CN117815366A - Preparation of composite nano particles containing sulfur element and bFGF and application of composite nano particles in wound repair - Google Patents
Preparation of composite nano particles containing sulfur element and bFGF and application of composite nano particles in wound repair Download PDFInfo
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Abstract
The invention discloses a preparation method of a composite nanoparticle containing sulfur element and bFGF and application of the composite nanoparticle in wound repair. According to the invention, the PEG atomization effect is used for combining sublimed sulfur and bFGF together, so that the composite nano particles containing sulfur element and bFGF are constructed, the effect of penetrating through skin barriers is improved, the proliferation and migration of fibroblasts and vascular endothelial cells related to tissue regeneration can be promoted, good repairing effects are achieved on wound surfaces of normal mice and difficult-to-heal wound surfaces of diabetic mice, toxic effects of internal organs are not caused, and the biological safety is high. Therefore, the composite nanoparticle can be used in wound repair.
Description
Technical Field
The invention relates to the field of wound repair, in particular to preparation of composite nano particles containing sulfur element and bFGF and application of the composite nano particles in wound repair.
Background
In the process of body injury and rehabilitation, damaged tissue repair and regeneration is a current problem. Although many significant advances have been made in research in regenerative medicine at present, there are still many problems to be solved. Some tissues that are otherwise capable of regeneration, such as skin, may also present damage repair barriers in certain situations. The skin is the largest organ of the human body and plays various roles. In order to combat many mechanical, chemical and biological injuries from the outside, including damage caused by heat, friction, radiation, pathogenic micro-organs, and toxic chemicals and materials, the skin needs to repair itself rapidly upon injury. Repair of skin lesions in mammals involves a complex series of processes, divided into four phases, hemostasis, inflammatory response, cell proliferation and tissue remodeling, requiring involvement of a variety of different cell/tissue types. Patients with metabolic basal diseases, such as chronic diseases in which insulin secretion is insufficient in vivo, resulting in hyperglycemia and thus causing disturbance of carbohydrate and fat metabolism. Because of the high blood glucose concentration maintained in the patient for a long period of time, the high sugar microenvironment can lead to the failure of normal physiological functions of various cells involved in tissue regeneration, such as fibroblasts, vascular endothelial cells, neutrophils and macrophages, and the failure of skin tissue regeneration. Clinically, diabetes is difficult to heal, and finally only amputation can be performed. The difficult-to-heal wound surface of diabetes brings great economic burden to the patient and the country, becomes a worldwide public health problem seriously threatening the health of human beings, and the problems still lack effective strategy to solve at present, and relieves the pain of the patient.
Sulfur is a key element of the oxygen group element, and has good biological properties such as antioxidation, antibiosis and the like because the outermost electron is easy to give out, so that the sulfur has been widely used for treating various skin diseases including wound healing, acne, rash and psoriasis clinically. Although elemental sulfur has high activity, direct application to the skin sometimes exhibits lower bioactivity due to the skin barrier, the nano-form of sulfur is considered to be a more active and effective form. On the other hand, basic growth factor (bFGF) is widely used as an active factor in clinic for tissue regeneration, but bFGF is easily degraded and inactivated by various enzymes in vivo and microorganisms outside, and exogenous bFGF is easily degraded by various enzymes and bacteria in the microenvironment of the wound surface difficult to heal of diabetes, so that the bFGF cannot exert biological activity. Wound repair is a complex and precise regulation and control process, different signals are required to be regulated and controlled, and various cells are coordinated to participate in the repair process, so that the research about the application of biological materials to wound repair is reported, and most of the biological materials are used for regulating and controlling a certain cell or a certain cell type, which is far from sufficient for wound repair. Therefore, there is a need to develop a novel drug for coordinating multicellular activity for wound repair, particularly diabetic refractory wounds.
Disclosure of Invention
The primary aim of the invention is to overcome the defects and shortcomings of the prior art and provide a preparation method of composite nano particles containing sulfur element and bFGF.
The invention also aims to provide the composite nano-particles containing the sulfur element and the bFGF, which are prepared by the method.
It is still another object of the present invention to provide the use of the composite nanoparticle containing elemental sulfur and bFGF.
