Co-delivery Of Doxorubicin And SATB1-shRNA By Novel Thermosensitive Magnetic Cationic Liposomes In Gastric Cancer Therapy

Gastric cancer is one of the most common malignant tumors with high incidence and poor prognosis. Chemotherapy is an important treating strategy for gastric cancer, but traditional chemotherapy always has limited treating efficacy and caused great side effects, which is mainly due to the lack of targeting and controlled release. Traditional chemotherapy is a systemic drug administration, which can not achieve therapeutic concentrations in the tumor site, while the relatively high systemic blood concentrations will cause many side effects, moreover multidrug resistance (MDR) would significantly reduce its therapeutic effects. Liposome (LP) is a desirable nano drug or gene vectors, and it can be divided into magnetic liposome (ML), thermo sensitive liposome (TSL) and cationic liposome (CL), etc. This study intends to integrate the characteristics of the above liposome and design a thermosensitive magnetic drug and gene co-delivery system which not only combine magnetic targeting and controlled release, but also drug and gene co-delivery. Firstly, we will design it from thermosensitivity, magnetic response and gene delivering efficiency, then we’ll load doxorubicin (DOX) and plasmid encoding SATBl-shRNA into it, we’ll detect the thermal release rate of DOX, SATB1gene silencing efficiency, magnetic targeting DOX and SATB1-shRNA delivering efficiency, in vitro cytotoxicity, apoptosis activity and in vivo anti-tumor effects for gastric cancer.Part I Screening of the liposomal formulation and characterization of thermo sensitivity of Novel Thermosensitive Cationic LiposomesObjective:To screen the prescription of thermosensitive cationic liposome which can be used for drugs and gene delivery, and identify its zeta potential and heat-sensitivity.Methods:DPPC was to be as the main lipids with the adding of cholesterol and a variety of cationic materials to design three liposomal formulation. TSL:DPPC.Cho1=80:20, CL: DPPC:DC-Chol:Chol=80:10:10, TSCL:DPPC:DC-Chol:DOAB:Chol=80:5:5:10(molar ratio); Calcein and PBS loaded liposomes were prepared in each formulation by thin film hydration method; we analyzed the particle size, polydispersity and zeta potential of the liposomes by a particle sizing instrument, and then identified the via Calcein release assays and DSC.Results:CL and TSCL were positively charged, while TSL was electrically neutral; TSL, CL and TSCL all can achieve Calcein thermal release at42℃; CL had a early Calcein release at37℃, while the TSL and TSCL remained stable at37℃; DSC results show that the Tm of TSL is41.6℃, CL is38.2℃, and TSCL is40.8℃.Conclusions:Combining themosensitivity and zeta potential, we can determine TSCL formulation, which is DPPC:DC-Chol:DOAB:Chol=80:5:5:10simultaneously have good cationic and thermosensitive characteristics Part II Preparation of thermosensitive magnetic cationic liposome and characterization of its magnetic response abilityObjective:On the basis of TSCL, we prepare TSMCL by encapsulating Magnetic FluidFe3O4, and identify their morphology and magnetic response.Methods:Based on liposomal formulation DPPC:DC-Choi:DOAB:Chol=80:5:5:10, and Magnetic FluidFe3O4as the core, we used thin film hydration method to prepare TSMCL, and analyzed the particle size, polydispersity and zeta potential of the Magnetic FluidFe3O4, TSCL and TSMCL by a particle sizing instrument; then observed the morphology of them by TEM; We detected the magnetic field response ability of Magnetic FluidFe3O4by VSM; By recording the heating curve of Magnetic FluidFe3O4and TSMCL in AMF, we calculated the SAR and evaluated their AMF response ability.Results:Compared with TSCL, the diameter of TSMCL had significantly increased, but no obvious difference in zeta potential; Magnetic FluidFe3O4displayed characteristics of nano magnetic Magnetic, and TSCL and TSMCL with typical liposome morphology. VSM showed that Magnetic FluidFe3O4was superparamagnetic, and heating curve showed that Magnetic FluidFe3O4and TSMCL can be heated from25℃to42℃within20min, SAR of Magnetic FluidFe3O4was25.4W/gFe, TSMCL was28.3W/gFe.Conclusions:Magnetic FluidFe3O4had nano particle size, good polydispersity, and the desirable magnetic field response ability both in permanent magnetic field and AMF; TSMCL would have magnetic