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This study was aimed to develop non-toxic, high transfection efficiency polyethyleneimine(PEI) cationic nanoparticles. The exosyndrome of PEI cationic nanoparticles was measured by zeta sizer, ultraviolet and visible spectroscopy. The condensation ability and the resistance to DNaseI of pEGFP-N1/PEI and pEGFP-N1/PEI modified polyethylene glycol(PEG) were evaluated by agarose gel electrophoresis. The cell toxicity of polyethyleneimine cationic nanoparticles was measured by using MTT test. The orthogonal design was used to optimize the transfection efficiency with the N/P ratio, the grafting ratio and the gene dosage as the factors. The experimental results showed that pEGFP-N1/PEI nanoparticles have lower cell toxicity, better composite ability and better resistance to DNAseI. The highest transfection efficiency of PEI cationic nanoparticles was 91% by using the PEI nanoparticles with the N/P ratio 40:1 and gene dosages 6 microg/well. PEI cationic nanoparticle modified by PEG effectively transferred DNA to hepatoma carcinoma cells and it is a non-toxic, with high transfection efficiency, and a promising non-viral carrier for gene delivery. The transfection efficiency will be improved by optimizing the experiment condition.
Subject(s)
Humans , Carcinoma, Hepatocellular , Genetics , Pathology , Cell Line, Tumor , Gene Transfer Techniques , Liver Neoplasms , Genetics , Pathology , Nanoparticles , Chemistry , Polyethylene Glycols , Chemistry , Polyethyleneimine , Chemistry , Transfection , MethodsABSTRACT
Objective To synthesize the hydrophobic supraparamagnetic ironic oxide(SPIO) loaded and hydrophilic SPIO loaded polymeric nano-vesicles and to investigate the feasibility of using hydrophobic SPIO loaded and hydrophilic SPIO loaded polymeric nano-vesicles to display the tumor in MRI in vivo through animal experiments. Methods The polymeric nano-vesicles were prepared from poly (D, L-lactic acid) (PDLLA) and poly (ethylene glycol) (PEG) by a multiple emulsion/solvent evaporation method.The hydrophobic SPIO and hydrophilic SPIO were loaded in the polymeric nano-vesicles respectively.Eighteen nude mice models with human colorectal carcinoma xenograft were established. They were divided equally into three groups (n = 6). The three groups of nude mice models were injected with water-soluble SPIO, hydrophobic SPIO loaded and hydrophilic SPIO loaded vesicle via the mice caudal vein respectively.Dynamic MRI scan were performed in all the mice models. T2WI signal intensity and T2 relaxation time were measured in the tumor, liver and muscle by using T2 mapping software. ANOVA of repeated measurement was used to analyze if there were significant differences of signal intensity changes among the three groups, while Bonferroni method was used for pair-wise comparison. Results On T2 WI, tumors showed decrease in signal intensity after hydrophobic or hydrophilic SPIO loaded polymeric nano-vesicle injection, while no signal intensity decrease was found in the tumor after water-soluble SPIO administration. The maximum percentage of signal intensity decrease in tumor caused by hydrophobic SPIO loaded and hydrophilic SPIO loaded vesicle were 11.00%, 11.40%, respectively. There was statistical significant difference of signal intensity changes among these three groups (F = 10. 96, P ＜ 0. 01). The decrease in signal intensity in the groups with hydrophilic or hydrophobic SPIO loaded polymeric nano-vesicles injection were more pronounced as compared with that of water-soluble SPIO (P ＜ 0. 05), but there was no significant difference in signal intensity decrease between the groups of hydrophilic and hydrophobic SPIO-loaded polymeric vesicles injection (P ＞0. 05). The three agents could lead to signal intensity decrease in the liver. The maximum percentage of signal intensity decrease in liver caused by water-soluble SPIO, hydrophobic SPIO loaded and hydrophilic SPIO loaded vesicle were 32. 85%, 52. 77%, 56. 89%, respectively. There was statistical significant difference between these groups (F = 161.18, P ＜ 0. 01) . The groups of injecting hydrophilic and hydrophobic SPIO loaded polymeric nano-vesicles had the more obvious signal decrease than the one with water-soluble SPIO (P ＜ 0. 01). Hydrophilic SPIO loaded polymeric nano-vesicles exhibited more signal intensity decrease than hydrophobic SPIO loaded polymeric nano-vesicles (P ＜ 0. 01). All three agents could not lead to T2WI signal decrease in the muscle, and there was no significant difference in signal change on T2 WI among three groups (F = 0. 59, P ＞ 0. 05). Conclusion SPIO loaded polymeric nano-vesicles can cause significant T2WI signal loss in human colonic carcinoma on MR imaging in vivo. It can be used as tumor imaging contrast agents.
