preparing functionalized nano-sulfur according to claim 1, characterized in that: The reaction conditions are as follows: first, the temperature is raised from 30°C to 70°C within 30 minutes, maintained at 70°C for 30 minutes, and then lowered from 70°C to 30°C within 30 minutes.

3. The method for preparing functionalized nano-sulfur according to claim 1, characterized in that: The amount of sublimed sulfur used is calculated based on 1 to 5 mL of PEG200 per milligram of sublimed sulfur.

4. The method for preparing functionalized nano-sulfur according to claim 3, characterized in that: The amount of sublimed sulfur used is calculated based on 1 mL of PEG200 per milligram of sublimed sulfur.

5. The method for preparing functionalized nano-sulfur according to claim 3, characterized in that: The dialysis described herein is performed using a dialysis bag with a molecular weight cutoff of 1000 kDa for 72–120 hours. The dialysate used in the dialysis was PBS buffer solution.

6. A functionalized nano-sulfur, characterized in that: It is prepared by the method described in any one of claims 1 to 5.

7. The functionalized nano-sulfur according to claim 6, characterized in that: The functionalized nano-sulfur is a spherical nano-sulfur with a particle size of 50–100 nm.

8. The use of the functionalized nano-sulfur as described in claim 6 or 7 in the preparation of products for wound repair.

9. The application according to claim 8, characterized in that: The wound repair mentioned includes wound repair for diabetic patients or animals.

10. The application according to claim 8, characterized in that: The product in question is a medicine.

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