| Cationic-material-based nonviral gene vectors,such as cationic polymers,lipids,and proteins,have shown great promise in effective intracellular delivery of therapeutic DNA and mediating specific RNA interference in vitro and in vivo.Serum albumin has been widely used as a material for nano-and microparticulate drug carrier systems as it is considered to be nontoxic,nonantigenic and biodegradable.If the anionic side-chain carboxylic groups are modified with a cationic amino group,cationic serum albumin is obtained,which has low cytotoxicity and good biocompatibility since the cationization does not destroy the protein structure and its properties.Cationic human serum albumin(CHS A)was obtained by surface modification of HSA with ethylenediamine through an amide linkage.Slight changes were made to the previously described method for adjusting the pI of CHSA.HSA then was characterized by SDS-PAGE electrophoresis,IR spectroscopy and circular dichroism proved that CHSA contained more free amino groups and conformations of protein was more loose than HSA,but The whole structure remains essentially unchanged.By measuring the zeta potential of CHSA in buffers with different pH values,the pI of the CHSA was determined as about 8 and 9.The CHSA has an average 102.51±3.11 and 92.71±1.37 free amino-group measured by TNBS,which means that on average about 42.51 ±3.11 and 32.71 ±1.37 ethylenediamine groups were newly linked to one protein molecule.By simply mixing,CHSA and nucleic acid could form nano-sized particles in a wide range of N/P ratios through electrostatic interaction.The agarose gel electrophoresis and Hoechst 33258 method showed that CHSA could successfully block the migration of DNA.When the N/P>4,the encapsulation efficiency could reach more than 90%,and it could protect the DNA against the substitution of anions such as heparin and the degradation of DNase I As well as the effects of serum.The particle size and zeta potential of the complex were also related to the N/P ratio.The larger the N/P ratio was,the higher the zeta potential and smaller the size was.The TEM image confirmed the uniform round shape and narrow size distribution of the complexes.Circular dichroism indicatied that the complex also maintained the structure of the original HSA.The results of the characterization showed that the addition of NLS could not be considered in the formulation.To achieve gene co-delivery,the CHSA/HDM2 siRNA complex was prepared.Agarose gel electrophoresis showed that the complex also could block the migration siRNA and compress the siRNA.The particle size is larger and the zeta potential is smaller than the pDNA complex.The cytotoxicity of CHSA as a vector was studied by MTT assay showed that the concentration of 500 ug/mL or less,the cell without cytotoxicity and survival rates were more than 100%.Similar to cell cytotoxicity,the levels of IL-12 and IFN in mice were not significantly different between the CHSA group and the saline group under experimental conditions.The proliferation inhibition of CHSA/NLS/P53 and CHSA/HDM2 siRNA on tumor cells in vitro was more than 30%when N/P ratio was more than 10.The cell-uptake of CHSA/NLS/pDNA was time-dependent.The uptake rate of CHSA/NLS/pDNA complex in HepG2 cells was higher than that in A549 cells,and the high pI protein uptake was higher than that of low pI.After incubation for 0.5 hr,CHSA-2 complex was significantly ingested in cells.The main uptake mechanism of the complex in both cells was energy-dependent and cathepsin-mediated endocytosis.The intracellular tracking and localized by laser scanning confocal microscopy(LSCM)showed that the complex was found in the endosomes after 30 min and showed up in the cytoplasm and the nucleus after 2 hr.The in vitro transfection of the complex was investigated by fluorescence intensity of green fluorescent protein using laser scanning confocal microscopy and the activity of luciferase with chemiluminescence method.The results showed that the transfection efficiency of the complex was related to the N/P ratio.With the increase of N/P ratio,the transfection efficiency increased,while the N/P ratio increased to 30,the transfection efficiency decreased.The expression of HDM2 protein was detected by Western blot.The results showed that the silencing efficiency increased and the effect was better than that of Lipofectamine2000 when N/P increased.The best combination ratio of CHSA/NLS/P53 and CHSA/HDM2 siRNAs was 60%:40%.The size was 189.6 ± 2.45 nm,the zeta potential was 18.7±0.67 mV.The electron microscopy showed that the shape of the complex was regular and spherical.The results of Western blot showed that the combination of the complexes had the most expression of P53 protein.The ability of induced apoptosis was detected by Annexin V-FITC/PI staining assay and Caspase activity.The results showed that the complex could successfully activate the mitochondrial-mediated and death receptor-mediated apoptosis pathway downstream of P53,and then induced apoptosis,its induction of apoptosis is also stronger than Lipofectamin2000.The BABL/C mice survival rate of P53 and HDM2 siRNA was significantly higher than that of the control group(P<0.05).The survival rate of the CHSA complex was 44.42%compared with the saline group.In summary,we designed and optimized the cationization degree of CHSA used as a DNA and siRNA co-delivery carrier for treating cancer cells.CHSA could bind nucleotide via electrostatic interactions and form nanosized complexes that significantly enhanced the cell-uptake efficiency and lysosome escape.Therefore,when therapeutic gene,P53 and HDM2 siRNA,was delivered by CHSA,a notable transfection and silencing effect was found both in vitro and in vivo.In addition,with careful control of the pI,CHSA also showed a low toxicity and immunogenicity.Collectively,CHSA-based nanoparticles may be a promising strategy for practical and effective gene co-delivery to treat lung metastatic cancer.Further research is needed for better understanding and optimization of this system. |
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