Construction Of Photocontrolled "Self-degradable" Nanoparticle Hydrogel System For Blocking YAP Signal Of Vascular Smooth Muscle Cells

Background Arteriovenous Fistula(AVF)is a prerequisite for the smooth implementation of hemodialysis patients,while vascular pathway dysfunction caused by intimal hyperplasia stenosis is an important factor for the increase of patient mortality,at present,there is no effective prevention and treatment measures in clinical practice.Therefore,how to prevent and cure AVF intimal hyperplasia and stenosis and improve the patency rate is a hot and difficult point in the field of blood purification in recent years.In the field of basic research,researches on the mechanism of intimal hyperplasia of AVF are increasing,but there is no scientific and effective means to prevent and cure AVF stenosis.The reason lies in the lack of specific targets,targeted intervention drugs and limited clinical drug use and so on.Phenotype switch of vascular smooth muscle cells(VSMCs)are well-recognized pathological cellular events that lead to neointima formation of AVF,However,the underlying mechanisms are not known.In recent years,biomechanical studies have confirmed that mechanical receptors on the membrane of VSMCs can transmute mechanical signals into biochemical signals,thus playing a role in regulating phenotypic transformation of VSMCs and affecting vascular homeostasis.Among the numerous mechanical receptors,Hippo-YAP signaling pathway has received extensive attention.Hippo-YAP signaling pathway can regulate the proliferation and migration of VSMCs and participate in the process of vascular injury and remodeling.Based on this,it is not difficult to speculate that Hippo-YAP signal is involved in the intimal hyperplasia of AVF.the clinical use of verteporfin(VER)as a YAP inhibitor has great potential to improve the intimal hyperplasia of AVF.In addition,there are some shortcomings in traditional vascular therapy,such as low bioavailability of drugs in vivo and not giving priority to affecting diseased cells,resulting in less enrichment of drugs at the target and prone to unnecessary systemic side effects.The development of nano-carrier technology provides a new solution strategy for the treatment of vascular injury diseases,so the development of innovative extravascular drugs and nanomaterial carrier system has a clinical prospect.Purpose 1.To investigate whether YAP in VSMCs is involved in the process of intimal hyperplasia in arteriovenous fistula of rats with chronic kidney disease;2.To Design and synthesize ROS-sensitive polymer materials,and to construct the nanoparticle/hydrogel system loaded with verteporfin.3.To explore the effects of remotely regulating the release of drugs from nanoparticle/hydrogel system on the proliferation and migration of VSMCs,and to systematically study the biological mechanism of the system at the cellular level.4.To demonstrate the stability and safety of nanoparticle/hydrogel system in vivo transport,and to explore the potential value of this system and controlled release drug delivery strategy in clinical application.Methods 一、Expression of YAP in the neointima of arteriovenous fistula and its role in the proliferation of vascular smooth muscle cells.1 AVF model of common carotid artery and external jugular vein was established in mice with chronic kidney disease.AVF tissues and normal blood vessel tissues were collected,and the intimal of AVF and the expression of YAP and SMA-α in the intimal of AVF were evaluated by HE and immunohistochemical staining.2 A scratch experiment was conducted to verify that the YAP inhibitor verteporfin(VER)could inhibit the migration of VSMCs;VSMCs with overexpression of YAP were constructed,and the effects of VER on the protein expressions of YAP,SMA-α,PCNA and CTGF in VSMCs were detected by Western Blot.二、Construction and characterization of nanoparticle/hydrogel system loaded with verteporfin.1.The ROS-sensitive hyperbranched polyphosphate material(TK-hb PPE)was synthesized,and the carrier material was characterized by ~1H NMR.Drug loaded nanoparticle TKhb PPE/VER was prepared by mono-emulsification method and blended with PLGAPEG-PLGA thermosensitive hydrogel(PPP)to construct the nanoparticle/hydrogel system TKhb PPE/VER@PPP.2.Characterization of the drug-loaded nanoparticle/hydrogel system:(1)The size and morphology of nanoparticles TKhb PPE/VER were detected by dynamic light scattering and transmission electron microscopy;(2)The content of VER in TKhb PPE /VER@PPP system was determined by ultraviolet spectrophotometer and fluorescence spectrophotometer,and the encapsulation efficiency was calculated.(3)The production of reactive oxygen species(ROS)by TKhb PPE/VER under different light conditions was detected by SOSG detection kit,and the drug release behavior under different light conditions was compared.三 、 The effect of blocking YAP signal by TKhb PPE/VER@PPP system on VSMCs proliferation.1.Cell uptake experiment: Nanoparticle hydrogel mixtures were quickly added to the upper chamber of Transwell.The mixture was first placed in an empty 24-well plate and placed in an incubator until gelatinized.The upper chamber of Transwell was irradiated by 660 nm red light,and then the upper chamber was transferred to the well plate containing cells for further culture for 4 h.The content of VER in VSMCs was detected by flow cytometry and the localization of VER in cells was observed by laser confocal.2.Cytotoxicity and apoptosis assay: VSMCs cell viability was determined by CCK 8 method.Apoptosis of VSMCs was detected by Annexin V-FITC/PI kit and Death/Live staining.3.Scratch experiment was conducted to verify the inhibitory effect of VER released into VSMCs by drug-loaded nanoparticles in the hydrogel system on VSMCs migration.and a VSMCs cell model with overexpression of YAP was constructed to detect the effect of VER released under red light on the expression of YAP,SMA-α,PCNA and CTGF protein.4.VSMCs were irradiated with 660 nm red light of 0.05 W /cm2 for 5 min,and cell viability was determined by CCK 8 method.Western Blot was used to detect the effects of red light irradiation on the protein expressions of YAP,SMA-α,PCNA and CTGF in VSMCs,to explore the safe behavior of cells under low power irradiation.