Transdermal Delivery Of Heparin Using Low Frequency Sonophoresis In Combination With Sponge Spicules

In this thesis,we studied the use of low-frequency sonophoresis(LFS)in combination with sponge Haliclona sp.spicules(SHS)to deliver low molecular weight heparin for the treatment of venous thrombosis.The research results are as follows:1.Confirmed the use of LFS in combination with SHS in a proper order,referred to as cSoSp(combined Sonophoresis and Spicules),can greatly enhance the transdermal drug delivery in an excellent synergistic manner.In the transdermal experiment in vitro,the topical application of cSoSp significantly enhanced the skin absorption of Fluorescent-Dextrans(4000 Da,FD-4K),as a model drug of low molecular weight heparin(LMWH),the skin absorption rate of FD-4K was as high as 9.5%± 1.6%after 16 hours,which was significantly higher than SHS combined with LFS group(0.8%± 0.1%,p<0.001)and was 27.9 ± 10.4-fold higher than control group(0.3%± 0.1%,p<0.001),4.5 ± 1.0-fold higher than 10 mg SHS group(2.1%± 0.3%),p<0.01,7.3±1.4-fold higher than 2 mg SHS group(1.3%± 0.1%,p<0.001)and 7.3± 3.1-fold higher than the LFS group(1.3%± 0.5%,p<0.001).And the synergy index(SI)of cSoSp was 4.1 ± 0.6,which was much higher than cSpSo group(SI=0.4 ± 0.1),the combined use of Dermaroller and LFS group(SI=1.9 ± 0.4)and the combined use of LFS and Dermaroller group(SI=1.7 ± 0.3).Futhermore,the full-thickness porcine skin treated with different penetration enhancing strategies was sectioned at 10 μm thickness and imaged by using a confocal microscope,we can find that the cSoSp can lead to significant enhance skin deposition of FD-4K in skin,especially in skin deep layers,the barrier of stratum corneum is completely broken.Also the cSoSp dramatically increased the transdermal flux of FD-4K(188.6±93.8 ng/cm2h-1)compared to other penetration enhancing methods.After that we observed the effect of application sequence of LFS and SHS on the skin surface by scanning electron microscopy and local staining penetration experiment,we successfully explained the synergistic mechanism of cSoSp.In sum,the combined use of SHS and LFS has a good delivery effect for hydrophilic biological macromolecules.2.Analyzed the therapeutic effect on venous thrombus of cSoSp by the delivery of LMWH.The venous thrombus was induced by thrombin injection.Then LMWH was delivered to both superficial venous thrombosis(SVT)and deep venous thrombosis(DVT)in rabbit marginal ear vein for theatment by different skin delivery strategies and intravenous injection.In the case of SVT treatment,cSoSp decreased the thrombus size by 95.2 ±3.8%in 48 h and significantly(p<0.05)pronounced the thrombolytic effect of LWMH compared to other skin delivery strategies.In the case of DVT treatment,cSoSp decreased the thrombus size by 77.6±2.6%in 48 h,which was almost comparable(p>0.05)to intravenous injection.3.Evaluated the application safety of cSoSp.Skin irritation and toxicity study using guinea pig indicated that the utilization of cSoSp was nonirritating without any morphological change in keratinocytes,although it can induce the immune cells infiltration over 240 h,the cSoSp has good application security.The topical application of LFS in combination with SHS(cSoSp)offers a promising strategy to enhance the transdermal delivery of hydrophilic macromolecules,and low molecular weight heparin was successfully delivered for the treatment of venous thrombosis.This novel transdermal drug delivery method not only provides a safe and efficient strategy for enhancing the transdermal delivery of hydrophilic biomacromolecules such as heparin,but also provides a promising method for the treatment of thrombotic diseases.