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Minocycline-loaded Multifunctional Drug Delivery Systems For Targeted Spinal Cord Injury Therapy

Posted on:2019-03-31Degree:DoctorType:Dissertation
Country:ChinaCandidate:X J WangFull Text:PDF
GTID:1314330545952857Subject:Pharmacy
Abstract/Summary:PDF Full Text Request
Spinal cord injury(SCI)is a common and devastating disease with high mortality and disability rate,affecting the quality of patients' and their families' life seriously.With the rapid development of economy,the morbidity increases year by year.Currently,therapeutic strategies in clinic mainly focuses on surgery for relief of oppression to spine cord and methylprednisolone for impeding further progression of secondary injury.However,controversy around its real neurologic efficacy has plagued the use of methylprednisolone due to severe side effects,narrow therapeutic window and limited functional recovery in SCI patients.With the gradual deepening of understanding in SCI,novel therapeutic stratiges such as cell transplantation for replacing the lost nerve tissue and tissue engineering utilized as bridge for spine cord repair and neural regeneration have made promising progress presently.However,massive cell loss and apoptosis induced by secondary injury are not inhibited basically,and that might result in extremely limited neurological recovery and behavior improvement possibly.Multi-targets therapy which could suppress secondary injury and promote neural regeneration or repair simultaneously might be an effective strategy for SCI therapy.Minocycline hydrochloride(MC.HCl),an available broad-spectrum antibiotic clinically with neuroprotective activity,could inhibit multiple secondary injury mechanisms via its anti-inflammatory,anti-oxidant and anti-apoptotic properties,thus improving the kinematics of SCI animals significantly.Besides,MC.HCl exhibited encouraging outcomes in Phase ? clinical trial.However,long-term and systemic administration of MC.HCl with high dose might cause liver and kidney toxicity even death.Nano-drug delivery system(NDDS)was an effective strategy to improve the bio-distribution and reduce the adverse effects of drug.Additionally,multi-targets therapy could be achieved through the functionalization of the carrier materials.Therefore,NDDS based on polysialic acid(PSA)with capacity of promoting neural regeneration and polyethylene glycol(PEG)with neuroprotective activity were constructed respectively for targeted delivery of minocycline and multi-targets therapy of SCI.Polysialic acid(PSA)was a critical large cell-surface carbohydrate during central nervous system development and capable of promoting precursor cell migration,axon path-finding and synaptic remodeling,thus playing vital role in tissue repair,regeneration and long extension of axons.Therefore,a functional NDDS based on PSA was designed firstly.PSA was hydrophobically modified with octadecylamine(ODA)to form amphiphilic conjugate(PSA-ODA,PSO)via acylation reaction,and PSO with different ODA feeding ratios were prepared.The synthetic PSO conjugate could self-assembly into micelle.With ODA feeding ratio increased from 5%to 20%,critical micelle concentrations(CMC)of PSO decreased from 126.1 to 37.3 ?g/mL,and the corresponding particle size decreased from 149.00 ± 7.21 to 84.40 ± 8.42 nm,respectively.PSO micelles with ODA feeding ratios of 10%and 20%could encapsulate hydrophobic minocycline effectively with encapsulation efficiency over 70%and drug loading up to 12%.The minocycline-loaded PSO micelles(PSM)exhibited sustained drug release behavior and the release could be prolonged to 72 h.Drug release from PSM micelle with ODA feeding ratio of 20%was relatively slow,which was unfavorable for SCI therapy,thus PSO and PSM with ODA feeding ratio of 10%were utilized for further study.BV2 and primary microglia cells were used for investigation of the cytotoxicity and cellular internalization capacity of PSO conjugate.The results showed that after incubation with PSO conjugate(concentration ranging from 1 to 1000 ?g/mL)for 48 h,cell viabilities were over 90%,indicating the extremely low cytotoxicity of PSO conjugate.Meanwhile,PSO micelle displayed excellent and time-dependent cellular internalization capacity towards both cells.Lipopolysaccharide(LPS)was used to activate BV2 and primary microglia cells for evaluation of anti-inflammatory capacity of PSM micelle.The results displayed that after LPS-activated cells incubated with MC.HCl and PSM for 24 h,levels of TNF-?,IL-6 and NO decreased apparently.Protein levels of iNOS and Iba-1 down-regulated obviously.Besides,no significant difference was observed between MC.HCl and PSM group.In hydrogen peroxide-induced oxidative stress model,survival of SH-SY5Y cells increased and calcium concentration inside cells decreased remarkably after PSM and MC.HCl pre-treatment.Correspondingly,proportion of apoptotic and necrotic cells decreased distinctly.No significant difference between MC.HCl and PSM group was observed.While,PSO exhibited neither anti-inflammatory nor neuroprotective capacity basically.SCI model was established via weight-drop method using female SD rats.PSO displayed massive accumulation in the lesion site and equally distribution in white and gray matter in SCI rats.After administration of MC.HCl and PSM for 24 h,concentrations of TNF-?,IL-6,IL-1,MDA and NO in the lesion site reduced obviously when compared with Saline group.Expression level of iNOS and cell apoptosis decreased apparently as well,indicating that secondary injury was inhibited effectively.After consecutive administration for 7 days,MC.HCl,PSM and PSO resulted in obvious locomotor function improvements of SCI rats during the 12 w observation period,in which PSM conferred superior therapeutic