Development Of SiRNA-loaded Lipid-based Nanocarriers For HCC Treatment And Research On Their High-efficient Delivery Mechanisms

China has an annual increase of 350,000 new hepatocellular carcinoma(HCC)patients,accounting for about half of the world's new cases.In clinical practice,most of the diagnosed patients are in the advanced stage and require anti-tumor drugs.Traditional cytotoxic anti-tumor drugs such as doxorubicin and fluorouracil still have problems such as low selectivity,serious side effects and drug resistance in clinical treatment of HCC.In recent years,small interference RNA(siRNA)-based gene therapy has gained the world's attention and become a promising new class that can silence the disease-causing genes with high eff-icacy and specificity and can be used for the treatment of malignant tumors.Due to the structure of siRNA,it is very vulnerable to the nucleases in serum.In addition,the large molecular weight,hydrophilic characteristic and negatively charged surface make siRNA molecules impossible to pass through the semi-permeable cell membrane directly.It is therefore necessary to deliver siRNA molecules by using nanocarriers.Among various siRNA delivery vehicles,lipid-based non-viral delivery systems are favored for their safety and biocompatibility.Approvement for the marketing of the world's first lipid-based siRNA delivery vechile made by US FDA in August 2018 further demonstrating the potential of lipid delivery vehicles for siRNA delivery and HCC treatment.Until August 2018,the US FDA approved marketing of the world's first siRNA drug(Onpattro),becoming a milestone in the official application of siRNA drugs.According to the clinical trial reports of siRNA drugs,the most important factor that limiting the clinical translation of siRNA drugs is the overall in vivo siRNA delivery efficacy and the off-target issue.With the rapid development of siRNA delivery systems in recent decades,up to date,the efficiency of siRNA delivery vectors primarily depends on their ability to overcome the barriers in cell level,while addressing off-target problems primarily relies on their ability to target tumors and eliminate side effects after off-target.In this regard,two novel lipid-based siRNA delivery systems were designed and constructed to achieve eff-icient delivery of siRNA and address off-target problems.In order to improve the efficiency of siRNA delivery and eliminate the side effects after off-target,lipid-based liquid crystalline nanoparticles with phase transition properties was designed and constructed to effectively overcome the cell barrier during siRNA delivery,while eliminating thes side effects by taking advantage of synthetic lethality.After being taken up into the endosomes by cells,the developed lipid-based liquid crystalline nanoparticles can undergo phase transformation by utilizing the hydrolysis property of the constituent molecules.The phase transition process can further facilitate high-eff-icient membrane fusion between these developed lipid-based liquid crystalline nanoparticles and endosome membrane,leading to the releasing of contents.In addition,these lipid-based liquid crystalline nanoparticles encapsulate siRNA through its water solubility,and the nanoparticel itself does not have strong positive charge.This allowed siRNA molecules to desorb from delivery materials after endsomal escape and be incorporated into the RNA interference machinery.We then used cyclin-dependent kinase 1-siRNA(CDK1-siRNA)to investigate the integraty and activity of siRNA delivered by these lipid-based liquid crystalline nanoparticles at both cellular and animal levels.Results showed that the developed lipid-based liquid crystalline nanoparticles encapsulated with CDK1-siRNA could effectively inhibit the proliferation of HepG2 hepatoma cells and the growth of ectopic transplanted tumors,while did not affect the physicological activity of normal liver cells.By using the phase transition property of the novel lipid-based liquid crystalline nanoparticle,we have skillfully solved the low-eff-icient and side-effect issues that faced by most lipid-based siRNA delivery vehicles.However,since the surface of the lipid-based liquid crystalline nanoparticle is not modified,there still exist disadvantages such as poor targeting of tumor tissues and the problem of off-targeting cannot be fully solved.In this regard,this work designed and prepared hybrisomes in order to further improve the tumor targeting ability of nanocarriers on the basis of achieving efficient siRNA delivery and eliminating side effects after off-targeting.The hybrisomes are obtained by removing the contents of exosomes that derived frome Sk-hepl cells and hybridizing with DPPC phospholipids.The application of exosome membrane can make hybrisomes inherit the natural nucleic acid delivery behavior of exosomes and its tumor "homing" ability,while the addition of DPPC(Dipalmitoylphosphatidylcholine)phospholipids can improve the stability of nanocarriers.The hybrisomes can achieve desirable siRNA encapsulation and protection through electroporation.In addition,the hybrisomes are stable and safe enough for siRNA delivery.Both macrophage uptake and immune factor analysis demonstrate that hybrisomes have excellent long-circulation property to avoid blood clearance.Moreover,biodistribution results of hybrisomes indicate their desirable tumor "homing" ability.Studies also show that hybrisomes will be sorted and ultimately delivered to the Golgi apparatus after taken up by recipient cells.It is consistent with the fate of the exosomes in their doner cells.This exosome membrane-mediated specific delivery route overcomes the intracellular barrier for siRNA delivery by avoiding the lysosomal pathway.Finally,we also evaluated the antitumor eff-icacy of hybrisomes that encapsulated with CDK1-siRNAat both cellular and animal levels.Results show that CDK1-siRNA molecules delivered by hybrisomes still have biological activity at both cellular and animal levels.These hybrisomes can effectively silence the CDK1 gene expression,exhibiting excellent anti-tumor effect and sparing normal liver cells.So far,in view of the low delivery eff-iciency and off-target problem of non-viral siRNA delivery vectors,lipid-based liquid crystalline nanoparticles and hybrisomes have been designed to achieve high siRNA delivery efficacy and overcome off-target issue.This work provide new ideas and new methods for the rational design of lipid-based siRNA delivery systems,which offers new possibilities for the clinical translation of siRNA drugs for liver cancer treatment.