| The hepatocarcinoma is one of the most difficultly treated malignant tumors. It isurgent to develop new therapeutic protocols, which can inhibit the revivification ofcancer ceils after operations and raise the living standard of mataphase and latehepatocarcinoma patients. Gene therapy continues to hold exciting promises in treatingmany diseases. However, gene therapy depends on efficient and safe devices for thedelivery of nucleic acid into the target cells. Therefore, the construction of safe andeffective intracellular gene delivery system is not only a hot research field in MolecularBiology but also offers a new challenge for pharmaceutical researchers.The common used delivery systems in gene therapy are virus vectors andnon-viral vectors. Virus vectors may have autoimmune responses and lead the host to beinfected with the virus or activate the oncogenes. Among non-viral vectors, cationicliposomes were studied more extensively. Cationic liposomes also known as 'fusion'liposomes are positively charged on the surface and their components are similar withthe cell membrane, which made them easily combination with the negatively chargedcells. The transfectivity of cationic liposomes in vivo is not as efficient as in vitrobecause their surface is net positively charged which might lead to aggregation withplasma protein and accumulation in lung tissue.Recently, Cholest-5-en-3-ol-(3β)-[2-[[4-[(carboxymethyl)dithio]-1-iminobutyl]amino]ethyl] carbamate (CHETA, C36H61N3O4S2), one of the cholesterol derivatives,was used to prepare 'Procationic liposomes'. Many researchers put interests on it for itsfacility to drug delivering in intra-cells because there are disulfide reductant factors suchas protein disulfide isomerase (PDI) or thioredoxin reductase (TrxR). CHETA consists of a hydrophobic cholesterol tail group (X) and a hydrophilichead group(Y+-S-S-Z-), of which the head group incorporates both positively andnegatively charged regions connected by a disulfide bond. The disulfide bond issusceptible to cleavage (X-Y+-S-S-Z-→Y+-SH+HS-Z-) by components of theintracellular membrane such as TrxR. Procationic liposomes were neutral or negativelycharged in physiological environment. However, when brought into contact with cellularmembranes or intemalized by cells, their surface charge became positive. Therefore,procationic liposomes integrated cationic liposomes and anionic liposomes, which mightovercome their shortage and make use of their advantage. However, this drug deliversystem is lack of targeting characteristic.To achieve targeted cell delivery, receptor-mediated targeting was generally usedin recent years. For axample, in liver cells there are asialo glycoprotein receptor,transferrin receptor; in non-parenchymal cells membrane there are mannose receptor,and low-density lipoprotein receptor. According to the findings of Meng chao Tian,content of transferrin receptor are much higher in hepatoma cells than in normal tissues,and hence it was expected that the uptake of the procationic liposomes should beimproved by incorporating Tf with it.In this study, we constructed procationic liposome of CHETA by CCDoptimization strategy, using LacZ and EGFP as reporter gene, HSV-TK as model drug,and protamine sulfate as the condensing agent to form PLPD that further associated withTf targeting to hepatoma cells.Plasmid DNA was isolated and purified from E. Coli (DH 5-a) using the QiagenGiga Endo-free plasmid purification kit. DNA concentration and purity were quantifiedby UV absorbance at 260 nm and 280 nm on a GBC UV cintra 10e Spectrophotometer.The structural integrity and topology of purified DNA was analyzed by agarose gelelectrophoresis.PLPD and Tf-PLPD were prepared into lyophilized injection to improve theirphysical and biological stability. The lyophilization of PLPD and Tf-PLPD did not resultin obvious change in morphology, particle size, Zeta potential, pH and transfection efficiency in vitro with good redispersibility.In vitro transfection experiments, we use easily detect LacZ and EGFP as reportergene and make a comparative study on the expression efficiency of cationic liposomeswith naked DNA and Cationic liposomes LPD. When LacZ was used as the reportergene, the transfection efficiency of PLPD was lower than that of Tf-PLPD. They are12.18±3.8 mUβ-galactosidase/mg protein and 24.26±2.6 mUβ-galactosidase/mgprotein, respectively. The latter is 2 times fold of naked DNA. In the serum of S.D. rat, both PLPD and Tf-PLPD keep stable. The results also indicate that the transfectionefficiency (TE) of Tf-PLPD and PLPD showed no significant difference in the presenceor absence of serum (10%, v/v), while that of LPD was markedly inhibited by serum.Without serum in culture, the TE of Tf-PLPD is equivalency with LPD; With serum inculture, the TE of Tf-PLPD was higher than LPD. Compared to LPD, the PLPD andTf-PLPD had much less cytotoxicity to three hepar Cell lines (including HepG2, SMMC7721 and Chang's normal heptocyte). When EGFP was used the reporter gene, the result of transfection efficiency is similar with above. Furthermore, procationicliposomes could target TK gene to HepG2 cells and the TK/GCV system had shown thecell growth inhibition effect and bystander effect.In summary, the transferrin modified pro-cationic liposomes had been preparedand showed some advantages as following: firstly, the in vitro transfection efficiency ofit is comparable with LPD but superior to naked DNA; secondly, it had much lesscytotoxicity to cells than cationic liposomes; thirdly, the transfection efficiency was notaffected by serum; lastly, it could be prepared into lyophilized injection. Such deliverysystem could make full use of advantages and also overcome the shortages of cationicliposomes which are easily to integrate with the plasma component in body cycle. It mayprove to be a useful strategy to delivery therapeutic genes into hepatoma cells. |
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