| [Objective] Explore the FITC-TAT-mediated magnetic liposomes and character-ization biocompatibility of FITC labeled TAT-PEG magnetic cationic liposomes with Schwann cells in vitro, and analyze its ability to through the membrane by co-culture of Schwann cells in vitro, observe the local gathering in the rat spinal cord tissue after spinal cord injury, research The targeting abilityin the injured spinal, to provide experimental evidence with theliposomes mediating drugs for the treatment of spinal cord injury in the future.[Method] In the study, long cycle and superparamagnetic TAT-magnetic nano liposomes were prepared by reverse phase evaporation. Transmission electron--microscopy, particlesize and Zeta potential analyzer measured particle size and basic characterization of liposomes. By unilateral ligating saphenous nerve of Wistar rats to activate Schwann cells, and then isolation, culture and purification of Schwann cells were performed in vitro. At last the purity of Schwann cells were identified by S-100 antibody and DAPI staining.The Schwann cells and two types of liposomes (TAT-type, noTAT-type) were incubated together. The Schwann cells were divided into two groups:the control group (co-culture with TAT-liposome for 1 hour) (59μg/ml) 0.5ml, the experimental group (co-culture with noTAT-liposome for 1 hours) (59μg/ml) 0.5ml. Observing the uptake of liposomes by Schwann cells under fluorescence microscope was undertaken.Sixteen of female Wistar rats were subjected to IMPACTOR MODEL-Ⅱto establish a T10 spinal cord injured model. The animals were randomly divided into two groups:24 hours after injury, respectively, by tail vein injection of FITC-magnetic nano-liposomes (1mg/kg), the control group received nano-liposomes without TAT, experimental group to join the TAT-magnetic nano-liposomes.By injecting FITC labeled new liposome (1mg/kg) into tail vein, rats were killed 1 hour later. Harvested the spinal cord was subjected to quick 5μm frozen sections.The sections of spinal cord, liver, spleen and kidney were also observed under fluorescence microscope. Sixteen of female Wistar rats were randomly divided into two groups:control group without treatment for spinal cord injury, only to take the same dose of anesthesia, the experimental group to establish spinal cord injury model.Two groups of animals were injected in the tail vein of the TAT-magnetic nanoliposomes (1mg/kg), the time point selected as the experimental group was 24 hours after injury, control group was 24 hours after anesthesia adding the time required for production models. rats were killed 1 hour later. Harvested the spinal cord was subjected to quick 5μm frozen sections.[Results]1. Magnetic liposomes were spherical lipid lanes, better dispersion, smaller particle size, higher external Zeta potential. There were no significant difference between TAT-nanoliposomes group and noTAT group in the particle size and Zeta potential.2. Activated Schwann cells were isolated, cultured. The purity was more than 95%.3. Cellular uptake experiment show that:TAT-liposomes gathered inside Schwann cells,and most gathered around the nucleus.The noTAT-liposomes can not be observed in Schwann cells.By comparing the FITC fluorescence microscope in two groups,the average optical density(average optical density, AOD) showed a significant difference.4. In the blue fluorescence under fluorescence microscope, FITC made the magnetic nano-liposomes showeing green fluorescence. In the rat spinal cord tissue,TAT-liposomes gathered more around the injured spinal cord side, neuronal cells present in the cytoplasm of a large number of fluorescent particles, gathered into a sheet showing the trend of the nuclear.noTAT-liposomes were then distributed less in damage side of the spinal cord. Both AOD values calculated, the result was statistically significant.5. By excitation with blue in fluorescence microscope, in the no TAT group,in the liver, spleen, kidney, bright green fluorescence are visible.The TAT group is relatively weaker with green fluorescence.6. By the blue fluorescence excitation, Spinal cord tissue had less green fluorescence in normal rats.Spinal cord gathered a large number of green fluorescencein injured spinal cord rats.Calculating values of the two AOD, the results have statistical significance.[Conclusion] This study proved that TAT-mediated nanoliposomes can be poured into the damaged spinal cord, get into neurons, and have the targeting characteristics of gethering to damaged spinal cord,provided a feasible basis for becoming a load carrier of drugs for curing spinal cord injury. |
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