The aim of the invention is achieved by the following technical scheme:
a preparation method of composite nano particles containing sulfur element and bFGF comprises the following steps:
(1) Adding sublimed sulfur into PEG400, stirring and mixing at 30-45 ℃ (preferably 30-40 ℃) and transferring to a high-temperature closed reaction kettle, firstly raising the temperature from 30-45 ℃ to 80-120 ℃ within 15-30 minutes, maintaining the temperature at 80-120 ℃ for 1-2 hours, then lowering the temperature from 80-120 ℃ to 25-45 ℃ within 20-40 minutes, taking out, adding water, and dialyzing to obtain a nano sulfur particle solution;
(2) Dropwise adding a basic fibroblast growth factor (bFGF) solution into the nano sulfur particle solution obtained in the step (1), stirring and reacting at 25-35 ℃, and dialyzing (removing free bFGF and independent nano sulfur) after the reaction is finished to obtain the composite nano particles containing sulfur elements and bFGF.
The dosage of the sublimed sulfur in the step (1) is calculated according to the proportion of 0.5-2 mL PEG400 of sublimed sulfur per milligram; preferably 1ml peg200 per mg of sublimed sulfur.
The reaction conditions described in step (1) are preferably: the temperature was raised from 40℃to 100℃over 30 minutes, maintained at 100℃for 1 hour, and then lowered from 100℃to 30℃over 30 minutes.
The water in step (1) is preferably distilled water.
The dialysis in the step (1) is to use a dialysis bag with a molecular weight cut-off of 1000 kDa; preferably, the dialysis is carried out for 72 to 120 hours by adopting a dialysis bag with the molecular weight cut-off of 1000 kDa; more preferably, the dialysis is carried out for 72 hours using a dialysis bag having a molecular weight cut-off of 1000 kDa.
The dialysate used in the dialysis described in steps (1) and (2) is a PBS buffer solution.
The concentration of the basic fibroblast growth factor (bFGF) solution in the step (2) is 100-300 IU/mL; preferably 200IU/mL.
The concentration of the nano sulfur particle solution in the step (2) is 1-3 mu mol/L; preferably 2. Mu. Mol/L.
The volume ratio of the nano sulfur particle solution to the alkaline fibroblast growth factor solution in the step (2) is 1:3.
the reaction time in the step (2) is 6-12 hours; preferably 6 hours.
The dialysis in the step (2) is to use a dialysis bag with a molecular weight cut-off of 2000 kDa; preferably, the dialysis is carried out for 72 to 120 hours by using a dialysis bag with the molecular weight cut-off of 2000 kDa; more preferably, the dialysis is carried out for 72 hours using a dialysis bag having a molecular weight cut-off of 2000 kDa.
A composite nanoparticle comprising elemental sulfur and bFGF prepared by the method of any one of the above.
The particle size of the composite nano particles containing the sulfur element and bFGF is 50-150 nm.
The application of the composite nano-particles containing the sulfur element and the bFGF in preparing products for wound repair.
The wound repair comprises the wound repair of normal people or animals, or the wound repair of diabetics or animals (the repair of diabetic refractory wound).
The products include pharmaceuticals and the like.
Compared with the prior art, the invention has the following advantages and effects:
1. according to the invention, sublimed sulfur and bFGF are combined together through the PEG atomization effect, so that a novel sulfur-containing and bFGF complexing agent (composite nano particles) is constructed, the effect of penetrating through skin barriers is improved, and the novel sulfur-containing and bFGF complexing agent plays an important role in wound healing, in particular in wound repair of diabetes refractory.
2. The novel sulfur-containing element and bFGF composite nano-particles constructed by the invention can promote proliferation and migration of fibroblasts and vascular endothelial cells related to tissue regeneration through regulating and controlling two signal paths of FGFR and Hippo, thereby achieving the effect of coordinating activities of a plurality of cells by a drug molecule for repairing wound surfaces.
Drawings
FIG. 1 is a diagram showing the optimized result of the preparation process of the composite nano-particles containing sulfur and bFGF in the present invention; wherein, A is a transmission electron microscope image of sulfur-containing element and bFGF composite nano particles prepared under different PEG molecular weight conditions and an influence image of the transmission electron microscope image on proliferation of fibroblasts (HSF) and vascular endothelial cells (HUVEC); b is a transmission electron microscope image of the sulfur-containing element and bFGF composite nano-particles prepared at different reaction temperatures and an influence image of the transmission electron microscope image on proliferation of fibroblasts (HSF) and vascular endothelial cells (HUVEC); c is a particle size distribution diagram and a transmission electron microscope diagram of sulfur-containing element and bFGF composite nano particles prepared under different reaction feeding ratios, and an influence diagram of the particle size distribution diagram and the transmission electron microscope diagram on proliferation of fibroblasts (HSF) and vascular endothelial cells (HUVEC).
