KILLING THROMBUS WITH
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1 KILLING THROMBUS WITH D. Dash Department of Biochemistry Institute of Medical Sciences Banaras Hindu University
2 Thrombus has two components: (1) Protein Component composed of Insoluble Fibrin Clot (2) Cellular Component composed of Aggregated Platelets
3 Arterial Thrombosis Scanning electron micrograph of thrombus (RBC entrapped within aggregated platelets)
4 Two components of Anti-Thrombotic Measures: (1) Fibrinolytic Drugs (e.g. Streptokinase, Urokinase & Alteplase) (2) Anti-Platelet Drugs (e.g. Aspirin, Clopidogrel & Ticagrelor)
5 Fibrinolytic therapy is associated with serious bleeding complications! (Off-Target Effects) Requires consistent physician supervision & monitoring!
6 F I B R I N O G E N Products of Thrombin cleavage site A B α C-terminal D α β Thrombin cleavage site Fibrinopeptides A & B β α E Fibrin N-terminal α β α β D α C-terminal A B F I B R I N
7 Formation of Fibrin polymer Fibrin monomer (Soluble) Fibrin polymer (Clot / Thrombus) (Insoluble) Stabilized fibrin polymer (Insoluble) A B Fibrinogen Fibrinopeptides D Vulnerable for thermal ablation C-terminal Thrombin cleavage site Thrombin Aα α Bβ β E N-terminal α Aα Bβ β Spontaneous FXIIIa D C-terminal Fibrinopeptides A B Fibrinolytics
8 PHOTOTHERMAL THERAPY NIR LASER IRRADIATION (808 NM) GOLD NANORODS / GRAPHENE DEATH OF CANCER CELLS
9 Set-up: In vitro Photothermal Abalation of Fibrin Clot Mirror Thermal camera Laser source Laser Beam Gold nanorods Laser-irradiated Gold Nanorods H E A T Sample Tube (containing Clot) Sample holder stage Fibrin Clot (Before) Rarefication of fibrin clot after laser irradiation Fibrin Clot (After)
10 RISE IN TEMPERATURE IS A FUNCTION OF GNR CONC. Gold Nanorods (GNRs) (Axial diameter: nm; Longitudinal diameter: nm) RISE IN TEMPERATURE ( C) OF SOLUTION WITH INCREASE IN GNR CONCENTRATION Infra red (IR) thermal images of GNR samples in microplate wells (Left Panel) or centrifuge tube (Right Panel) exposed to the NIR laser at power density of 1.05 W/cm 2. Within the panel cursor represents spot temperature and the vertical pseudo-color bar signifies temperature intensity from high (yellow) to low (dark blue).
11 PHOTOTHERMAL ABLATION OF FIBRIN CLOT Lysis = 28% Lysis = 20% Turbidity assay (purified Fg) Lysis = 17% Fluorescence Assay (10% Alexa Fluor 488-labeled fibrin) Drabkin s Assay Lysis = 29% Lysis = 27% Lysis = 34% Lysis = 29% Methylene Blue Assay Photothermal ablation of clot (a) Turbidity assay (purified Fg) (b) Fluorescence assay in both purified Fg and PPP (c) and (d) Drabkin s assay in purified Fg and PPP (e) corresponding histogram (f) and(g) Methylene Blue assay in both systems and (h) corresponding histogram. Each histogram is a representative of five different sets of experiments.
12 Fluorescence intensity (Arbitrary unit) PHOTOTHERMAL ABLATION OF THROMBUS UNDER FLUID SHEAR Venous Shear (500 s -1 ) Arterial Shear (1500 s -1 ) Laser(a ) (a) (b) (c) (d) ) ) Control Control Laser Lysis = 55.39% Lysis = 39.13% Control Laser- irradiated Control Laser- irradiated Venous Shear Arterial Shear
13 Normalized Recovery Normalized Recovery FRAP (Fluorescence Recovery after Photobleaching) analysis by Confocal Microscopy (a) (b) FLUORESCENCE RECOVERY KINATICS Control (c) (d) NIR Laser-treated Bleached area Time (sec) Faster fluorescence recovery represents greater degree of molecular dynamism in laser-treated fibrin clot. FRAP analysis of 10% Alexa 488-labeled fibrin strands. (a) Before iteration, (b) photobleached with 100% laser (488 nm) power. (c and d) chronological events demonstrating fluorescence recovery at 2% of excitation laser power. White Arrows indicates the region of interest.
