| Objects: To synthesize a novel triblock monomethyl poly(ethylene glycol)-poly(l-lactide)-poly(trimethylene carbonate)copolymer and prepare polymeric nanomicelles loaded with zonisamide and prednisone(MPEG-PLLA-PTMC/ZNS-PDN).Study the characterization of the physical and chemical properties of the nanomicelles and antioxidative effects in vitro.Investigate the treatment of MPEG-PLLA-PTMC/ZNS-PDN nanomicelles in hemisection spinal cord injury of SD rats.Method:(1)The MPEG-PLLA-PTMC copolymer was synthesized successfully by ring-opening polymerization with catalysis.And MPEG-PLLA-PTMC/ZNS-PDN nanomicelles were praperd by film hydration method.Several tests were conducted to evaluate MPEG-PLLA-PTMC/ZNS-PDN micelles,including size distribution,zeta potential,drug loading and entrapment efficiency,morphology,crystallography,and in vitro release behavior.(2)The CTX TNA2 cell line were exposed to 500μM H2O2 and treated with free ZNS+PDN(dissolved in DMSO),MPEG-PLLA-PTMC/ZNS-PDN and blank MPEG-PLLA-PTMC micelles(M)at different concentrations(0-50 μM).The cortical neuron cells were separated from embryonic SD mice.The antioxidant activity and cytotoxicity of MPEG-PLLA-PTMC/ZNS-PDN were tested by MTT assay.Specific proteins,including glial fbrillary acidic protein(GFAP)and neuroflament(NF),were identified by immunohistochemical staining.(3)Sixty male SD rats were randomized into five groups: Group 1: sham operated group,shame surgery included general anesthesia,skin incision,and laminectomy but without spinal hemisection;Group 2: control group,rats were received 0.9% Normal Saline;Group 3: MPEG-PLLA-PTMC/ZNS-PDN nanomicelle treated group;Group 4: MPEG-PLLA-PTMC nanomicelle treated group,and Group 5: free ZNS-PDN treated group,respectively,each group 12 SD mice.Each group received them treatment at 6 hours and 4 days after operation.Mice were examined with MRI at the 7th、30th days spinal cord injury intertion status were observed.After7d、 14 d、30d,we applied Hematoxylin-Eosin(HE),immunohistochemistry(NF 、 GFAP 、 Nrf2 、 Tunel),Basso,Beatlie,Bresnahan(BBB)Locomotor Rating Scale to evaluate the recovery of injured spine.Resuts:(1)The 1H-NMR results indicate that the triblock MPEG-PLLA-PTMC copolymers were prepared successfully,and its molecular weight was 3900.The MPEG-PLLA-PTMC/ZNS-PDN micelles were characterized in detail.The MPEG-PLLA-PTMC/ZNS-PDN particle size distribution of the prepared was presented with a mean particle size of 50 nm.The FT-IR 、 XRD results indicated that MPEG-PLLA-PTMC/ZNS-PDN nanomicelles had been prepared successfully.And MPEG-PLLA-PTMC/ZNS-PDN nanomicelles also show high entrapment efficiency and satisfactory sustained-release behavior.(2)After drug treatment for 24 h and 48 h,the absorbance of the cells in the MPEG-PLLA-PTMC/ZNS-PDN group was significantly increased compared to the DMSO and the control group(P<0.05),indicating that MPEG-PLLA-PTMC/ZNS-PDN nanomicelles may provide a neuroprotective effect against oxidative stress in nervous tissue.In MPEG-PLLA-PTMC/ZNS-PDN treated groups,the axon lengths of neurons were obviously elongated compared to those in the control group after 7 days.In addition,the number of branch points was also increased in the MPEG-PLLA-PTMC/ZNS-PDN treated groups.(3)Except control group,every group have some changes after 2W with MRI,but spinal cord injured part was cured MPEG-PLLA-PTMC/ZNS-PDN,other groups were partly healed after 30 d.Spinal cord injury part were observed with HE staining,Except sham group,every group white matter and grey matte was destoried partly,nerve fiber broken and nerve celles deaded.after 7d and 14 d,but the group of MPEG-PLLA-PTMC/ZNS-PDN recovered better than other groups,glial scar was less than others.BBB scores revealed that the MPEG-PLLA-PTMC/ZNS-PDN treated group showed significant differences in BBB scores of the injured side(p<0.05)at 7 days,1 weeks,1 months,2 months,and 3 months after the spinal cord was hemi-dissected.At last,the immunohistochemistry revealed that MPEG-PLLA-PTMC/ZNS-PDN micelles treated group had higher expression of NF and Nrf2(P<0.05),but lower GFAP and apoptosis than other groups at 14 days post SCI.(P<0.05),Conclusion:(1)A novel pharmaceutical preparationsm MPEG-PLLA-PTMC/ZNS-PDN nanomicelles had been prepared successfully,and possessed good physical and chemical properties,which could be used in vivo.(2)The MPEG-PLLA-PTMC/ZNS-PDN nano micelles have a strong ability of promoting neuron axon elongation and the ability of antioxidative stress in vitro.(3)MPEG-PLLA-PTMC/ZNS-PDN nanomicelles showed good neuroprotection after hemisection spinal cord injury. |
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