FIG. 2 is a graph of the morphological characterization result of the sulfur-containing element and bFGF composite nano-particles prepared by the present invention; wherein A is a particle size distribution diagram of the sulfur-containing element and bFGF composite nano particles; b is a transmission electron microscope image of the sulfur-containing element and bFGF composite nano-particles; c is an atomic force microscope image and a quantitative analysis result image of the sulfur-containing element and bFGF composite nano-particles.
FIG. 3 is a graph showing the activity regulation and control of the sulfur-containing element and bFGF composite nano-particles prepared by the invention on various cells related to tissue regeneration; wherein A is the proliferation of composite nano particles to fibroblasts and vascular endothelial cells by adopting a clone formation experiment; b is to measure the migration effect of the composite nano particles on fibroblasts and vascular endothelial cells by adopting a scratch experiment; c is to adopt Edu kit to determine proliferation of composite nano particles to fibroblast; and D is to measure the proliferation effect of the composite nano particles on vascular endothelial cells by adopting a Edu kit.
FIG. 4 is a graph showing the promotion of regeneration of zebra fish tail fins by the sulfur-containing element and bFGF composite nanoparticle prepared by the present invention (in the figure, dpa represents days after tail fin excision (day past amputation)).
FIG. 5 is a diagram showing the repair of a wound surface of normal mouse skin by the sulfur-containing element and bFGF composite nano-particles prepared by the method; wherein A is an experimental flow diagram; b is the repair and healing condition of the skin wound surface of a normal mouse; c is the healing area of the skin wound surface of a normal mouse; d is the staining result of the HE section of the wound tissue of the normal mouse.
FIG. 6 is a diagram showing the repair of a skin wound of a diabetic mouse by the sulfur-containing element and bFGF composite nanoparticle prepared by the method of the present invention; wherein A is an experimental flow diagram; b is the condition of repairing and healing the skin wound surface of the diabetic mice; c is the healing area of the skin wound surface of the diabetic mice; d is the staining result of the HE section of the wound tissue of the diabetic mouse.
FIG. 7 is a graph showing the in vivo biosafety evaluation result of the sulfur-containing element and bFGF composite nanoparticles prepared by the present invention; wherein A is the influence of sulfur-containing element and bFGF composite nano particles on the development of embryos and young fishes of zebra fish; b is the survival condition of young zebra fish and the length and heart rate of the young zebra fish in the development process; c is the staining result of the organ HE section of the mouse; d is a graph of the change in body weight of the mice.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The reagents and starting materials used in the present invention are commercially available unless otherwise specified.
Sublimed sulfur (purity 99.99%), polyethylene glycol (PEG 200, PEG400, PEG 800), and basic fibroblast growth factor (bFGF) were all purchased from Sigma-Aldrich (Shanghai).
Example 1
1. Optimization of preparation process of novel sulfur-containing element and bFGF composite nano-particles
1.1 in order to obtain novel sulfur-and bFGF-containing nanoparticles with stable nanostructure and repair promoting activity, we optimized the preparation process as follows:
(1) PEG of different molecular weights
a. Accurately weighing 10mg of sublimed sulfur, and respectively weighing 10mL of PEG200, PEG400 and PEG800. The weighed sublimed sulfur is dissolved in PEG and magnetically stirred at 30-40 ℃ for 30 minutes, then the solution is transferred to a high temperature closed reaction kettle, the muffle furnace is programmed to rise temperature (from 40 ℃ to 100 ℃ in 30 minutes), the temperature is maintained at 100 ℃ for 1 hour, and then the muffle furnace is programmed to drop temperature (from 100 ℃ to 30 ℃ in 30 minutes). The high temperature closed reaction vessel was taken out, the reaction solution was transferred to a beaker, and an equal volume of distilled water at 4℃was immediately added. After dialysis for 72 hours with a dialysis bag with a molecular weight cut-off of 1000kDa (dialysis solution is PBS buffer solution), nano sulfur particles are obtained.
b. 30mL (with the concentration of 2 mu M) of the obtained Nano sulfur particle solution is taken, 10mL of bFGF solution (containing 2000IU of bFGF) is dropwise added into the Nano sulfur solution, after the dropwise addition of the bFGF solution is finished, the solution is magnetically stirred and reacts for 6 hours at the temperature of 25-35 ℃, after the reaction is finished, a dialysis bag with the molecular weight cut-off of 2000kDa is adopted for dialysis for 72 hours (the dialysis solution is PBS buffer solution), and free bFGF and independent Nano sulfur are removed, so that novel composite Nano particles (Nano-S@bFGF) containing sulfur and bFGF are obtained.
