| Spinal cord injury(SCI)is one of the most devastating diseases with no cure available,and mostly leads to an irreversible loss of neural function.The secondary injury is the major pathological process that aggravates the tissue damage and hinders the recovery,mainly by the heightened inflammation related damage.Therapeutic agents have been investigated to counter the secondary injury,but the therapeutic effect is frequently limited by the poor accumulation,the short retention and the lack of controlled release of therapeutics in lesion tissue.Nanomedicine has shown potential in solving these problems.In this paper,the recent progress of nanomedicine was reviewed,especially the nanomaterials based tissue-specific treatment,and a scar tissue targeting polymer micelle was built for SCI treatment.A scar tissue-targeting prodrug polymer micelle was built for spinal cord injury therapy.This study has been divided into three major parts:(1)The fabrication of the scar tissue-targeting prodrug polymer micelle(cPAM);(2)The scar tissue-targeting micelle cPAM promoted the shift from M1polarization to M2 polarization of microglia;(3)The scar tissue-targeting micelle cPAM promoted the neural recovery of mice spinal cord injury.Objective:To build an injured spinal cord tissue-targeting prodrug polymer micelle based on the lesion microenvironment,which could improve the delivery efficacy of neural protective components,suppress the secondary damage,benefit the preservation of normal neural tissue,and the neural function recovery after spinal cord injury.Methods:Section I:The injured spinal cord tissue targeting terapeptide(cysteine-alanine-glutamine-lysine,CAQK)was synthesized,whose targeting efficiency to injured spinal cord was identified in vivo.A scramble terapeptide(CGKK)was also synthesized as control.The apocynin prodrug(APEG-PAPO,PAPO)was synthesized,then CAQK was introduced to the PAPO to endow the polymer(CAQK-PAPO)the injured tissue-targeting ability.PAPO and CAQK-PAPO could self-assemble into micelles of PAM and cPAM,whose characterizations were also determined,including morphology,size,zeta potential,stability,degradation profile,and biocompatibility.Importantly,the injured spinal cord tissue targeting efficacy of cPAM was evaluated in vivo again.Section II:The microglia cell line of BV2,activated by LPS and IFN,was used to mimic the neural inflammation condition.After treatment with PBS,cPAM or esterase-degradation products of cPAM(De-cPAM),the population of M1 and M2polarized BV2 was assessed by flow cytometry.In addition,the specific markers of M1and M2 polarized BV2 were also determined by western blot and rt-PCR.The cellular ROS content was determined using DCFH-DA probe in vitro.On the other hand,a T9spinal cord contusion mice model was also performed and treated with PBS,APO,PAM or cPAM i.v.And the polarization of microglia in vivo was evaluated by immunohistochemistry,and the tissue ROS content was determined using DHE probe.Section III:A T9 spinal cord contusion mice model was performed to test the therapeutic effects of cPAM on the neural function recovery.SCI mice were divided into five groups,and treatment with PBS,APO,PAM,cPAM or cPAM(after blockade with CAQK).The locomotor functional recovery was assessed according to BMS scoring system,and the foot-ink test.The tissue integrity and axon damage were evaluated by H&E staining,LFB staining,and immunohistochemistry staining.To assess the organ toxicity of cPAM,the morphology of major organs was also observed,including heart,liver,and spleen.Results:Section I:The results of IVIS and histochemistry observation showed an enhanced accumulation of Rhod B-labeled CAQK in lesion spinal cords when compared to Rhod B-labeled CGKK.The results of ~1H NMR indicated the amphipathic molecules of CAQK-PAPO and PAPO were successful synthesized,which were also found to self-assemble into stable micelle with homogeneously round shape,observed using TEM.The result of DLS indicated the size of cPAM was at about 22 nm,and the zeta potential was-9 m V.Both micelles of PAM and cPAM were found stable in PBS or FBS containing medium.cPAM exhibited good biocompatibility,with low cytotoxicity on primary neuron.Importantly,the cPAM exhibited an improved accumulation and prolonged retention in lesion tissue when compared to control after i.v injection.Section II:The treatment with cPAM significantly reduced the M1 polarized population and the protein level of M1 marker of BV2,and increased the M2 polarized population and the M2 marker after inflammatory stimulation.In addition,the cellular ROS content was significantly decreased in cPAM group,revealed by the lower fluorescent intensity.On the other hand,the results of immunohistochemistry revealed an increased i NOS positive staining microglia in SCI mice.The treatment with cPAM remarkably decreased the i NOS positive microglia,and increased the Arg-1 positive microglia,while the PAM did not.In addition,the cPAM effectively decreased the tissue ROS content,indicated with lower oxidative-HE signal intensity,while the PAM treated group did not.Section III:The result of BMS scoring revealed a sustained increase in cPAM treated mice,significantly higher than other groups at 4 weeks post injury.While there was no significant difference between the mice treated by PAM or cPAM(after blockade with CAQK)with the PBS group.The H&E staining showed a significantly less necrotic tissue area,compared with other control.In addition,the result indicated a higher LFB stained area in lesion segment in cPAM treated mice when compared with other control.Thus,the result of immunohistochemistry staining showed significantly more NF200positive axon in lesion segment in cPAM treated mice when compared with other control.The H&E staining of heart,liver,and spleen revealed no obvious change after cPAM treatment.Conclusion:In conclusion,an injured spinal cord targeting prodrug polymer micelle was successfully built,which induced an effective targeting treatment of SCI via its robust scar binding effect,making the scar tissue a drug releasing platform for a sustained treatment.The micelle of cPAM exhibited good biocompatibility,degradation into APO in response to esterase activity,and a CAQK dependent scar tissue targeting effects after systemic administration,with an enhanced tissue accumulation and prolonged retention. |
