An Experimental Research On The Brain-targeted Drug-loaded Nona-particles For Treatment Of Glioma And Refractory Epilepsy

Partâ… Experimental Research on the therapeutic effect ofRGD-containing peptide-modified poly(aspartic acid)ion complex micelles against malignant gliomaAim:To design and synthesize an RGD-containing peptide-modifiedpoly(aspartic acid)ion complex micelles (RGD-PIC),which is stableagainst salt concentration variation and pH value variation,biocompatible for neurons,and therapeutic against malignant glioma,hence offering experimental basis for the designing and synthesis oftargeted drug-loading nano-micelles for treatment of refractory epilepsy.Methods:Starting with Mal-PEG-OH and a cyclic RGD peptide,we conjugatedthe RGD-PIC.EmployingCCK-8assay,thebiocompatibilitywithneuronsandrecognition as well as killing effect against glioma cells were testedin vitro in cultured rat hippocampus-derived neurons and U87 cell line,a malignant glioma cell line.We also produced the quantum dot-markedRGD-PIC for visualizing the cellular distribution of RGD-PIC in cells.Then RGD-PIC was peripherally administered to a malignant glioma modelof rat,generated by stereo-tactic injection of C6 cell line,anothermalignant glioma cell line,into the caudate nucleus.Quantum dot trackingtechnique was employed to determine the targeted delivery effect andimmuno-fluorescence technique was used to detect caspas-3 expression inboth glioma areas and non-glioma areas.Results:The RGD-PIC micelles weresuccessfully synthesized,demonstrating homogeneous size and sphericshape.When co-cultured with cells,RGD-PIC showed selective accumulationin U87 cells,while the same phenomenon was not observed in neurons.Moreover,RGD-PIC significantly influenced the cell viability of U87cells,while leaving neurons unaffected.In vivo data showed targetedaccumulation of RGD-PIC and increased expression of capase-3 in gliomaarea compared with non-glioma area.Conclusion:The RGD-PIC we designedand synthesized could selectively kill glioma cells in vitro and targeteddeliver to gliomacells in vivo,probably causingglioma cell apoptosis.Partâ…¡ Expression and distribution of multi-drug transporterP-gp in a limbic refractory epilepsy model of ratAim:To clarify the differentiating expression of P-gp in different ratorgans of normal,non-refractory epileptic and refractory epilepticrats,and the distribution mode of P-gp in the brain,secreting andabsorbing organs of refractory epileptic rat.Methods:Lithium-pilocarpine were used to induce epilepsy in SD rats and epilepticrats resistant to phenotoin was screened out.Western blot,RG-PCR andimmuno-fluorescent staining were applied to test the expression of P-gpand its mRNA—mdr-1,and the expression and distribution of P-gp.Results:Significantly higher cortical and hippocampal expression of P-gpand its gene was observed in refractory epileptic rats,compared withnormal and non-refractory epileptic rats (P＜0.05).Cerebral P-gp wasmainly localized on the micro-vascular endothelial cells,while the P-gpexpression in liver,small intestine and kidney were found on cells otherthan vascular endothelial cells.Conclusion:P-gp over-expression washigher in refractory epileptic rats,as compared with normal andnon-refractory epileptic rats.P-gp was distributed on micro-vascularendothelial cells only in brain,while not in liver,kidney or smallintestine,leading to the possibility that P-gp can play as a targetmolecule for targeted drug delivery against refractory epilepticpatients.Partâ…¢Synthesis,measurement and biocompatibility of P-gpMAbnano-structured materialAim:To conjugate a structurally stable nanoparticle with P-gpMab on itssurface and AEDs in the core by active targeting on the basis of the uniquesurface effect of nanopartilces,and measure its physical and chemicalcharacteristics and biocompatibility with central nervous system.Methods:Starting with Mal-PEG-OH,under the catalytic effect of Stannousoctanoate,the amphiphilic block copolymer MePEG-PCL,and Mal-PEG-PCL.P-gp MAb was thiolated and incorporated to form the P-gpMAbnano-structured material (PNM).Scanning Electronic Microscopy was usedto detect its