(2) Different reaction temperatures
The method is similar to the step (1), and the difference is that: the molecular weight of the PEG used was 400 (PEG 400), the muffle furnace was programmed to warm (from 40℃to 50℃at 100℃at 150℃in 30 minutes) and then maintained at that temperature (50℃at 100℃at 150 ℃) for 1 hour, followed by the muffle furnace being programmed to cool (from that temperature to 30℃in 30 minutes).
(3) Feed ratio of different sulfur to bFGF
The method is similar to the step (1), and the difference is that: the molecular weight of PEG used was 400 (PEG 400), the amount of sublimed sulfur was 10mg, and the amount of PEG400 was 5mL, 10mL, 20mL.
1.2 further by assessing its proliferative activity on wound healing-related fibroblasts (HSF) and vascular endothelial cells (HUVEC). The method comprises the following specific steps:
fibroblasts (HSF) and vascular endothelial cells (HUVEC) were purchased from ATCC, and after digestion of cells in log phase, the cells were seeded into 96-well plates at 3500/well, each group was provided with 6 multiplex wells. After 24 hours, the DMEM complete medium in the wells was aspirated, and 100 μl of DMEM medium containing 0.05% (v/v) Fetal Bovine Serum (FBS) per well was changed to starve for 24 hours. Then, 90. Mu.L of the drug dissolved in DMEM containing 0.05% (v/v) FBS (the novel sulfur-containing element and bFGF composite nanoparticle prepared by PEG of different molecular weights or at different reaction temperatures, described above, had a final concentration of 10. Mu.M+2000 IU/mL, i.e., 100nM sulfur-containing element and 2000 IU/mL) was changed, and after the cells were treated for 48 hours, 10. Mu.L of CCK8 solution was added to the wells using DMEM medium without the drug as a control, followed by further incubation in a cell incubator for 2 hours. And finally, detecting the absorbance at 450nm by using an enzyme-labeled instrument, calculating the proliferation rate of the cells, and repeating the calculation of the average value for three times.
The results are shown in FIG. 1: the preparation is carried out by adopting PEG with different molecular weights, and the nano particles are relatively complete and uniform under the condition of PEG400 (figure 1A) is found through transmission electron microscope observation; the preparation is carried out at different reaction temperatures, and the nano particles obtained at the temperature of 100 ℃ are obtained by observation through a transmission electron microscope and have a relatively complete spherical structure, and the temperature is too high or too low to enable the nano particles to have a complete spherical structure (shown in figure 1B); the preparation is carried out by adopting a feeding ratio, and the nano particles obtained under the condition that the feeding ratio of sulfur to bFGF is 1:1 are observed by a transmission electron microscope, and have a complete spherical structure and are uniformly dispersed (figure 1C). Thus, through the above process optimization, we determined that the preparation conditions for preparing the novel sulfur-and bFGF-containing composite nanoparticle were: PEG400 was used at a feed ratio of 1:1 and a reaction temperature of 100 ℃. Further through the proliferation activity of the wound healing-related fibroblasts (HSF) and vascular endothelial cells (HUVEC), the novel composite nano-particles containing sulfur and bFGF prepared by the optimized post-process have the best proliferation promoting effect on the fibroblasts and the vascular endothelial cells compared with other preparation conditions (figure 1).
2. Preparation and characterization of novel sulfur-containing and bFGF composite nano-particles
2.1 preparation of novel Sulfur-and bFGF-containing composite nanoparticles
Accurately weighing 10mg of sublimed sulfur, and measuring 10mL of PEG400. The weighed sublimed sulfur was dissolved in PEG400 and magnetically stirred at 30-40 ℃ for 30 minutes, then the solution was transferred to a high temperature closed reaction kettle, the muffle furnace was programmed to warm (from 40 ℃ to 100 ℃ in 30 minutes), maintained at 100 ℃ for 1 hour, and then cooled (from 100 ℃ to 30 ℃ in 30 minutes). The high temperature closed reaction vessel was taken out, the reaction solution was transferred to a beaker, and an equal volume of distilled water at 4℃was immediately added. After dialysis for 72 hours with a dialysis bag with a molecular weight cut-off of 1000kDa (dialysis solution is PBS buffer solution), nano sulfur particles are obtained.
