The Building And Evaluation Of Tumor PH-senstive Gene Delivery System

International cancer alliance released data show that global cancerhappens severely. Morbidity and mortality rates present rising trend. Theincidence of cancer in China is nearly half of the world. At present our countrycancer incidence is285/10,000.There are six people were diagnosed withmalignant tumor every minute a day on average. In our country, the femalebreast cancer has become one of the most common malignant tumor and has arapid growth of2%～3%every year.At present, the anti-tumor drugs hassome disadvantages such as poor targeting effect, high toxicity and lowoutcomes.Liposome is a micro capsule, since it has a similar structure of biologicalmembrane bilayer, the mainly internalization way is by meshy endodermissystem after enters the body. The drug targeted gathered in the liver, spleen,lungs and other tissues. But liposome as a drug carrier still has somedisadvantage, such as poor targeting effect and poor stability. pH-sensitiveliposome become unstable structure when in low pH environment. ThepH-sensitive liposome will rupture when internalized to the cell plasmamembrane or with other plasma membrane fusion. The drug will releasebefore achieved lysosome and avoid the degradation of the lysosome, deliverdrugs to cells more effectively. Due to the tumor interstitial fluid pH is lowerthan the surrounding normal tissues, the pH sensitive liposome for tumortherapy effect has great clinical value.Objective:Due to the tumor cells are slightly acidic environment, we designed apeptide (pHLIP) which can be inserted into the cell membrane, throughsynthetic DSPE-PEG–pHLIP, realized the target of the tumor cellmembrane. H8is a polycation, and can assist small molecules into cells.in this study, we use the stearic acid and H8formed the stearylated-octahistidine,through static adsorption formed the SA-H8/siRNA complexes.Theefficiency of the delivery of siRNA is enhanced.Methods:On the basis of the literature and preliminary experiment, we found asynthesis method of stearylated-octahistidine. At room temperature, stirring32h in dark and filling with N2.We confirmed the product by MS.The reaction of DSPE-PEG and pHLIP was stirred at room temperaturein dark and filling with N2.through measure the residual sulphur content todetermine the end of the reaction. We confirmed the product by MS. Afterreading the relevant literature, liposome was prepared using film dispersionmethod, respectively use coumarin and siRNA as model drug, size andencapsulation rate were studied as index, the preparation technology ofliposomes was optimized. The appearance, particle size distribution, zeta andshape as indx, we studied the stability of liposome. We use the hunman breastcancer cells (MCF-7) as the cell model, incubated respectively in pH6.5andpH7.4slightly medium environment with the compression load liposome andordinary liposome. The cells were processed by PBS. We use flow cytometryinstrument to analyze the uptake of the liposome.Result:Through MS analysis, we confirmed the synthetic SA-H8and DSPE-PEG–pHLIP.They are white flocculent solid after freeze drying. Therespectively production rate were48.9%and52.2%.For the preparation ofcoumarin6liposomes, we got uniform particle size with yellowish hair creamand evenly mixed suspension. The liposomes were relatively stable. Theenvelopment rate was91.0%. For the preparation of siRNA liposomes, whenobserved under the transmission electron microscopy, we saw the particleswere uniform distributed and light in the middle.Flow cytometry analysis results show that, compared with ordinaryliposome, pHLIP modified carrier for cells to absorb has a significant pHresponse characteristics, under the condition of the environment,pH6.8intake rate is significantly higher than pH7.4.Conclusion:We confirmed that, when Coumarin6and siRNA as model drugs, SA-H8cationic polymer as siRNA carrier, DSPE-PEG-pHLIP modified thesurface of the liposome, liposomes were prepared using film dispersionmethod, the stability of liposomes is good. For breast cancer MCF-7cells asmodel, pHLIP peptides modified the surface as a leading membrane material,at low pH value, the intake rate of human breast cancer cells was obviouslyhigher than that without modified liposomes, DSPE-PEG-pHLIP promotethe cell’s intake of siRNA.