14 Our Experimental Strategy of Thrombolysis NIR-Laser Separation of Fibrin Strands & Restoration of Blood Flow
15 Photothermal Lysis in Mouse Model Step I- Thrombus induced in Femoral vein by FeCl 3 injury. Step II- Fibrin-targeted GNR injected through caudal vein. NIR-Laser Irradiation Step III- Thrombus irradiated with NIR- laser.
16 Colour Doppler Scan for Blood Flow in a Murine Femoral Vein Mouse-1 Mouse-1 Mouse-1 pulse wave Pulse Lost No Laser Pulse Lost Mouse-2 Mouse-2 Mouse-2 pulse wave Pulse Lost NIR Laser Irradiation Doppler Scan before Clot Formation showing Routine Blood Flow Doppler Scan after Clot Formation showing Occluded Blood Flow Doppler Scan Showing Restoration of Blood Flow after Laser- Irradiation in Mouse 2
17 Can Low-Dose Streptokinase Therapy (not associated with bleeding complications) synergize with Photothermal Therapy Towards effective Thrombolysis?
18 Colour Doppler Scan for Blood Flow in a Murine Femoral Vein Mouse-1 Mouse-1 Mouse-1 pulse wave Pulse Lost Only Streptokinase (Low Dose) Pulse Lost Mouse-2 Mouse-2 Mouse-2 pulse wave Pulse Lost Streptokinase (Low Dose) + LASER Doppler Scan before Clot Formation showing Routine Blood Flow Doppler Scan after Clot Formation showing Occluded Blood Flow Doppler Scan showing Restoration of Blood Flow after Laser & Low Dose Streptokinase
19 Hematoxylin-Eosin Stained Transverse Sections of Mice Femoral Veins No Laser Laser Magnification 10x Streptokinase (Low Dose) Streptokinase (Low Dose) + LASER
20 PHOTOTHERMAL THERAPY Targeted to lesion site Hemorrhagic complications associated with Streptokinase therapy is minimized, when low dose Streptokinase (Chemotherapy) is combined with Photothermal Therapy (MULTIMODAL APPROACH)
21 PUBLICATION Singh, N., Varma, A., Verma, A., Maurya, B.N. & Dash, D. (2016) Nano Res. 9: (Relief from vascular occlusion using photothermal ablation of thrombus with a multimodal perspective) (Impact Factor: 8.893) (cited by Nature India )
22 PATENT Patent Application No. # 3168/DEL/2014, dated on A FIBRIN-TARGETING DEVICE WITH NIR-ACTIVE NANOMATERIALS FOR IMPROVED THROMBOLYSIS EMPLOYING PHOTOTHERMAL (PT) METHOD
23 LAB MEMBERS
24 ACKNOWLEDGEMENT DST Nanomission DBT ICMR Tata Innovation Fellowship
25 THANK YOU!
26 THERE S PLENTY OF ROOM AT THE BOTTOM Richard P Feynman (American Physicist) (1959 Speech)
27 Nano World Deals with structures in the length scale of about 1 to 100 nm (1 nm = 10-9 m) (1 nm is roughly the width of 10 hydrogen atoms)
28 Examples of Nanomaterials Gold nanoparticles / nanorods Silver nanoparticles Carbon nanotubes (single-walled and multi-walled) Graphene Nanodiamond Magnetic (Fe 3 O 4 ) nanoparticles Quantum dots (Nanoscale Semiconductors), and so on.
29 PART-2 PHOTOTHERMAL ABLATION OF THROMBUS USING GOLD NANORODS
ROZAIMAH ZAIN-HAMID. Department of Pharmacology and Therapeutics Faculty of Medicine, Universitas Sumatera Utara
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