Taking 30mL (with the concentration of 2 uM) of the obtained Nano sulfur particle solution, dropwise adding 10mL of bFGF solution (containing 2000IU of bFGF) into the Nano sulfur solution, magnetically stirring and reacting for 6 hours at the temperature of 25-35 ℃ after the dropwise adding of the bFGF solution is finished, dialyzing for 72 hours by using a dialysis bag with the molecular weight cutoff of 2000kDa (the dialysis solution is PBS buffer solution) after the reaction is finished, and removing free bFGF and independent Nano sulfur to obtain novel composite Nano particles (Nano-S@bFGF) containing sulfur and bFGF.
2.2 characterization of morphology
The experiment firstly adopts a PEG atomization method to prepare nano sulfur, and then adopts different sulfur and bFGF proportion to construct novel composite nano particles containing sulfur and bFGF. Characterization of the nanotopography by a Markov particle sizer and a transmission electron microscope shows that when the ratio of sulfur to bFGF is 1:1, the novel composite nanoparticle has a regular nanosphere structure and is uniformly dispersed (fig. 2A and 2B). Further observation of the Nano-morphology by an atomic force microscope revealed that the atomic force data at the time of Nano-s+bfgf (1:1) were consistent with the data of transmission electron microscopy (fig. 2C). The data prove that the novel sulfur-containing and bFGF-containing composite nano-particles with the particle diameters of 50-150 nm are successfully prepared.
3. Novel sulfur-containing element and bFGF composite nano-particles for promoting multicellular activity related to tissue regeneration
In order to verify that the constructed novel sulfur-containing element and bFGF composite nano-particles regulate and control the multi-cell activity related to tissue regeneration. Human fibroblasts (HSF) and human vascular endothelial cells (HUVEC) are adopted for research, and through a cell clone formation experiment, an EdU cell proliferation experiment and a cell scratch experiment, the specific steps are as follows:
(1) Cloning formation experiments
Fibroblasts (HSF) and vascular endothelial cells (HUVEC) were purchased from ATCC, and after digestion of cells in logarithmic growth phase, the cells were inoculated into 6-well plates, the cell density was 500-1000 cells/well, and the novel sulfur-and bFGF-containing composite nanoparticle (200 nM+500IU, i.e., sulfur element concentration was 200nM, bFGF content was 500IU; the same below) prepared in step 2.1 was treated with a DMEM complete medium-dissolved drug, wherein a drug-free DMEM medium was used as a control, to form colonies. The cell culture medium was then removed and the colonies were fixed. Colonies were stained with crystal violet for about 20 minutes and washed 3 times with distilled water. The number of cell clones was photographed and counted. The experimental setup was repeated three times.
(2) Cell scratch assay
After digestion of the fibroblast cells (HSF) and vascular endothelial cells (HUVEC) in the logarithmic phase, appropriate amounts of cells were inoculated into 12-well plates, and the cell densities were allowed to confluent overnight. The next day, the cells were gently washed 3 times with PBS to remove the scraped cells, and the cells were changed to DMEM-complete medium-dissolved drug, i.e., the novel sulfur-and bFGF-containing composite nanoparticle (200 nm+500 iu) prepared in step 2.1 above, wherein the DMEM medium without drug was used as a control, and then placed in a cell incubator for continuous culture. The cells were observed for migration by placing the 12-well plate in a microscope at a specific location at 0, 24, 48 hours and photographed. The experimental setup was repeated three times.
