Construction Of Electro-responsive Micelle-based Drug Delivery System And Application For Epilepsy Therapy

Objective Epilepsy is one of the common chronic neurological disorders caused by abnormal discharge of brain neurons,which has various seizure types.Different anti-epileptic drugs(AEDs)such as phenytoin(PHT),diazepam(DZP),carbamazepine(CBZ),etc.are in application for clinical treatment.However,due to the drawbacks of conventional administration(e.g.,low bioavailability,neurotoxicity,etc.),the drug delivery system(DDS)with electro-responsive and brain-targeted ability is needed for improving the therapeutic efficacy of multiple AEDs for various types of epilepsy and reducing the side effects.In this study,TPGS-Fc,which is synthesized by D-a-tocopherol polyethylene glycol succinate(TPGS)and ferrocene carboxylic acid(Fc-COOH),is assembled with poloxamer 407 to prepare the electro-responsive and brain-targeted micelles(named as TFP).TFP can release drugs after the abnormal electric stimulation for on-demand therapy,increasing the therapeutic efficacy of various AEDs such as PHT,DZP and CBZ.Meanwhile,fluorescence probes-loaded TFP was applicable for epileptic foci imaging.Our micelle provides the practical and theoretical basis of new platform construction for the treatment and imaging of epilepsy and other central neural disorders.Methods 1.In the first part,TPGS-Fc was synthesized by esterification reaction and characterized by proton nuclear magnetic resonance and matrix-assisted laser desorption/ionization time of flight mass spectrometry.Three kinds of mixed micelles were prepared by TPGS-Fc and different copolymers(poloxamer 407,poloxamer 188 and solutol HS 15).They were named as TFP,TFP 188 and TFHS15,respectively.The morphology,particle size and ζ-potential were characterized by transmission electron microscopy and dynamic light scatter,and the fluorescence intensity was detected by fluorescence spectrometer.The molecular models were established to simulate and compare the binding energy between TPGS-Fc and PHT,DZP or CBZ through density functional theory(DFT)calculation,clarifying the mechanism of TFP for universal drug delivery.Before and after responding to the electric stimulation,the drug loading and drug releasing ability of PHTloaded TFP(TFP@PHT),DZP-loaded TFP(TFP@DZP)and CBZ-loaded TFP(TFP@CBZ)were detected by high performance liquid chromatograph(HPLC).2.In the second part,the ability of cellular internalization of different micelles was investigated by cellular uptake experiment.In vitro blood-brain barrier(BBB)model was used to evaluate the capacity of different micelles to cross the BBB.Due to the competitive binding to transferrin receptors,the mechanism of cellular internalization of TFP was determined.The inhibition to P-glycoprotein(P-gp)efflux pump was investigated by adenosine triphosphate(ATP)assay kit.Brain-targeted capability of cyanine5.5-loaded TFP(TFP@Cy5.5)in vivo was detected by IVIS Spectrum animal imager.The pharmacokinetics of TFP@CBZ were evaluated by HPLC.3.In the third part,the therapeutic effects of TFP@PHT,TFP@DZP and TFP@CBZ were evaluated by seizure stage,latency to seizure,number of seizures,EEG,etc.in acute,continuous and chronic epileptic mice models.The drug releasing ability of TFP@Cy5.5 was evaluated in the epileptic kindling-induced mice brain.The capability of epileptic foci imaging of TFP@Cy5.5 was detected in the kindling-or kanic acid(KA)-induced mice models.The biosafety of TFP,TFP@PHT,TFP@DZP and TFP@CBZ was investigated by rotarod test,open field test,serum biochemical detection,immunofluorescence staining and hematoxylin and eosin staining.Results 1.In the first part,TPGS-Fc was successfully synthesized,and three kinds of micelles consist of TPGS-Fc were prepared with poloxamer 407,poloxamer 188 and solutol HS 15.The micelles were 24.4±1.3 nm(TFP),10.3±0.9 nm(TFP188)and 131.3±5.0 nm(TFHS15),respectively,and the size of TPGS-Fc