Treatment Of Spinal Cord Injury With Photodynamic Therapy Mediated By Upconversion Nanoparticles

[Objective] To observe the killing effect of the nanoparticles(UCNPs-PEG-M540) to the astrocytes acquired from spinal cord of newborn SD rat in vitro after exposure to the980nm near infrared (NIR) laser. The experiment in vivo is to explore the effect of photodynamic therapy (PDT) mediated by UCNPs-PEG-M540in reducing glial scar formation and promoting functional recovery after rat’s spinal cord injury.[Method] The technique of solvothermal was applied to synthesize the core-shell shape nanoparticles(NaYF4:Yb3+,Er3+/NaYF4) and the PEG and M540were bonded on particles surface. After the nanoparticles were exposed to the980nmNIR, Diphenylisobenzofuran (DPBF) was used to detect the generation of singlet oxygen (1O2). The spinal cord from newborn SD rat were acquired after anesthesia and then cut into pieces, the tissue was then placed in culture flasks containing. The methods of differential adhesion and thermostatic shaking were used to purify the cells. After cell proliferation and passage in vitro, GFAP staining was used to identify the astrocytes. The cell growth feature was also observed. After2generation of cell passage, the astrocytes were collected and cultured with particles for24h. MTT was applied to observe the cell survival after exposed to the NIR and changes of the cell structure with electron microscope. Meanwhile, rat’s SCI model was made at T10with NYU Impactor-II machine with10g×50mm weight drop. Eighty spinal cord injured rats were divided into4groups randomly and evenly, which were DMEM group; M540group; UCNPs-PEG group; UCNPs-PEG-M540group. One week after spinal cord injury, the graft as above was injected into the injured spinal cord respectively. BBB score was carried out at the3rd day and at weekly intervals postinjury.12weeks later,8rats in each group were chosen out to carry out10%BDA anterograde tracing mark. Then,2weeks later, the animals were sacrificed, the spinal cord tissue with injured lesion was taken out and fast cryostat sections(5μm) was made followed by BDA developing, GFAP and NF200immunochemistry staining. Western blot was used to quantify the GFAP expression in the injured lesion.The pictures were processed by the software Image pro plus5.0and ImageJ, the IOD value of positive response area was counted to compare among four groups.[Result] Transmission electron microscopy (TEM) showed that the bare core nanoparticles substantially present the hexagonal shape and relatively uniform particle size. Particle size analyzer indicated the diameter of the particle was around28nm. After the shell was claded, the shape of the nanoparticle with shell-core structure was still hexagonal, which was confirmed by X-ray diffraction (XRD) analysis and diameter was around43nm. Fourier transform infrared spectroscopy demonstrated that PEG were successfully coated on the surface of the particles. Generation of singlet oxygen was detected by Energy dispersive X-ray spectroscopy (EDX). The cultured astrocytes can stablely pass over4generation after purified. The purity was around94.78%with similar morphology of fibroblasts. The cells showed positive GFAP staining and the doubling time of2-3days. After the cells co-cultured with particles and laser radiation, MTT analysis indicated that the cell viability dramatically decreased. Electron microscopy showed that the nanoparticles were taken up by astrocytes and finally the cells showed apoptotic or necrotic performance. From the4th week after injury, BBB score in UCNPs-PEG-M540group was significantly higher than other3groups (p<0.05). BDA anterograde tracing showed more regenerated nerve fibers pass through the injured site in UCNPs-PEG-M540group, while little in the other three groups (p<0.05). This was also confirmed by IOD value of positive area in cross section. The IOD value of positive reponse area of NF200staining in UCNPs-PEG-M540group was the highest among the4groups (p<0.05). While, GFAP staining was the lowest (p<0.05). Results of Western Blot showed the gray value of GFAP in UCNPs-PEG-M540group was the smallest among four groups (P<0.01), which means that UCNPs-PEG-M540group has the smallest glial scar formation.[Conclusion] With the radiation of980nmNIR, UCNPs-PEG-M540can effectively kill the astrocytes when co-cultured together. The UCNPs-PEG-M540can also effectively reduce the glial formation, promote the axonal regeneration and functional recovery after rat’s spinal cord injury.