(3) Edu detection of cell proliferation
After digestion of the fibroblast cells (HSF) and vascular endothelial cells (HUVEC) in logarithmic growth phase, the cells were resuspended in DMEM complete medium and seeded into 48-well plates at a density of 4000 cells/well, and left to stand in a 37 ℃ thermostatic cell incubator overnight until the cells adhere to the walls. The next day the medium in the wells was aspirated, and the drug dissolved in the complete medium, i.e. the novel sulfur-and bFGF-containing composite nanoparticle prepared in step 2.1 above (200 nm+500 iu), was treated for 48 hours with DMEM medium without drug as control. Edu (5-ethynyl-2' -deoxyuridine) was then diluted with DMEM complete medium and added to the wells to a final concentration of 10 μm in the wells and incubation was continued for 2 hours at 37 ℃. The medium was aspirated off and washed 2 times for 3 minutes in PBS. Cells were fixed at room temperature for 30min with 4% paraformaldehyde added to each well. The PBS was washed 3 times for 5 minutes each. Per well, add permeate (PBS containing 0.5% (v/v) Triton X-100) and incubate at room temperature for 15 minutes. The PBS was washed 2 times for 5 minutes each. Edu reaction was prepared and added to the wells and incubated at room temperature for 30 minutes in the dark. The PBS was washed 3 times for 5 minutes each. Then adding Hoechst reaction solution, dyeing for 10 minutes at room temperature in dark, and washing for 3 times by PBS for 5 minutes each time. Finally, the cells are observed under a fluorescence microscope, photographed and counted. The experimental setup was repeated three times.
As shown in fig. 3, the constructed novel sulfur-containing element and bFGF composite nanoparticle was found to significantly promote proliferation and migration of fibroblasts and vascular endothelial cells. Experimental results show that the constructed novel composite nano particles containing sulfur and bFGF can coordinate various cell activities related to tissue regeneration and exert the effect of promoting tissue regeneration.
4. Novel sulfur-containing element and bFGF composite nano-particle for promoting regeneration of zebra fish tail fin
In order to further research the efficacy of promoting tissue repair of the novel sulfur-containing element and bFGF composite nano-particles in vivo, a zebra fish tail fin regeneration model is adopted for verification, and the specific steps are as follows:
zebra fish are conventional commercial AB-series wild-type zebra fish, are grown up in the laboratory according to the conventional method, then adult zebra fish are selected, and after 0.2g/mL of tricaine (purchased from Sigma-Aldrich) is used for anesthesia, the tail fin near the back side is cut into sections by a surgical knife along 70% of the tail fin end to the root in the direction parallel to the root under a microscope. The tail fin near the abdomen was left untreated and was left as a control. The zebra fish after operation is fed into a small cell containing 50mL of system water, and is dosed once every other day (the novel sulfur-containing and bFGF composite nano particles prepared in the step 2.1) with the concentration of 200nM+500IU, PBS is used as a blank control group, meanwhile, the regenerated length of the zebra fish tail fin is recorded every 2 days, the total recorded length is 8 days, and the regeneration rate of the tail fin is counted. The experimental setup was repeated three times.
As shown in fig. 4, it was found that the speed of tail fin regeneration of adult zebra fish treated with the novel sulfur-containing element and bFGF composite nanoparticles was significantly accelerated and statistically significantly different, and the novel sulfur-containing and bFGF nanocomposite prepared by in vivo preliminary verification of the zebra fish tail fin regeneration model had the effect of promoting tissue regeneration.
5. Novel sulfur-containing element and bFGF composite nano-particle for promoting wound repair of mice
In order to further verify the efficacy of the novel sulfur-containing element and bFGF composite nano-particles in promoting wound repair. We constructed a model of loss of full cortex in the back of mice, subcutaneously injected (50 uL) at two fixed points around the wound every other day, euthanized mice by day eight and HE section stained with the repaired wound tissue. The method comprises the following specific steps:
balb/c mice (purchased from the laboratory animal center in Guangdong province) with a body weight of 18-22g at 6-8 weeks of age were selected, 6 in each group. After pentobarbital sodium anesthesia (purchased from Sigma-Aldrich (Shanghai)), a full-skin injury model was established on the back of the mice using a skin perforator with a diameter of 6 mm. After the back skin wound injury model is established, two injection points are fixed around the wound surface, and 50uL of drug molecules (the concentration of the novel sulfur-and bFGF-containing composite nano particles prepared in the step 2.1 is 200nM+500IU) are injected subcutaneously, wherein equal volume of PBS is used as a blank control, and bFGF (500 IU) is injected as a control. Once every other day, and collecting corresponding wound healing data. And at the same time, on the eighth day, wound tissue material is obtained, paraformaldehyde is fixed, paraffin blocks are prepared, and tissue sections are made. The histomorphology of the nano sulfur on the wound surface tissue repair of the skin tissue is observed through HE staining.
As shown in fig. 5, the wound healing speed after the drug treatment is significantly accelerated and statistically significantly different compared with that of the blank control group, and further, we found that the skin tissue newly formed after the drug treatment has no hollow of dermis layer and more complete epidermis structure and complete hair follicle structure through HE section of the wound tissue. In conclusion, the novel sulfur-containing element and bFGF composite nano-particles constructed by the method have a good repairing effect on the wound surface of a normal mouse.
