| Liver cirrhosis, or fibrosis, the ultimate pathological feature of allforms of chronic hepatic damage, is responsible for much morbidityand mortality worldwide, and there are currently no approved antifibrotic therapies for liver cirrhosis. siRNA has been pproved to be able to silence genes in human and has the potential to cure human diseases. Thus, we aim to establish an anti-hsp47/gp46siRNA liposome deliver system for liver fibrosis in this study, and our ultimate goal is to develop a siRNA drug for liver fibrosis.We established a siRNA liposome deliver system which targets liver fibrosis by both lipid film-extrusion formulation method and ethanol based self-assembling method (SNALP), and optimized it.By ethanol based self-assembling method, anti-FVII siRNA liposome deliver system which targets the hepatocytes for cardio vascular disease (CVD) has been reproduced. The effects of PEGylation on gene siliencing of anti-FVII siRNA liposomes and anti-hsp47/gp46in vitro and in vivo have been investigated, as well as the mechanisms.(1) An anti-hsp47/gp46siRNA liposome deliever system has been established by extrusion formulation method.Without PEG in this system, we formulated anti-hsp47/gp46liposomes with the Z-ave of217-234nm, PDI0.118-0.200, Zeta potential20-30mV and siRNA encapsulation efficiency greater than90%by adjusting lipids/siRNA concentration. There was no cytotoxicity seen from this formulation, however the siRNA recovery efficiency was low (25-36%), as well as the gene sicilencing efficacy in vitro. With PEG in this system, lipid/siRNA concentration has remarkable effect on decreasing the particle size and PDI in this system. The liposomes were with a Z-ave of154-169nm, a PDI0.104-0.163, a neutral surface charge and a siRNA recovery efficiency71-83%. But the cytotoxicity was seen after adding PEG, and the in vitro gene silencing efficacy was low. We also investigated the effect of adding different molar ratio of PEG, and found that lower Z-ave (213-154nm) and PDI (0.136-0.104) can be induced by higher molar ratio of PEG in this system, but the gene silencing efficacy was low and cytotoxicity has been seen.(2) The anti-hsp47/gp46siRNA liposome deliver system by SNALP method has been established and optimized. The devices for this sytem in small, medium and large scales have been designed and assembled, which provides the possibility of scaling up for this system.We have formulated the anti-hsp-47/gp46liposomes by SNALP method with high gene silencing efficacyin vitro (40-60%on hsp-GFP co-expression cell lines,80%gp46mRNA silencing efficiency on immortalized people hepatic stellate cells), Z-ave under100nm, PDI under1.000, siRNA recovery efficicency greater than80%and encapsulation efficiency greater than90%, which can remain stable at least in a month if stored in4℃.We have established fast DMN induced high gp46expression rat model, in which the gp46gene expression can reach3.5times as normal rats in a week. The gene silencing of the liposomes we formulated was around45%in this model.(3) We produced anti-FVII siRNA liposome deliver system by SNALP method. The anti-FVII siRNA liposomes we formulated with10mole%PEG had a Z-ave of65-68nm, PDI of0.25, siRNA recovery of80%, encapsulation efficiency of94%and neutral surface charge. The FVII gene in C57BL/6mice was61%silenced by ithi liposome,while the FVII protein expression was silenced11%(lmg/kg·bw),38-69%(5mg/kg·bw) and75-88%(10mg/kg·bw) in mice.(4) In SNALP system, with increasing PEG-C-DMA moar ratio, the Z-ave of the siRNA liposomes was dramatically decreasing (0.5mole%,129-141nm;10mole%,65-73nm); the PDI was increasing (0.5mole%,0.05nm;10mole%,0.25nm); siRNA recovery efficiency was70-90%; the encpsulation efficiency was greater than90%; Zeta potential colse to OmV. And these siRNA liposomes were stable in a year when stored in4℃.In anti-FVII siRNA liposome deliever system, the FVII gene silencing efficacy in primary mice hepatocytes was decreasing with increasing PEG-C-DMA molor ratio(0.5mole%,98%(RNA level);10mole%,60%(RNA level)). The same trend was seen in mice FVII gene silencing study (0.5mole%,90%(RNA level);10mole%,20%(RNA level)). Mechanism study showed that the higher distribution of liposomes in hepatocytes was not the reason for the higher gene silencing efficaycy from anti-FVII siRNA liposome with lower PEG-C-DMA molar ratio. The liposomes with lower PEG-C-DMA molar ratio had a better endosomal disruption ability since they had a better hemolysis ability un pH5.5.In anti-hsp47/gp47siRNA liposome deliever system, the hsp47/gp46gene silencing efficacy in hsp-GFP co-expression cell lines and primary rat HSC was decreasing with increasing PEG-C-DMA molor ratio. However, the trend in rats was not exactly the same.In total, we have established an anti-hsp47/gp46siRNA liposome deliver system, with favorable physicochemical properties, great in vitro gp46gene silencing efficacy (greater than95%in primary rat HSC and a gene silencing of40-50%in vivo. The PEGylation effect on it was also investigated, which would help in anti-fibrosis siRNA drug development. |
PDF Full Text Request