was 46.8±0.2 nm.The results of in vitro electro-responsive release showed that more probes about 35.1 ± 7.7%,7.4± 0.1%and 13.1 ±7.0%were released from TFP group,TFP 188 group and TFHS15 group under electrical field at 30 min than from those without electrical stimulation,which proved that TFP had the highest electro-responsive release ability.The results of DFT calculation showed that TPGSFc had higher binding energy with PHT,DZP and CBZ than TPGS.After electrical stimulation,the amount of PHT,DZP and CBZ released from TFP were 62.7±8.1%,38.8±6.3%and 64.8 ± 7.3%,respectively,while those groups without stimulation were 31.0±3.0%,17.3 ± 4.9%,38.4 ± 2.3%,respectively.In vitro drug release of TFP@PHT,TFP@DZP and TFP@CBZ after electrical stimulation demonstrated the electro-responsive drug release property of TFP.2.In the second part,the results of in vitro cellular uptake and BBB permeability showed that the fluorescence intensity in TFP group was the highest.It was demonstrated that TFP could penetrate the BBB by transferrin receptor-mediated transcytosis and P-gp efflux pump inhibition by ATPase suppression in bEnd.3 cells.In vivo imaging showed that the fluorescence intensity of TFP@Cy5.5 was higher than free Cy5.5 in mice brains,verifying the brain-targeted capability of TFP@Cy5.5.Pharmacokinetic results showed that TFP@CBZ could increase the maximum concentration of CBZ and prolong the blood circulation time compared with free CBZ.3.In the third part,compared with free PHT,TFP@PHT significantly prolonged the latency to seizure and reduce the seizure stage,the mortality and seizure severity of the acute epilepsy mice model,verifying the enhanced therapeutic efficacy of PHT.The results of the kindling-induced epilepsy mice model showed that TFP@Cy5.5 could respond to the abnormal discharge and release Cy5.5,enhancing the fluorescence intensity in epileptic foci.The analysis of epileptic foci imaging showed improved fluorescence intensity along with the increased severity of epilepsy,indicating the on-demand drug releasing capacity of TFP.In the status epilepsy(SE)mice model,compared with DZP,TFP@DZP prolonged the latency to SE and generalized seizure(GS)and reduced the number of GS,the mortality and severity of seizure effectively,demonstrating the enhanced therapeutic efficacy of DZP.In the chronic epilepsy mice model,TFP@CBZ reduced the number and latency of GS and suppressed the severity of seizure compared with CBZ,indicating the improved therapeutic efficacy of CBZ.The fluorescence imaging of kindling-or KA-induced epilepsy mice models showed that the fluorescence intensity of TFP@Cy5.5 in epileptic foci was higher than in the normal brain.Finally,the biosafety of TFP,TFP@PHT,TFP@DZP and TFP@CBZ was proved.Conclusion In our study,an electro-responsive brain-targeted micelle drug-delivery system named TFP was constructed to solve the defects of epilepsy therapy(side effects,poor efficacy and etc.).AEDs with different molecular structures could be loaded in the system through various non-covalent bonds,such as hydrogen bonding and hydrophobic interaction,and could be delivered to the brain because of its highly efficient brain-targeted ability.Meanwhile,AEDs could be released from the system due to its electro-responsive capability in epileptic seizure,thus increase the concentration of drugs rapidly in brain.When loaded with a fluorescence probes,the micelle was able to be disassembled and released the probes after abnormal electric stimulus.The fluorescence imaging of epileptic foci was performed due to the aggregation-induced quenching feature of the probes.Our micelle provides a new strategy for electro-responsive drug release for different types of epilepsy therapy,and also provides a new scheme for epilepsy foci imaging.Furthermore,it paves a theoretical and practical basis for the construction of platforms of imaging and treatment for other diseases in central neural system.