PH/thermosensitive Dual-Responsive Hydrogel Based Sequential Delivery For Site-specific Limb Ischemia Treatment

Background and objective:Ischemic disease of lower extremity is a devastating disease that seriously threatens the viability of ischemic limbs and even leads to limb loss,with high morbidity and mortality.The current conventional treatment strategies have obvious limitations and cannot meet satisfactory treatment effect.Therefore,a more ideal treatment strategy is urgently needed.Therapeutic angiogenesis has the potential to induce angiogenesis,which can provide effective treatments for limb ischemia.Although therapeutic angiogenesis has made some progress in research,there are also obvious problems to be solved,such as unstable drug and protein activity,low cell survival rate and uncontrollable action time.Nano drug delivery system has successfully attracted the strong interest of scientific researchers and becomes one of the research hotspots of medicine and translational medicine because of its excellent properties,such as good drug loading ability for both hydrophilic and hydrophobic drugs,maintaining the stability of drugs and cells,controllable drug release behavior and good biocompatibility.For instance,degradable hollow mesoporous silicon nanoparticles（dHMSNs）,as one of the best carriers to make up for therapeutic angiogenesis,have shown great advantage in high efficiency,sustained,controlled-release and targeted drug delivery,which is a drug delivery system with great clinical application prospect.On the other hand,hydrogel,as a three-dimensional network structure system formed by polymer with hydrophilic groups,has also attracted growing attention in drug delivery,tissue engineering,and many other fields.Intelligent hydrogels not only have the characteristics of ordinary hydrogels,but also are sensitive to external stimuli.They can make corresponding phase changes according to the change of the external microenvironment and realize targeted,controllable and on-demand intelligent reactivity release of drugs,which have gradually become a new research hotspot.Due to the lack of suitable nanocarriers,drug release targeted the acute phase of cell protection and chronic phase of angiogenic cannot be satisfied.In conclusion,dHMSNs and intelligent responsive hydrogels are used to build a composite nano-drug delivery system.The hollow mesoporous silicon nanoparticles can solve the contradiction between the coexistence of hydrophilic and hydrophobic drugs.The intelligent responsive hydrogel can sensitively respond to the changes of the microenvironment.Ultimately,the system can achieve orderly,controlled and on-demand drug release in an intelligent manner for the treatment of limb ischemia.Research contents:（1）After referring to several pieces of literature,we synthesized successfully dHMSNs and the stimulus-responsive hydrogel（FHSgel）,and carried out a series of characterizations to explore their physicochemical properties.The results showed that dHMSNs had higher specific surface area,which could load more drugs.And the appropriate pore size could meet the needs of sustained drug release.The results also showed that dHMSNs had good biodegradability and could be used as one of the ideal carriers for drug delivery.FHSgel had an injectable ability,which met the requirements of transdermal administration and in situ targeting.Characterization experiments showed that FHSgel had a highly mesh-like three-dimensional structure,which could realize the controlled release of drugs with different particle sizes.Meanwhile,the rheological experiment showed that FHSgel had good temperature sensitivity and phase transition reversibility,and was sensitive to p H-response.It could realize the dual response of p H and temperature,making it possible to respond intelligently to the change of microenvironment.（2）Firstly,the hydrophobic drug diallyl trisulfide（DATS）was loaded on dHMSNs to construct DATS@dHMSNs（Dd）.And then the hydrophilic drug Melatonin（M）and DATS@dHMSNs were co-wrapped in FHSgel to construct the final system M/Dd-FHSgel.In vitro drug release was studied by Ultraviolet-visible（UV-vis）spectroscopy and intracellular fluorescence assay.The results suggested that DATS@dHMSNs could slowly and sustainably release DATS,and under the action of reduced glutathione（GSH）,H₂S could be released slowly compared with free DATS,achieving the effect of sustained drug release.The p H-and temperature-responsive drug release results of the system showed that the system had a good dual-response to p H and temperature,releasing melatonin more rapidly as the p H decreased and DATS@dHMSNs more slowly as the temperature increased,thereby realizing intelligent,orderly,on-demand and controllable drug delivery.（3）Through in vivo and in vitro experiments,it was verified that both dHMSNs and FHSgel had good biocompatibility.In vitro cell experiment had verified that the system could protect acute damaged cells well,showing a good cell protection effect.Its mechanism was further explored,and the results showed that it could play a role through directly scavenging free radicals,anti-apoptosis and anti-oxidation.In addition,in the simulated chronic ischemia environment in vitro,this system could significantly promote angiogenesis.Further study of its molecular biological mechanism showed that H₂S released from this system could up-regulate the phosphorylation of p38 and ERK1/2 in endothelial cells,promoting the proliferation and migration of endothelial cells as well as the formation of lumen.Finally,the therapeutic effect and biological safety of the system were verified in vivo experiments.The results also showed that the system had good cell protection and angiogenesis promotion effect,and could effectively protect ischemic limbs.Conclusion:Herein,we developed an injectable p H/thermosensitive dual-responsive FHSgel encapsulated melatonin and DATS@dHMSNs,which could exert cell protection effects in the acute phase and pro-angiogenic effects in the chronic phase in ischemic limbs.This smart hydrogel based nanoplatform could respond to the changes of ischemic microenvironment in different phases and conduct corresponding sequential drugs release simultaneously.Further in vivo and in vitro experiments have shown that this dual-responsive sequential drug delivery could improve the treatment efficiency of limb ischemia considerably,which thus could effectively reduce the disability rate.