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Objective To formed ranoscale ultrasound contrast agents loading PFOB by selfassembly of amphiphilic block copolymer for ultrasonic imaging in vivo and in vitro.Methods The biodegradable copolymer-poly(ethylene glycol)-b-poly (D, L-lactic acid) (PEG-PDLLA) self-assembled to form PFOB-loaded nanomicelle and nanovesicle with different PFOB concentration by solvent volatilization.The configuration and particle-sizing of resulting nanoparticles was determined,and their ultrasonic imaging in vitro and contrast-enhanced ultrasonography on subcutaneous tissue by focal injection in vivo were observed.Results In transmission electron microscope images, these micelles and vesicles appeared uniformly spherical with smooth surface.All the size distributions were narrow and mean diameters were from 404.3 to 475.8 nm using laser particle-sizing analyzer.In vitro and in vivo experiment showed that,the higher PFOB concentration, the more remarkable effect of ultrasound imaging.Especially, nanovesilces'ultrasonography effect was much better than nanomicelles' under same conditions.Conclusions PFOB-loaded nano-micelles and nano-vesicles prepared by solvent volatilization and self-assembly of PEG-PDLLA can obviously enhance ultrasound contrast and nanovesicle behaves better than nano-micelle.
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Objective To evaluate the tumor targeting characteristic of the Folate-SPIO-DOX-Micelles by in vitro studies,and to test the feasibility of monitor tumor targeting using it and clinical MRI.Methods The polymeric micelles,Folate-SPIO-DOXO-Micelles were prepared.The in vitro tumor cell targeting efficacy of these folate modified and DOX or SPIO-loaded micelles (Folate-SPIO-DOX-Micelles)was evaluated by observing the cellular uptake of micelles by human hepatic carcinoma cells(Bel 7402 cells) which overexpressed folate surface receptors. Cell suspensions were incubated with Folate-SPIO-DOXO-Micelles for 1 h.Prussian blue staining was performed to show intracellular irons.Flow cytometry was used to further quantify the cellular uptake of the nanoparticles into Bel 7402 cells.MRl was performed to show the signal intensity changes by using T2 WI sequences at a clinical 1.5 T MR system.Results Prussian blue staining showed much more intracellular iron in cells incubated with Folate-SPIO-DOX-Micelles than the cells incubated with the non-targeting SPIO-DOX-Micelles.As revealed by flow cytometry,the mean fluorescence intensity of cells in the folate group and the non-folate group were 117.88 and 46.33,respectively.The T2 signal intensity in MRI of cells treated with the folate targeting micelles decreased significantly (when the concentration of SPIO in cell culture medium was 5,10,20,40,and 80 μg/ml,respectively,T2 signal intensity decreased by -5.02%,-23.58%,-45.89%,-70.34%,and -92.41%,respectively).In contrast,T2 signal intensity did not show obvious decrease for cells treated with the folate-free micelles (when the concentration of SPIO in cell culture medium was at 5,10,20,40,and 80 μg/ml,respectively,T2 signal intensity decreased by -3.77%,-2.16%,-2.18%,-2.74% and -19.77%,respectively).Conclusion The polymeric micelles,Folate-SPIO-DOX-Micelles has good targeting ability to the hepatic carcinoma cells in vitro,and the cell targeting events of the micelles can be monitored by using a clinical MR scanner.