GABA- LOADED HALLOYSITE NANOTUBE SYSTEM: RELEASE CHARACTERISTICS and EFFECT on SEIZURE PARAMETERS in EPILEPTIC RATS
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1 GABA- LOADED HALLOYSITE NANOTUBE SYSTEM: RELEASE CHARACTERISTICS and EFFECT on SEIZURE PARAMETERS in EPILEPTIC RATS Dr. Gülsel YURTDAŞ KIRIMLIOĞLU, Prof.Dr. Yasemin YAZAN, Prof.Dr. Kevser EROL, Res.Assis. Çiğdem ÇENGELLİ 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
2 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
3 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME generalized or paroxysmal focal reaction of brain convulsion provoked by inhibition of GABA synthesis, blockage of release or postsynaptic reaction
4 Major inhibitor neurotransmitter in central nervous system -COOH and NH 2 groups Regulation of impulse transmission, induction of hypotensive, diuretic and tranquilizer effects 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
5 Tubular structure Good biocompatibility and very low cytotoxicity Two-layered aluminosilicate Outer diameter nm, length µm 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
6 Incorporation of GABA into halloysite - nanosized - nontoxic - biocompatible - highly specific and high affinity formulations Providing GABA entrance into brain Modifying GABA release from formulations Minimizing serious side effects Enhancing patient compliance 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
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8 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME Loading Methods Under vacuum (100 mmhg) 1:1 molar ratio Heat treatment (80ºC, 2 hr) Heat treatment + Washing + Centrifugation 1:1 molar ratio Heat treatment + Washing + Centrifugation 1:2 Molar ratio Heat treatment + Frezee- drying 1:1 Molar ratio
9 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
10 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME Column Cihaz Schimadzu- 20A 4.6 x 100 mm, 3 µm C 18 Inertsil CDS column, particle size 100 Å Oven Temperature 30 C Mobil Phase Methanol:Na 2 HPO 4 buffer (40:60) (ph 6.7) Dedector Wavelength Fluorescence dedector 280 nm 450 nm Flow Rate 0.8 ml.min -1 Injection vvlume 27 µl
11 Dialysis bag ( Da) ph 7.4 phosphate buffer Validated HPLC method 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
12 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME Eskişehir Osmangazi University Scientific Ethical Committee (Protocol No: 265/2012) Wistar rats Group Code Group 1 Group 2 Group 3 Treatment PBS (ip) GABA solution (100 mg.kg -1 ) (ip) HNT-GABA H1 (100 mg.kg -1 GABA) (ip)
13 Pentylenetetrazole (70 mg/kg) induced epileptic model Latency to myoclonic jerks and incidence of generalized tonic clonic seizures with loss of righting reflex noted for 30 min Rats decapitated under ether anaesthesia Brains removed immediately and Stratum corsatum isolated GABA concentration in Stratum corsatum ELISA kit 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
14 As a result of the analyses
15 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME CHARACTERIZATION HNT-GABA VAC HNT-GABA H1 HNT-GABA H2 HNT-GABA H3 HNT (pure) Particle size (nm) ± ± ± ± ±23.71 PI 0.67 ± ± ± ± ± 0.06 Zeta potential (mv) ± ± ± ± ± 0.09 ph value 7.28 ± ± ± ± ± 0.00 PI: Polydispersity index, SE: Standard error
16 CHARACTERIZATION- HNT-GABA VAC HNT- GABA-H1 HNT-GABA H2 HNT-GABA H3 HNT (PURE)
17 CHARACTERIZATION-
18 CHARACTERIZATION-
19 CHARACTERIZATION-
20 GABA loading of nanotube formulations is shown in the Table. 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME DETERMINATION of GABA in HNTS % GABA Loading ± SE HNT-GABA H ± HNT-GABA H ± HNT-GABA H ± 0.106
21 % CUMULATıVE PERCENT OF GABA RELEASE IN VITRO RELEASE STUDY TıME (HOUR) GABA HNT-GABA H1 HNT-GABA H2 HNT-GABA H3 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME
22 % Cell Viability 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME CYTOTOXICITY HNT-GABA H1 PURE HNT GABA Control
23 IN VIVO STUDIES Seizure parameters GABA Solution PBS (ph 7.4) HNT-GABA H1 Latency ± SD (min) ± ± ± Ending time of the convulsion ± SD (immobility) (min) Duration of severe convulsion ± SD (min) ± ± ± ± ± ± Time of death ± SD (min) :42 - Mortality ratio (%) 4/7 (57.1) 3/7 (42.8) 0/7 (0) PBS: Phosphate buffer saline, SD: Standard deviation HNT-GABA H1 Groups Latency ± SD Ending time of the convulsion Duration of severe (min) ± SD (immobility) (min) convulsion ± SD (min) PBS * * ** GABA solution * ** ** SD: Standart deviation, p > 0.05: No significant difference, *: p < 0.05, **: p < 0.01, ***: p < 0.001
24 GABA Concentration (µg.g-1) 4TH INTERNATIONAL CONFERENCE & EXHIBITION ON NEUROLOGY AND THERAPEUTICS, ROME GABA AMOUNT in Stratum Corsatum PBS GABA HNT-GABA H1
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26 HNT-GABA H1 Nanosize Tubular shape Succesful incorporation of GABA Sustained release manner Low cytotoxicity PTZ induced epileptic rat model Brain delivery and seizure inhibition Retardation of latency time Decrease in ending time of convulsion Decrease of duration of severe convulsion Reduction of mortality ratio
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