6. Novel sulfur-containing element and bFGF composite nano-particle for promoting wound repair of diabetic mice
In addition to normal skin wound repair, wound repair disorders due to metabolic disorders in vivo are also a key scientific problem in the field of tissue regeneration. Wherein the wound surface difficult to heal of diabetes is a clinically troublesome wound surface repair problem, so db is adopted in the part of the wound surface difficult to heal - /db - Diabetes mouse model evaluation we constructed novelWhether the sulfur-containing element and bFGF composite nano-particles have a repairing effect on refractory wounds caused by diabetes. The method comprises the following specific steps:
db-/db-diabetic mice (purchased from the laboratory animal center in Guangdong province) of 30-35g body weight 6-8 weeks old were selected, 6 in each group. After pentobarbital sodium (purchased from Sigma-Aldrich (Shanghai)) had been anesthetized, a full-skin injury model was established on the back of diabetic mice using a skin perforator with a diameter of 6 mm. After the diabetic refractory wound injury model is established, two injection points are fixed around the wound surface, and 50uL of drug molecules (the concentration of the novel sulfur-and bFGF-containing composite nano particles prepared in the step 2.1 is 200nM+500IU) are injected subcutaneously, wherein equal volume of PBS is used as a blank control, and bFGF (500 IU) is injected as a control. Once every other day, and collecting corresponding wound healing data. And at the same time, the wound tissue is obtained on the tenth day, paraformaldehyde is fixed, paraffin blocks are prepared, and tissue sections are made. The histomorphology of the nano sulfur on the wound surface tissue repair of the skin tissue is observed through HE staining.
As a result, as shown in fig. 6, we found that the blank group had ulcers on the tenth day of wound surface after every other day of administration, which was consistent with the clinical characteristics of diabetic foot. When the novel sulfur-containing element and bFGF composite nano particles are adopted for treatment, the wound surface of the diabetic mice is found to be free from ulceration and gradually healed, and the quantitative analysis of the wound healing rate finds that the healing of the difficult-to-heal wound surface of the diabetes is obviously accelerated after the treatment of the medicine. Further we stained the wound surface with HE, found that the dermis thickness of the wound surface tissue after drug treatment was improved in the integrity of the epidermis. In conclusion, the novel sulfur-containing element and bFGF composite nano-particles constructed by the method can obviously improve the repair of the wound surfaces difficult to heal due to diabetes.
7. Novel in-vivo safety evaluation method for sulfur-containing element and bFGF composite nano particles
In vivo biosafety is a key indicator of biomaterial application. In order to evaluate the in vivo biosafety of the constructed novel sulfur element and bFGF composite nano-particles, the in vivo acute toxicity of the zebra fish juvenile fish is evaluated by adopting a zebra fish development model, and the specific steps are as follows:
(1) Zebra fish developmental toxicity evaluation:
zebra fish are conventional commercial AB-series wild zebra fish, are cultivated to adult in the laboratory according to a conventional method, then the adult male zebra fish and the adult female zebra fish are taken for spawning, fish eggs are collected, and the fish eggs are placed in 24 pore plates, wherein 10 fish eggs are arranged in each pore. Adding 1mL of the novel composite nanoparticle solution containing sulfur and bFGF, which is prepared in the step 2.1 and has the concentration of 200nM+500IU, into an orifice plate, setting 3 composite holes, taking photo and counting survival rate of the young zebra fish at 0 hour, 24 hour and 48 hour respectively by taking PBS as a control, counting the body length and heart rate of the young zebra fish at 48 hours, and repeatedly calculating an average value for three times to evaluate the developmental toxicity of nano sulfur to the zebra fish.
(2) In vivo toxicity evaluation of mice:
balb/c mice (purchased from Experimental animal center in Guangdong province) with a body weight of 18-22g at 6-8 weeks of age were selected. After pentobarbital sodium anesthesia, the injection is carried out three times, once every other day, the single drug dose is 50uL (the novel sulfur-and bFGF-containing composite nano-particles prepared in the step 2.1 are 200nM+500 IU), and the weight change is counted; mice were euthanized after day eight, blood biochemical indicators were detected, and their organs were HE sectioned to see if structural lesions were present.