morphological characteristics.Infraredspectrumstudyand ¹H NMR spectrum study were employed to test the chemical groups of thesynthesized PNM.DSL was applied to test its stability against pH valuevariation and salt concentration variation.PHT delivery was assayed withHPLC.Biocompatibility with cultured neurons,astrocytes,microglialsand human brain micro-vascular endothelial cells (HBMEC)were determinedby CCK-8 assay.Results:The synthesis of PNM loaded with PHT wassuccessful.The resulted PNMgained conspicuous core-shell structure withan average diameter of 150 nm to 200 nm.Determined by DSL,the relativelight intensity of the PNMremained above 90% under the pH range (7.3-8.5).The 0% NaC1 and 0.9%NaC1 solution has not exerted different effect onthe relative light intensity of PNM,92.6% and 91.6% respectively (P＞0.05).The loading efficiency determined by HPLC turned out to be 21.45%.Fora long period after 9 hours of PEG-PCL-PHT,the delivery was slow andstable,with no abrupt delivery.Pulsing the HBMEC,microglial and neuronswith PNM (50mg/L)resulted in no significant reduction of cell viability.However,cell viability of astrocytes dropped to 88.90% on the fifth day(P＜0.05).Conclusion:The PNM we designed and synthesized is stableagainst salt concentration variation and pH value variation,has a typicalcore-shell structure,could deliver in a stable and slow way,and isbiocompatible for neurons,microglials,HBMECs and astrocytes within thefirst five days.However,further improvement is needed as treatment withPNM caused decreased cell viability in astrocytes.Partâ…£Experimental research on the therapeutic effect of PNMagainst refractory epilepsyAim:To test the therapeutic effect of the PNMwe designed and synthesizedagainst refractory epilepsy.Methods:Rat hippocampus-derived neuronswere cultured,pulsed with non-Mg²⁺solution to induce epileptoiddischarge,followed with PNM or control treatment,and patch-clampwhole-cell recording.Further,CCK-8assaywasconductedto test the cellviability of neurons,and annexin-V flow cytometric assay was employedto detect the apoptosis of neurons.Meanwhile,two limbic chronic epilepsymodels of rats,respectively induced by lithium-pilocarpine and maximalelectroshock,were generated andphenotoin-resistant rats were screened out.PNMwas injected intravenously and EEG was recorded to assess theepileptic discharge.Micro-dialysis was used to collect the cerebrospinalfluid of rats and passed on to HPLC for determination of PHT concentration.Blood sample was collected from eyeballs and sent for TNF-αELISA.Results:The frequency and amplitude of epileptoid discharge decreasedsignificantly in PNM (100μg/ml,72h)treated neurons,and EPSP wasimproved.Compared with PHT,PNM showed no additional influence on theviability of neurons,while PNM reversed the apoptotic ratio of neuronsfrom 75.3% to 30.2% after non-Mg²⁺treatment (P＜0.05).In vivo data showedthat PNM treatment improved EEG performance and heightened PHTconcentration in cerebrospinal fluid significantly,while exerted noadditional influence on excretion of pro-inflammatory factor TNF-α.Conclusion:The PNMwe designed and synthesized could inhibit epileptoiddischarge,lower the apoptotic ratio and heighten the cell viability ofnon-Mg²⁺epileptic cell model in vitro,showing no less therapeutic effectthanPHT.Increased PHT delivery in the epileptic lesion of rat brain andbetter therapeutic effects were found after intravenous injection of PNMto refractory epileptic rats.The unchanged TNF-αlevel compared withPHT group excludes the concern that antibody conjugation might raise theproblem of unexpected immune reaction.Overall Conclusion:The RGD modified RGD-PIC nanopartilce could selectively kill gliomacells in vitro and be targeted delivered to glioma cells in vivo,causingapoptosis of the receiving cells.P-gp could be used as a target fortargeted drug delivery for refractory epilepsy.The P-gpMAb modifiedPhenetoin-loaded Nanoparticle (PNM)is protective against Mg²⁺-inducedneuronal seizure discharge model of epilepsy and has targeted therapeuticeffect against refractory epilepsy model,while escaping the recognitionand elimination by antigen-presenting cells.