Synthesis Nano Delivery System Of SiRNA With Single-Wall Carbon Nanotube And Cell-Penetrating Peptide

BackgroundMalignant tumor is a major public health problem that the human will be faced with, and it has been reported in our nation that8550people have been diagnosed with malignant tumor and about7300people have been died from that, howeverit has a tendency to increase. The complexity of the pathogenesis of malignant tumor brings difficulties to the diagnosis and treatment, so the early diagnosis and treatment is still the best method of prevention of malignant tumor. Now, the treatment of malignant tumor is mainly traditional method, such as chemotherapy, radiotherapy and surgery. With the continuous development of new technology, some new treatment methods attract the interest of the researchers and gene therapy belongs to one of them. Gene therapy is that foreign genetic material (DNA or RNA) is delivered into the target cells to correct or compensate gene defect and abnormality and be against the gene-related disease. It is known that the occurrence and development of malignant tumor is related with some genes that are lower or high expression and inhibiting or promoting corresponding-gene expression can suppress tumor growth and invasion, promote apoptosis of tumor cells and ultimately cure tumor.In1998, Andrew Z Fire et al. found RNA interference when the test of antisense RNA inhibition was performed in caenorhabditis elegans and He was awarded the Nobel Prize for this great discovery. RNA interference also offer a new technology for gene therapy. RNA interference is that target gene is induced to be silenced via double-stranded RNA, for example, microRNA and siRNA. siRNA plays an important role in silence genes. SiRNA is21～25bp double-stranded RNA, and guides the degradation of specific mRNA afterr combining with RISC via complementary base-pairing. Delivering forgein siRNA into tumor to inhibit gene expression related to the occurrence and development of malignant tumor becomes one of the hot research of gene therapy. siRNA is synthesised by chemical synthesis method in vitro, so the specific sequences of siRNA can be designed according to the sequence of target genes. However, the intrinsic characteristics of siRNA are main obstacles to the development and adaptation of siRNA for gene therapy, for example, naked siRNA has a negative charge and is a macromolecule which means it is almost unable to cross cell membrane, and the naked siRNA is also easily degraded by RNase before its transportation into the cell. Even if the siRNA is endocytosed into the intracellular compartments by a normal delivery system, the endosome/lysosome trapping and degradation of siRNA is a big obstacle as siRNA could not silence the target mRNA in cytosol. Designing and synthesizing well carrier and delivery system of siRNA is a new subject of gene therapy for targeting tumors. The carrier and delivery system of siRNA possess the nature, as follow,①siRNA should be bonded steadily by carrier and delivery system;②the synthetic and assembly method should be no complicated;③The carrier and delivery system can cross the cell membrane into the cell;④endosomal escaping,⑤siRNA can be protect from degrading by RNase;⑥low toxicity;⑦siRNA can be released when it enters into cell.The development of of nano-carrier has raised hopes for the siRNA application. Nano-carrier is that drug, small molecules and genes are bonded by nano materials, in order to increase the biological membrane permeability, improve the bioavailability, adjust the release rate and change the distribution of the drug in the body. Designing and synthesizing nano-carrier of siRNA attract the interest of the researchers. Gold nanoparticles, silica nanoparticles and magnetic nanoparticles, lipid, polymer, aptmar and so on have been designed and synthesized to devered siRNA as nano-carrier. However, the synthesis methods of nano-carrier is complex, and multistep chemical and physical methods are often needed. Even if the nano-carrier was synthesized, those problem of siRNA was not solved all. These problems hindered the siRNA from applying in clinical. Looking for theappropriate nanomaterials and synthesizing carrier system via the simple method and delivering siRNA successfully into the target cells and efficiently silencing target gene is the focus of this study.Single-walled carbon nanotube (singe wall carbon nanotube, SWCNT) have been widely studied in the preparation of materials, biomedical and other fields. Previous reports have shown