effects compared with other groups.Long extension of axons through the glia scar and reconnection with caudal tissue were clearly observed in the lesion site of PSM group.Besides,MC.HCl,PSM and PSO could reduce volume of lesion cavity,increase the number of nerve fibers with BDA positive staining,alleviate demyelination occurred after SCI,make more axons survive the injury and inhibit the formation of glia scar at some extent at 12 w post-injury.Notably,regeneration of neurons was observed in lesion site of PSM group at 4 w post injury.Additionally,MC.HC1 exhibited certain liver and kidney toxicity reflected by higher concentrations of AST,ALT,BUN and Scr after consecutive administration for 7 days.While,the biochemical parameters of PSO and PSM group were similar to those of Control,indicating the bio-safety of the designed drug delivery system.Polyethylene glycol(PEG),acting as sealing agents,was capable of repairing the damaged membranes of cells,organelles and axons,inhibiting calcium influx,maintaining the normal function of cells and repairing the damaged axons,thus reducing cell death and apoptosis induced by secondary injury and exerting neuroprotective capacity.Based on the previous study,another targeted and functional drug delivery system based on PEG was designed.Firstly,poly-(lactic-co-glycolic acid)(PLGA)and sialic acid(SA)were utilized for hydrophobic and targeting modification of PEG via esterification reaction.The obtained copolymer PEG-PLGA(PP)and SA-PEG-PLGA(SAPP)could self-assembly into micelles with CMC at 14.40 ± 2.32 and 18.78 ± 3.28?g/mL,respectively.Both PP and SAPP micelles could encapsulate minocycline effectively with encapsulation efficiency always over 65%.The obtained minocycline-loaded micelles(PPM and SAPPM)displayed sustained drug release profiles and the release could extend to 72 h.Besides,SA modification had nearly no effects on physicochemical characterizations of blank and drug-loaded micelles such as particle sizes,morphology,drug loading,encapsulation efficiency and drug release behavior.BV2 cells were used for evaluation of the cytotoxicity and cellular internalization capacity of PP and SAPP copolymer.The results presented that PP and SAPP exhibited low cytotoxicity and excellent cellular internalization capacity.The anti-inflammatory activities of PPM and SAPPM were equivalent to that of MC.HCl reflected by apparently decreased levels of TNF-a,IL-6 and NO,and down-regulation protein levels of iNOS and Iba-1.The neuroprotective of MC.HCl and PEG were confirmed on glutamate(Glu)-induced SH-SY5Y excitotoxicity model.The results demonstrated that when Glu-induced cells were pre-treated by MC.HCl,PPM,SAPPM,PP and SAPP,cell survival increased obviously,calcium levels inside cells reduced and ratios of apoptotic cells decreased.The synergistic effect between MC.HCl and PEG made PPM and SAPPM conferred better neuroprotective capacity compared with other groups.HUVEC cells were activated by LPS to simulate the inflammatory endothelial cells following SCI and assess the cellular uptake of SAPP.The results exhibited that LPS-activated HUVEC could overexpress E-selectin,and SAPP displayed specific cellular internalization capacity towards LPS-activated HUVEC in short time via the specific binding bewteen SA and E-selectin.When partial E-selectin was blocked by free SA,cellular uptake of SAPP decreased apparently.The targeting efficiency in vivo and bio-distribution of SAPP were investigated in SCI rats.The results demonstrated that expression of E-selectin was elevated obviously in the lesion site of SCI rats.SAPP displayed targeted distribution in the lesion site via specific binding with E-selectin,and the fluorescence signal in per gram spine cord tissue was higher than that of liver at 12 h post administration,indicating the superior targeting efficiency.After administration of MC.HCl,PPM and SAPPM for 24 h,concentrations of TNF-?,IL-6,IL-1?,MDA and NO in the lesion site reduced obviously when compared with Saline group.Expression levels of iNOS and caspase-3 decreased apparently as well,indicating that secondary injury was inhibited effectively.SAPPM displayed better inhibitory effect compared with PPM group and no significant difference was observed between SAPPM and MC.HCl group.After consecutive administration for 7 days,MC.HCl,PPM,SAPPM,PP and SAPP could improve locomotor function of SCI rats obviously during the 12 w observation period,in which SAPPM conferred superior therapeutic effects compared with other groups.Besides,the lesion cavity was reduced significantly,more axons were preserved and demyelination was ameliorated obviously at 12 w post-injury.SAPPM still exhibited superior therapeutic effect compared with other groups,possibly resulting from targeting distribution and synergistic therapy between minocycline and PEG.Additionally,obvious pathologic changes were observed in liver and kidney of MC.HCl and PPM group,indicating the toxicity of MC.HCl and PPM.While,SAPPM,PP and SAPP treatment displayed no toxicity to the collected major organs.In conclusion,the functional drug delivery system designed in the study could achieve effective suppression of secondary injury through the targeted delivery of minocycline to the lesion site of SCI rats.Simultaneously,the residual PSA could promote neural regeneration and the residual PEG could exert its neuroprotective capacity,thus safe and effective therapy of SCI was achieved through the synergistic therapy between drug and the delivery systems.Our findings might provide new insight and strategy for SCI therapy in future.
Keywords/Search Tags:Spinal cord injury, Polysialic acid, Minocycline, Sialic acid, Synergistic therapy, Neuroprotection, Neural regeneration
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