As shown in fig. 7, the drug molecules were found to be non-toxic to the development of embryos and young fish of zebra fish and non-teratogenic compared to the blank group; further, by counting the body length and heart rate of the young zebra fish in the development process, no obvious influence of the medicine on the development of the zebra fish is found. Besides the toxicity evaluation of zebra fish, the toxicity effect of the novel sulfur-containing element and bFGF composite nano-particles on mice is evaluated, and the morphology of organs of the mice is observed through statistics of weight change and HE slicing of the mice, so that the constructed novel sulfur-containing element and bFGF composite nano-particles do not cause the toxicity effect of the organs in the mice. In conclusion, the biological verification of two modes of zebra fish and mice proves that the novel sulfur-containing element and bFGF composite nano-particles are not easy to cause toxicity in vivo and have higher biological safety.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the composite nano-particles containing the sulfur element and the bFGF is characterized by comprising the following steps:
(1) Adding sublimed sulfur into PEG400, stirring and mixing at 30-45 ℃, transferring to a high-temperature closed reaction kettle, firstly raising the temperature from 30-45 ℃ to 80-120 ℃ within 15-30 minutes, maintaining the temperature at 80-120 ℃ for 1-2 hours, then lowering the temperature from 80-120 ℃ to 25-45 ℃ within 20-40 minutes, taking out, adding water, and dialyzing to obtain a nano sulfur particle solution;
(2) And (3) dropwise adding the basic fibroblast growth factor solution into the nano sulfur particle solution obtained in the step (1), stirring and reacting at 25-35 ℃, and dialyzing after the reaction is finished to obtain the composite nano particles containing sulfur element and bFGF.
2. The method for preparing the composite nano-particles containing the sulfur element and the bFGF according to claim 1, wherein:
the conditions for the reaction described in step (1) are: the temperature was raised from 40℃to 100℃over 30 minutes, maintained at 100℃for 1 hour, and then lowered from 100℃to 30℃over 30 minutes.
3. The method for preparing the composite nano-particles containing the sulfur element and the bFGF according to claim 1, wherein:
the dosage of the sublimed sulfur in the step (1) is calculated according to the proportion of 0.5-2 mL PEG400 of sublimed sulfur per milligram;
the concentration of the basic fibroblast growth factor solution in the step (2) is 100-300 IU/mL;
the concentration of the nano sulfur particle solution in the step (2) is 1-3 mu mol/L;
the volume ratio of the nano sulfur particle solution to the alkaline fibroblast growth factor solution in the step (2) is 1:3.
4. the method for preparing the composite nano-particles containing the sulfur element and the bFGF according to claim 3, wherein:
the amount of the sublimed sulfur in the step (1) is calculated according to 1mL PEG400 of the sublimed sulfur ratio per mg;
the concentration of the basic fibroblast growth factor solution in the step (2) is 200IU/mL;
the concentration of the nano sulfur particle solution in the step (2) is 2 mu mol/L.
5. The method for preparing the composite nano-particles containing the sulfur element and the bFGF according to claim 1, wherein:
the dialysis in the step (1) is dialysis for 72-120 hours by using a dialysis bag with the molecular weight cut-off of 1000 kDa;
the dialysis in the step (2) is dialysis for 72-120 hours by using a dialysis bag with the molecular weight cut-off of 2000 kDa;
the dialysate used in the dialysis in the steps (1) and (2) is PBS buffer solution;
the reaction time in the step (2) is 6-12 hours.
6. The method for preparing the composite nano-particles containing the sulfur element and the bFGF according to claim 5, wherein:
the dialysis in the step (1) is dialysis for 72 hours by using a dialysis bag with the molecular weight cut-off of 1000 kDa;
the dialysis in the step (2) is dialysis for 72 hours by using a dialysis bag with a molecular weight cut-off of 2000 kDa;
the reaction time in step (2) was 6 hours.
7. A composite nanoparticle containing elemental sulfur and bFGF, characterized in that: is prepared by the method of any one of claims 1 to 6.
8. The composite nanoparticle containing elemental sulfur and bFGF as claimed in claim 7, wherein: the particle size of the composite nano particles containing the sulfur element and bFGF is 50-150 nm.
9. Use of the sulfur-containing element and bFGF composite nanoparticle of claim 7 or 8 in the preparation of a product for wound repair.
10. The use according to claim 9, characterized in that: the wound repair comprises the wound repair of normal people or animals or the wound repair of difficult healing of diabetics or animals.
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