multi-step chemical modifications of SWCNT which have then been applied in siRNA delivery systems. After chemical modification, SWCNT surfaces are functionalized with chemically activated groups, which enable binding to siRNA through covalent bonding or non-covalent bond. However, multi-step chemical modifications of SWCNT are not an easy route to siRNA delivery, and it is even more difficult to release the siRNA into the cytosol to silence the target mRNA efficiently, as the siRNA is covalently linked with the SWCNT moiety. To address these serious problems in SWCNT-based siRNA delivery systems, we (?) firstly developed a very simple approach to makea siRNA delivery system. We designed an artificial CPP with three segments. The sequence of CPP is NH2-VGAlAvVvW1W1W1WbA-GSG-PKKKRKVC-COOH, where the positively charged segment is PKKKRKVC-COOH, the link segment is GSG and the hydrophobic segment is NH2-VGAlAvVvW1W1W1WbA. Through simple two-step method, siRNA, CPP and SWCNT can be assembled into a carrier system successfully. Firstly, the double strand negative siRNA can interact with the positively charged segment of CPP to form a siRNA-CPP complex at room temperature; secondly, siRNA-CPP strongly adsorbs to the intrinsic SWCNT surface via hydrophobic interactions with the help of ultrasonication to produce a stable siRNA-CPP-SWCNT delivery system.The mammalian target of rapamycin (mTOR), is an evolutionarily conserved serine threonine kinase belonging to the phosphoinositide3-kinase related kinase family, which is frequently activated in human cancer. Rapamycin and rapamycin derivatives inhibiting mTOR can treat liver cancer, ovarian cancer, leukemia, kidney, liver, cell lung cancer, bladder cancer, ovarian cancer, breast cancer, non-small cell lung cancer, bladder cancer and another tumors. In our siRNA delivery system, the siRNA (sense sequence:CUG AGU ACG UGG AAU UUG AdTdT; anti-sense sequence:UCA AAU UCC ACG UAC UCA GdTdT) targets the mRNA of mTOR.Objective1. Design and synthesis siRNA nano-carrier, siRNA-CPP-SWCNT, set up the sample synthesis methods of nano-carrier system and study the particle size of carrier, morphology, stability and other properties.2. The special properties of nano-carrier, siRNA-CPP-SWCNT, in vivo and in vitro were studied further, such as, RNese degradation, cell toxicity, tracking siRNA in the cell and siRNA delivery efficiency.3. Design and synthesis mTOR siRNA, assemble them into nano-carrier, deliver into cell and study the target gene silencing and growth situation of malignant tumor cell.Method1. Synthesis the siRNA-CPP-SWCNT delivery system. Firstly, we designed an artificial CPP with three segments, such as the positively charged segment is PKKKRKVC-COOH, the link segment is GSG and the hydrophobic segment is NH2-VGAlAvVvW1W1W1WbA. For the formation of the siRNA-CPP complex,8nmol CPP was incubated with2nmol siRNA in1000mL of DEPC-treated water for30min at room temperature, afer vigorous mixing for lmin. The above siRNA-CPP solution and0.0005mg SWCNT swere mixed and then placed in an ice-bath and ultra-sonicated for60minutes under40W microwave output. Afterwards, this solution was centrifuged at10OOOg for30min to discard the unbound SWCNT. The supernatant solution was transferred to a dialysis lm and bathed in DEPC-treated water for48h to remove free siRNA, CPP and siRNA-CPP.2. Characterization of the siRNA-CPP-SWCNT delivery system. After Synthesizing nano-carrier, the particle size of carrier, morphology, and stability were study, Dynamic Light Scattering(DSL), Atomic Force Microscopy(AFM) and Agarose Gel Electrophoresis were used to token, measure and analyze thenano-carrier.3. Degradation of siRNA by RNese.2μ L of RNase (5U mL1) was used to degrade0.5mg of siRNA-CPP and siRNA-CPP-SWCNT at37℃for1h. After digestion, enough heparin (100000units per mL,10mL heparin per mg siRNA) was added to dissociate the siRNA from the delivery system. The content of siRNA in the above six samples were analyzed by agarose gel electrophores to know the degradation of siRNA4. Cell viability assay. Cell viability was measured by a Cell Counting Kit-8system, after the Hela cell was treated with siRNA-CPP and siRNA-CPP-SWCNT at different concentrations of siRNA and in different time.5. Trace siRNA in the cell. Fuorescent FAM (green emission,(ex:488nm)) was labeled on the5’ end of the siRNA to trace siRNA in the cell. Hela cells were planted for24h.The old medium was replaced with fresh medium consisting of naked-siRNA, siRNA-CPP and siRNA-CPP-SWCNT at aoncentration of100nM FAM-siRNA