Research And Application Of Sponge Haliclona Sp. Spicules(SHS) Mediated Nanomaterials In The Treatment Of Skin Diseases

The skin is a kind of biofilm barrier,and it is difficult for drugs with molecular weight greater than 500 Da to enter the deep layer of skin directly.The existing penetration promoting technologies,including microneedle technology,have some limitations,which cannot achieve the safe and effective delivery of drugs in the area and parts of the skin.Sponge Haliclona sp.spicules(SHS)is purified from sponge Haliclona sp..As a new micro needle technology,SHS has many unique advantages in the field of drug transdermal delivery.In this thesis,SHS combined with different nanomaterials to constructed a novel skin delivery system for the treatment of skin diseases,the specific research contents and results are as follows.1.SHS combined with cationic flexible liposomes(CFL,0.05%)could effectively deliver siRNA to skin cells and achieve local gene interference.(1)In the transdermal experiment in vitro,the combined use of SHS and CFL(0.05%)made siRNA have the highest skin penetration,the skin absorption rate of siRNA was as high as 61.18%±2.49%after 16 hours,which was significantly higher than SHS combined with CFL(1%)(48.19%±7.21%,p<0.05),dermaroller combined with CFL(0.05%)(5.34%± 0.72%,p<0.05),SHS alone(32.55%±8.90%,p<0.01),CFL(0.05%)alone(4.14%±0.73%,p<0.001)and control group(0.84%± 0.16%,p<0.001).(2)After that we evaluated the ability of CFL(0.05%)to deliver siRNA into cells to reduce protein expression.CFL(0.05%)had little toxicity to cells according to MTT assay.Confocal imaging showed that CFL(0.05%)could induce fluorescently labeled siRNA to enter cells,and quantitative results showed that the GAPDH protein knockout rate was as high as 41.09%±5.14%.(3)Finally,we demonstrated the ability of SHS combined with CFL(0.05%)to deliver siRNA to the skin.The combined application of SHS and CFL(0.05%)on mouse skin can cause a knockout rate of GAPDH protein as high as 29.21%± 1.41%in the entire application area,which was comparable to that from the subcutaneous injection of CFL@siRNA in the injection center area(26.17%± 6.25%,p=0.458).The above results show that the combined use of SHS and CFL(0.05%)is a promising delivery system for topical RNAi therapy.2.The surface of SHS was modified with mesoporous silica to form surface modified SHS with mesoporous silica(mSHS),which could carry and effectively transdermal delivery of insoluble photosensitizer protoporphyrin ?(Pp?)for the treatment of melanoma in mice.(1)Both scanning electron microscope and transmission electron microscope results show that a layer of mesoporous material was successfully modified on the surface of SHS.The specific surface area of mSHS is 14.86±3.36 m2/g,which is 33.23± 5.32-fold that of SHS.The drug loading of mSHS was studied using coumarin 6 as a model drug in vitro.(2)mSHS loading capacity was positively correlated with coumarin 6 concentration.When the concentration of coumarin 6 was 40 mg/mL,each milligram of mSHS can carried 52.56 ?g of coumarin 6.(3)Transdermal experiments were performed with mSHS@coumarin 6 in vitro,and the results showed that the transdermal content of coumarin 6 in the mSHS group was 62.59 ± 12.26-fold that of the SHS group and 75.68 ±14.82-fold that of the control group.(4)The lethality of protoporphyrin ? to B16 cells was measured.The results showed that when the concentration of protoporphyrin ? was between 0.1-0.75 ?g/mL,the cell survival rate decreased with the increase of protoporphyrin ? concentration.(5)We further verified the efficacy of mSHS loading Pp?(mSHS@Pp?)in the treatment of mouse melanoma in vivo.The results showed that after four times treatments with mSHS@PpIX,the mice melanoma can be completely cured without recurrence.This study can provide corresponding theoretical support and new ideas for treatment of skin cancer.In this thesis,we creatively combined SHS with different nanomaterials to constructed SHS combined with cationic flexible liposomes to mediated local gene interference and mSHS to mediate local photodynamic therapy for the treatment of mouse melanoma.These new transdermal drug delivery systems provide new ideas for gene therapy of skin diseases and the treatment of skin cancer,provide theoretical data support for SHS as a new medical material for the treatment of skin diseases.At the same time,it had also played a good role in the development and application of marine biological resources in China.