Gelatin Microspheres Containing Drugs Loading With Themo-Sensitive Hydrogel Improves Diabetic Wound Healing

Diabetes is a universal public health problem.The diabetes population was 415million around the globe in 2015,and,the data will rise to 642 million in 2040.The microscopic pathology of skin injury with diabetes mel itus was cell phenotypic abnormalities,including of reducing of potency of cell mitosis and losing the ability to respond to the surroundings.Ultimately lead to the proliferation ability was decreased of epidermal cels and fibroblasts around the skin wound site.Theycannot migrate to the wound site,and the skin wound with diabetes mel itus couldnot heal for a long time.MMP-9 was overexpression in these skin wound,and the high levels of MMP9have something to do with the delaying of re-epithelialization because of epidermal cell migration down-regulated by high levels of MMP-9.Currently,there are some deficiencies in the treatment of diabetic wound healing.Autoimmune problems,cannot achieve therapeutic effect in a short time,exogenous pathogenic microorganisms and excessive cost restricted these application.Curcumin is a highly safe drug molecule,extractedfromcurcuminlonga,which anti-inflammatory,anti-oxidation,antibacterial,anti-tumor and anti-diabetes.Curcumin has function of anti-inflammatory,anti-oxidative and promoting cell migration,which can promote the healing of skin wounds.Its water solubility,biological instability and bioavailability were greatly improved,after the nanocrystallization process of curcumin.However,curcumin is a double-edged sword,high doses of curcumin induces cell apoptosis.Therefore,controlled release under physiological condition and bioavailability increased of curcumin is the key point of this research.Curcumin nano-particles was obtained,which increase its bioavailability.And then it was encapsulated into gelatin microsphere,MMP9-responsive drug carrier acquisition.The release of drug molecules can be controlled by the level of expression of MMP-9 around the skin wound.The degradation products of gelatin microspheres are peptides containing RGD sequence which can promote cell migration.The combination of these drug delivery system and the thermoresponsive hydrogel,can form a hydrogel state at the body surface temperature.The recombination skin wound dressing was obtained after the above steps.In this study,the high-purity curcumin molecules was nanocrystallization,Its water-soluble and bioavailability get improved.Curcumin nano-particles was encapsulated into gelatin microsphere,and then blended with pluronic F127 and F68,and the recombination skin wound dressing was obtained.The skin wound dressing was applied to skin trauma model of diabetic mice.Firstly,the curcumin nano-particles was obtained.Its particle size distribution in50-100nm.It was spherical,same particle size,its water solubility were greatly improved.The phenomenonfluorescence self-quenching was apeared.Compared to curcumin,curcumin nano-particles has increased cytotoxicity to fibroblasts.Curcumin nanoparticles have a higher positive rate than the curcumin molecules in BJ and HaCaT cels in thecell endocytosis experiment.Secondly,curcumin nano-particles was encapsulated into gelatin microsphere by glutaraldehyde cross-linking.And thegelatin microspheres have a regular spherical shape with uniform particle size and without aggregation.Gelatine microspheres distributed in the range of 2.5 to 5μm was close to 30%,and greater than 10μm was20.19%.Compared to gelatin microsphere without curcumin nano-particles,the partical size of gelatin microsphere with curcumin nano-particles was no significant increase.Drug loading capacity and encapsulation efficiency of it respectively were94.82±0.52%and 24.75±3.18%.It has good responsiveness to MMP-9.And as the concentration of MMP-9 increases,the release of curcumin nano-particles was increased within a range of 1nM to 10nM of MMP-9.However,The release of it reached its maximum within a range of 20nM to 50nM of MMP-9.The release of curcumin nano-particles was increased following the elongation of time treated by MMP-9,and it got its peak was within 24 hours.Curcumin nano-particles released from gelatin microsphere,its cytotoxicity to fibroblasts and epidermal cels and ability to reverse the oxidative damage of these cels by hydrogen peroxide was superior to curcumin and inferior to curcumin nano-particles.Also it can inhibit of fibroblasts and epidermal cells apoptosis.Promoting cell migration of it was superior to curcumin and inferior to curcumin nano-particles.Thirdly,thermo-sensitive hydrogel can cover on a surface of skin injury.Pluronic F127 and F68 with various concentrations was dissolved in normal saline,thermo-sensitive hydrogel was obtained.Phase-transition temperature of normal saline with 20%F127 and 6%F68 was 32.97℃.When 4.04μg/mL gelatin microsphere with curcumin nano-particles was added into it,its phase-transition temperature was 32.5℃.In addition,the survival percentage of BJ and HaCaT cells were exceeded 80%,when these cels were incubation with F127 or F68 within a range 1 to 1000μg/mL.Lastly,normal saline with 20%F127 and 6%F68 and gelatin microsphere with curcumin nano-particles made up wound dressing.And,it was appied to skin trauma model of diabetic mice made modelby streptozotocin.Compared to thermo-sensitive hydrogel and curcumin,the healing time of ful-thickness cutaneous wounds on the mouse back with diabetes mel itus was shorten treated by gelatin microsphere with curcumin nano-particles.The 18^th day of wound healing,relative area of skin wound respectively were 38.89%,17.47%and 4.93%treated by thermo-sensitive hydrogel,curcumin or gelatin microsphere with curcumin nano-particles.The skin wound were no complete re-epithelialization on the 20^th day of wound healing.Re-epithelialization was initiated on the 10^th day and completely finished on the 20^thh day,while the wound was treated with gelatin microsphere with curcumin nano-particles.Curcumin gelatin microspheres have the effect of promoting the migration of epidermal cels and fibroblast cels to the wound.The distribution of collagen was improved,the expression levels of CD31 and Ki67 increased and the level of MMP9was decreased by immunohistochemistry and collagen staining.In conclusion,this topic is based on the gene expression of diabetic trauma model and the pharmacological action of curcumin molecule.Curcumin molecules was nanocrystallization,and then was encapsulated into gelatin microsphere,the drug delivery system that responded to MMP-9 was obtained.And the drug delivery was mixed with thermo-sensitive hydrogel,evolved into wound dressing.The hydrogels were formed on the surface of the skin wound at the normal surface temperature.Experimental result demonstrated that this composite drug delivery system can significantly accelerate the healing of the ful-thickness cutaneous wounds in diabetic mice.The expression levels of CD31,Ki67 and MMP9 had changed.This experiment provides a new way of thought for the treatment of wound healing wounds.