for12h. Then staining with Hoechst33342was performed to study cellular uptake and staining with LysoTracker Red was performed to study endosomal escape.6. The delivering efficiency of siRNA. Fuorescent FAM was labeled on siRNA. Hela cells were planted for24h. The old medium was replaced with fresh medium consisting of naked-siRNA, siRNA-CPP and siRNA-CPP-SWCNT at aoncentration of100nM FAM-siRNA for12h. The collected cell were analyzed and statistics by Flow Cytometer.7. Gene silencing efficiency, Gene silencing efficiency was detected by western blotting and Quantitative reverse transcription polymerase chain reaction(RT-qPCR). Hela cells were treatment with unbound siRNA, siRNA-CPP and siRNA-CPP-SWCNT at a concentration of100nM siRNA for12h. After12h incubation, the media was replaced and the incubation of the cells was continued for another48h. The cells were then harvested and lyzed with RIPA buffer to get total protein for western blotting. For another24h, The cells were then harvested, and total RNA was prepared using Trizol reagent. After reverse transcription mTOR mRNA, Real time PCR analysis was further examine the silencing of the mTOR expression at the mRNA level8. Growth situation of tumor. Cells were stained with annexin V-FITC and propidium iodide (PI). The percentages of apoptotic and necrosis cells were quantified using an Accuri C6flow cytometer. Hela cells were plated on growth for24h,after cells were treated with siRNA-CPP-SWCNT at a concentration of100nM siRNA for48h. Aftert collected, cell wer dyed following the manufacturer’s instructions and detected by flow cytometry.9. Statistical analysis. All data were presented as the mean standard error from at least three independent experiments. Statistical signi cance was tested using a Student’s t-test.Result1. siRNA nano-carrier was successful to synthesized via only two steps. Firstly, siRNA was complexed with CPP via electrostatic interactions form siRNA-CPP; secondly, the siRNA-CPP strongly adsorbed onto the surface of pristine SWCNT via hydrophobic interactions under ultrasonication. Through particle size analysis, morphological observation and stability study, we proved that siRNA-CPP strongly adsorbed onto the surface of pristine SWCNT form the steady complex siRNA-CPP-SWCNT.2. siRNA-CPP-SWCNT was exposed in the solution contained of RNese for1h and the content of siRNA after degradation was analyzed by agarose gel electrophoresis, which showed that siRNA was protect from being degraded by RNese with the help of both CPP and SWCNT.3. Nano-carrier, siRNA-CPP-SWCNT was low toxicity n the certain consistency and time.4. siRNA labeled green fluorescence (FAM-siRNA) was traced by Laser Scanning Confocal Microscope and we found that siRNA was successful to be delivered into intracellular and at the same time saw compared with the group of siRNA-CPP, the delivery efficiency was more higher in the group of siRNA-CPP-SWCNT. Flow Cytometer was used to study the delivery efficiency further and the result agreed with the Laser Scanning Confocal Microscope. The delivery efficiency in siRNA-CPP-SWCNT was98%(That98%of the cell were marked with fluorescence showed98%of the cell uptake siRNA)。In the colocalization of FAM-siRNA and lysosome, we found siRNA was also delivered into cell in the group of siRNA-CPP, but all was trapped in the lysosome; while siRNA was not only delivered into cell in siRNA-CPP-SWCNT, but also was seen in the cytoplasm because of escaping from endsome. It showed siRNA escaped from endsome and enter into cytoplasm with the help of both CPP and SWCNT.5. That siRNA delivered into cytoplasm can silence target gene mTOR was verified by western blotting and PCR. The result of two experiment showed the expression of mTOR was reduced through degradation of mTOR mRNA. About70%of mRNA was degraded and it also proved the nano-carrier was high-efficiency.6. The apoptosis assay of the malignant tumor after treated with siRNA-CPP-SWCNT was carried through Flow Cytometer. Compared with control, we found siRNA-CPP-SWCNT could inhibit the growth of cells and induce apoptosis and death. ConclusionIn our research, a very simple method was presented to make a stable siRNA delivery system, siRNA-CPP-SWCNT. A series of studies showed that siRNA-CPP-SWCNT could protect siRNA from being degrading by RNese, was low toxicity, cross the cell membrane, escape endosome/lysosome trapping and degradation. Finally, siRNA signicantly cleaved the mRNA of mTOR, inducing lower levels of mTOR expression and markedly triggering cell apoptosis.