targeting and thermal trigger ability. Part Ⅲ Transfection efficiency of thermosensitive magnetic cationic liposomeObjective:To identify the gene delivery capability of TSL, CL, TSCL and TSMCL, and optimize in vitro transfection conditions.Methods:Prepared PBS loaded liposomes according to the TSL, CL and TSCL, then prepare TSMCL with different encapsulated concentrations of Magnetic FluidFe3O4, TSMCL (0.5mg/ml) and TSMCL (1mg/ml); detected the particle size, polydispersity and zeta potential of liposomes by the same instrument; By gel retardation assay, we determined DNA adsorption ability of liposomes; loaded with pGPU6/GFP plasmid, we performed in vitro transfection experiments, GFP expression levels were detected by fluorescence microscopy and flow cytometry; we optimized the in vitro transfection conditions by N/P ratio, complex incubation time, Magnetic Fluid Fe3O4concentration and magnetic field induced time.Results:Particle size of TSMCL(1mg/ml) was significantly increased compared to that of TSMCL(0.5mg/ml)(P<0.05), liposome particle size increased significantly after adsorption of pDNA (P<0.05); Gel retardation experiments showed that TSL had no DNA adsorption capacity, while CL, TSCL and TSMCL had certain ability to adsorb pDNA; In vitro transfection experiments showed that TSL had no transfection capabilities, and CL, TSCL transfected had certain capacity; when N/P ratio was5, incubation time was6h, TSMCL was0.5mg/ml, and the magnetic induction time was30min, the highest transfection efficiency could be achieved.Conclusions:TSL had no transfection capabilities, and CL, TSCL and TSMCL had gene transfer ability; when N/P ratio was5, incubation time was6h, TSMCL was0.5mg/ml, and the magnetic induction time was30min, the highest transfection efficiency could be achieved. Part IV Preparation and characterization of thermosensitive magnetic cationic liposomes for co-delivery Doxorubicin and SATBl-shRNAObjective:To load DOX and plasmid encoding SATB1-shRNA into the TSCL and TSMCL, and to identify the themosensitive DOX release rate, SATB1gene silencing effect and magnetic targeting DOX and SATB1-shRNA delivering efficiency.Methods:The Ammonium sulfate gradient method was used to load DOX into liposomes, and loaded plasmid encoding SATB1-shRNA by electrostatic adsorption to construct DOX and SATB1-shRNA co-delivery system. We measured the particle sizes, polydispersity and zeta potential of liposomes; determined the DOX loading efficiency by spectrophotometer; DOX release rate was detected by DOX release assay, targeting SATB1gene silencing effect was evaluated by RT-PCR and Western-blot; Cellular uptake of SATB1-shRNA and DOX was showed by fluorescence microscopy.Results:DOX loading rate for TSCL-DOX was89±3%, TSMCL-DOX was78±8%, TSCL-DOX-shSATBl was85±7%, TSMCL-DOX-shSATB1was73±9%; DOX liposome were kept stable at37℃while realized thermosensitive release at42℃, and the loading of SATB1-shRNA did not significantly influence the thermosensitive release of DOX; SATB1-shRNA liposome had significant SATB1gene silencing activity; the cellular uptake of DOX and SATB1-shRNA can be enhanced by magnetic targeting.Conclusions:Thermosensitive magnetic cationic DOX and SATBl-shRNA co-delivery system had ideal DOX loading rate, DOX thermal release rate, good SATB1gene silencing effect and magnetic targeting DOX and SATB1-shRNA delivery capability. Part V Anti-tumor activities of thermosensitive magnetic cationic liposomes for co-delivery Doxorubicin and SATBl-shRNAObjective:To detect anti-tumor effects of Doxorubicin and SATB1-shRNA co-delivery system in gastric cancer.Methods:MTT assay was used to detect the cytotoxicity, and Annexin V-FITC and PI double staining to detect apoptosis; tumor-bearing mice to detect anti-tumor effect in vivo.Results:DOX, TSCL-DOX, TSMCL-DOX, TSCL-shSATB1, TSMCL-shSATB1, TSCL-DOX-shRNA and TSMCL-DOX-shRNA had cell viabilities of40.3±3.4%,73.6±3.5%,51.3±4.5%,79.2±6.9%,71.6±4.7%,35.0±3.2%and22.3±3.4%; apoptosis rates of them were22.3%,8.9%,13.4%,9.4%,17.4%,27.7%and32.4%; Tumor size of blank group, Free DOX group, TSMCL-DOX group, TSCML-shSATB1group, TSCL-DOX-shSATB1group and TSMCL-DOX-shSATB1were2.14±0.23cm3,1.08±0.13cm3,0.68±0.10cm3,1.43±0.21cm3,0.77±0.12cm3and0.44±0.05cm3.Conclusions:DOX and SATB1-shRNA co-delivery by thermosensitive magnetic cationic liposomes can lead to a synergistic anti-tumor effect.