Research On GA-PEG-PKAE-GO,a Liver-targeting Complex Vector Carrying Sirna

ObjectiveHepatocellular carcinoma(HCC)is one of the most common malignant tumors,which seriously endangers people’s health.In addition to traditional treatments(radiotherapy,chemotherapy,and surgical treatment),gene therapy with RNA interference(RNAi)has recently appeared.As is a process known as transfection,RNAi requires a vector to transport small interfering RNA(si RNA),which is regarded as a drug,into a cell.The virus-mediated delivery of si RNA has high efficiency of transfection,but there are some disadvantages such as immunogenicity and carcinogenicity.Compared to viral vectors,non-viral vectors have lower toxicity,lower immunogenicity,lower carcinogenicity,easier preparation and large-scale production.However,non-viral vectors have a lower transfection efficiency than ones based on viruses.Consequently,the attempt to improve the transfection efficiency of non-viral vector is the key and basis of gene therapy,and has become the hotspot of research.MethodsIn this study,a novel quadruple targeting vector,GA-PEG-PKAE-GO,was synthesized based on graphene oxide as the pedestal.The main structure of the carrier was GO(graphene oxide).GA(glycyrrhetinic acid)is a liver targeting ligand.PEG(polyethylene glycol)can improve the blood circulation of the quadruplex.PKAE is a high-molecular polymer,which binds si RNA with electrostatic interaction and GO with terminal pyrene groups by π-π stacking interaction.The vector’s capability of transfering si RNA was evaluated in vitro and in vivo.1.2-hydroxyethylacrlate was reacted with 2,2-dimethoxypropane to prepare KDA,which was reacted with diethylenetriamine via aza-Michael addition reaction to obtain PKAE.Acid-base titration was performed to measure the buffering capacity of PKAE.The ability of PKAE to bind/release si RNA was determined via agarose gel electrophoresis.2.GA-PEG-GO was synthesized in succession to GA-PEG,both with EDC/NHS reaction.PKAE was bound to GA-PEG-GO by π-π stacking interaction.The diameter,ζ-potential and PDI of GA-PEG-PKAE-GO was synthesized.The quadruplex’s photothermal effect was measured.The ability of the quadruplex to bind/release si RNA was determined via agarose gel electrophoresis.3.The following experiments were done using human hepatoma cell line Hep G2.The cytotoxicity of GA-PEG-PKAE-GO was determined by MTT assay.The transfection efficiency of the complex was determined by FAM-labeled si RNA.Endocytoplasmic escape and si RNA release were examined by double staining method with calcein and Lyso Tracker Red.The gene silencing ability of Bcl-2 si RNA transfected by GAPEG-PKAE-GO was determined: Bcl-2 m RNA transcription was measured by RT-PCR,Bcl-2 protein expression by Western blotting,cytotoxicity of tumor cells by MTT assay,and cell apoptosis by cytotoxicity with double staining with Annexin V and PI.4.The biodistribution of GA-PEG-PKAE-GO was examined using nude mice bearing tumor thriving from previously implanted Hep G2 cells.GAPEG-PKAE-GO that carries doxorubicin,which served as fluorescent marker,was administered to nude mice.The biodistribution of doxorubicin,which reflects the distribution of GA-PEG-PKAE-GO,was measured by fluorescence spectrophotometry.Results1.PKAE was synthesized.It bound si RNA tightly at physiological p H(7.4),while released si RNA efficiently at acidic p H(5.4).2.GA-PEG-PKAE-GO was prepared with a particle size of(266.1 ± 101.8)nm,a PDI of 0.380 and a ζ-potential of(46.9 ± 7.96)m V.It took 5 min for GA-PEG-PKAE-GO to increase the liquid temperature by 3.8°C under near infrared(NIR)radiation(808 nm,0.25 W).The result was significantly higher than the control group.The quadruplex binds si RNA at p H 7.4,whereas it release si RNA at p H 5.4 because of the hydrolysis of PKAE.3.MTT assay showed that the cell viability was not significantly different between the control group and the experimental group given the compound targeting vector GA-PEG-PKAE-GO.The transfection efficiencies of GA-PEG-PKAE-GO showed that GA-PEGPKAE-GO could transfer FAM-si RNA into cells.The fluorescence intensity and fluorescence emission of GA-PEG-PKAE-GO group with 808 nm NIR radiation were higher than those of GA-PEG-PKAE-GO group without radiation,which were also higher than those of Lipofectamine 2000 group.Compared to PEG-PKAE-GO group,the fluorescence intensity and fluorescence emission of GA-PEG-PKAEGO group were higher.Endosomal escape experiment showed that green fluorescence emitted by FAM-si RNA carried by GA-PEG-PKAE-GO coincided with that of red fluorescence emitted by endosomes/lysosomes labeled by Lyso Tracker Red.Along with time,the red fluorescence was attenuated and the green fluorescence was diffused throughout the cell,indicating that GA-PEG-PKAE-GO broke the endosome and released the si RNA into the cytoplasm under the acidic conditions of the endosome.RT-PCR and Western Blotting showed that the m RNA level and the level of Bcl-2 protein translation were significantly decreased after Bcl-2 si RNA transfection.MTT assay showed that the viability of Hep G2 cells was significantly decreased after Bcl-2 si RNA transfection.Flow cytometry analysis showed that Hep G2 cells showed apoptosis after Bcl-2 si RNA transfection.4.The distribution of GA-PEG-PKAE-GO in liver and tumor of nude mice was significantly higher than that of other organs.ConclusionThe quadruple targeting vector,GA-PEG-PKAE-GO,could deliver siRNA in vivo with targeting property,transfect si RNA into cells,release si RNA smartly into cytoplasm and ensure the efficiency of si RNA within cells.It may be a potential candidate for targeting drug(si RNA)delivery system.