四、Preliminary exploration of TKhb PPE/VER@PPP system in mice.1.Enrichment and content of TKhb PPE/VER@PPP system in mice vascular tissue:Nanoparticles/hydrogel TKhb PPE/VER@PPP were locally implanted into the blood vessels,and the skin was sutured after solidification into gel.(1)Small animal imaging technology detected the enrichment of nano-drug particles in tissue;(2)The samples were collected at different time points after red light irradiation to detect the content of VER in external jugular vein tissue samples.2.Safety evaluation of TKhb PPE/VER@PPP system on mice: TKhb PPE/VER was smeared on the normal external jugular vein of mice,and irradiated with 0.05 W/cm2 red light for 5 min.serum samples were collected at different time points to detect liver and renal function to evaluate the safety of this system on mice.Results 一、The high expression of YAP in the neointima of arteriovenous fistula was associated with the migration and proliferation of vascular smooth muscle cells.1 In mice with chronic renal disease,the intimal tissue of internal fistula was thickened and the lumen was narrowed 4 weeks after AVF,and there was high expression of YAP in the intimal tissue.2 YAP inhibitor VER can significantly inhibit the the expression of YAP,SMA-α and CTGF proteins in vascular smooth muscle cells,and inhibit the migration of cells.二 、TKhb PPE/VER@PPP nanoparticle/hydrogel system loaded with verteporfin was successfully constructed.1.The ROS sensitive polyphosphate material TK-hb PPE was successfully synthesized by 1H NMR.and TKhb PPE/VER nanoparticles were prepared by loading VER.The PLGA-PEGPLGA thermosensitive hydrogel was mixed with TKhb PPE/VER at different mass ratio at room temperature.and a stable TKhb PPE/VER@PPP system was formed at a mass ratio of 15:1.2.Dynamic light scattering and transmission electron microscopy showed that the size of TKhb PPE/VER nanoparticles was uniform and and the size was 300 nm.UV-vis absorption spectra showed that both nanoparticles and free VER had absorption peaks at 660 nm,which confirmed that VER were successfully loaded,and the entrapment efficiency was 30-35%.The SOSG probe found that the ROS produced by TKhb PPE/VER increased gradually with the extension of light time.The drug-loaded nanoparticles were irradiated with 660 nm red light at a light intensity of 0.05 W/cm2 for 5 min,It was found that the loaded VER drug could be released slowly,and the cumulative release reached 18% of the total drug dose after 24 h.三 、 The effect of blocking YAP signal by TKhb PPE/VER@PPP system on VSMCs proliferation.1.After irradiating the system,laser confocal observation showed that VER was located around and in the nucleus of VSMCs,and flow cytometry showed that VSMCs could absorb more VER.2.CCK 8 and Dead-Live staining results showed that red light irradiation of TKhb PPE/VER@PPP had a strong inhibitory effect on VSMCs cell viability and proliferation under different VER concentrations.Annexin V-FITC/PI cell apoptosis staining showed that after red light irradiation,there were more early and late apoptotic cells in the light group,However,the non-light group and the normal control group basically did not cause cell apoptosis,with a statistically significant difference(P < 0.001),indicating that red light irradiation can promote the release of VER into VSMCs,to inhibit cell proliferation and induce apoptosis.3.The results of cell scratch experiment showed that the scratch distance of VSMCs was decreased significantly after red light irradiation of TKhb PPE/VER@PPP system,indicating that the VER released by this system significantly inhibited the migration ability of VSMCs.Meanwhile,Western Blot showed that TKhb PPE/VER@PPP significantly decreased the protein expressions of YAP,SMA-α,PCNA and CTGF in VSMCs overexpressing YAP gene after light exposure.4.VSMCs were irradiated with 660 nm red light of 0.05 W /cm2 for 5 min,and cell viability was no statistically significant difference.At the same time,there was no difference in the expression of proliferation-related proteins detected at the protein level,which indicated that low-power red light irradiation had a good safety to cells.四、Application of drug-loaded nanoparticle/hydrogel TKhb PPE/VER@PPP system in mouse blood vessels.1.The results of in vivo imaging showed that drug-loaded nanoparticle hydrogel could gather locally in the left neck of mice,and the tissue of external jugular vein was taken at different time points to detect the content of VER.The results showed that 4 hours after irradiation,the content of VER in vein tissue extract increased gradually with time,and reached the peak at 24 hours,and a small amount of VER could still be detected until 72 h.2.TKhb PPE/VER@PPP was smeared on the normal blood vessels of mice and irradiated with red light,the blood indexes showed that there was no significant difference in liver and kidney function between the light group and the non-light group.indicating that this system had good safety for mice in a short term at a small dose.Conclusion 1.There was high expression of YAP protein in the neointima of the arteriovenous fistula of mice with chronic kidney disease,and YAP was involved in the proliferation and migration of vascular smooth muscle cells.2.The drug-loading nanoparticles TKhb PPE/VER were successfully prepared and characterized,and the drug-loading nanoparticle/hydrogel system TKhb PPE/VER@PPP was constructed.3.Under red light irradiation,the system triggered the drug-loaded nano-hydrogel system TKhb PPE/VER@PPP to release VER,inhibit the proliferation and migration of vascular smooth muscle cells,and down-regulate the expression of intracellular YAP protein.4.The system can be successfully retained in the blood vessels of mice,can regulate the release of VER by light control,and has good safety in vivo,which suggests that the method of peripheral vascular administration of TKhb PPE/VER@PPP system is feasible,and longdistance light administration can promote the disintegration of nanoparticles to release drugs,which provides a feasible scheme for local vascular administration to interfere with AVF fistula stenosis.