The Use of Gold Nanoparticles in Imaging and Targeted Drug Delivery Simona Dostálová 31. 7. 2015 Investice do rozvoje vzdělávání

Gold nanoparticles EPR effect & biocompatibility High surface area Easy to produce Unique optical properties (strong absorption, scattering in the visible-near infrared region) ↓ size = ↑ absorption; ↑ size = ↑ scattering Chemically inert High electron density Spheres/rods/shells/cages/cubes

GNPs in theranostics and biomedicine Light absorption in „water window“ (700-1200 nm) Chemical sensors/immunosensors/biosensor Imaging agent Photodynamic therapy Drug delivery vehicles siRNA, miRNA delivery Tumor targeting

The past Characterization of gold nanoparticles-modified CdTe quantum dots by scanning electron microscopy NanoCon 2013 3D-printed chip for detection of methicillin-resistant Staphylococcus aureus labeled with gold nanoparticles Electrophoresis Apoferritin as a targeted delivery system MendelNet 2013

The present Significant changes of metallotionein expression in tumors => Metal nanoparticle-based immunohistochemistry = immunodetection of metallothionein in tissue using gold-labeled antibodies and LA-ICP-MS

GNPs Tetrachloroauric acid HAuCl4 1mM [0.0197 g/50 mL] 10 mL Sodium citrate [0.265 g/10 mL] 0.25 mL stirring Heating in different conditions Microwave reaction system Shaking Power 300 W Ramp 10:00 Hold 12:00 IR 50°C Power 300 W Ramp 10:00 Hold 05:00 IR 100°C Power 300 W Ramp 05:00 Hold 05:00 IR 120°C SIZE: 2 nm – 40 nm

GNPs conjugation with HWR peptide L 1 2 3 4 + 1.25 µL HWR + 2.50 µL HWR + 5 µL HWR HWR peptide Gold NPs Added HWR (μl) Average size (nm) 32 1.25 50 2.50 90 5.00 150 1= HWR 1 mg/mL 2= GNPs + 1.25 µL HWR 3= GNPs + 2.50 µL HWR 4= GNPs + 5.00 µL HWR

Conjugation with antibodies Gold NPs Gold NPs Gold NPs + HWR Gold NPs + HWR + antibody 1:100 Gold NPs + HWR + antibody 1:1000 Gold NPs + HWR + antibody 1:10000 L 1 2 3 4 5 6 7 1= HWR 1:100 2= IgG 1:100 3= GNPs 4= GNPs + HWR 5= GNPs + HWR + IgG 1:100 6= GNPs + HWR + IgG 1:1000 7= GNPs + HWR + IgG 1:1000 Too low sensitivity => optimization on QDs

QDs conjugation with HWR L= Ladder 1= HWR [1mg/mL] 2= QDs 110°C precipitated 3= QDs 110°C + HWR [0.0208 mg/mL] Peptide concentration [0.0208 mg/mL] causes the saturation of available bonds on QDs.

QDs conjugation with HWR and antibodies L= Ladder 1= HWR [1mg/mL] 2= IgY [54 mg/mL] 3= QDs 110°C precipitated 4= QDs 110°C + HWR [0.0208 mg/mL] 5= QDs + HWR + IgY [13.675 mg/mL] The conditions of gel electrophoresis were optimized: - running buffer Tris Tricine with pH adjusted to 9, - loading buffer with mercaptoethanol (stabilizing agent) Optimal ratios of HWR and antibody were found L 1 2 3 4 5

ELISA

Dot blot LA-ICP-MS

Conclusion Gold nanoparticles have many uses in theranostics and biomedicine One of them - metal nanoparticle-based immunohistochemistry We optimized the binding of HWR peptide and antibodies We need to optimize the HWR-GNPs binding and detection of complex

Investice do rozvoje vzdělávání Acknowledgements Bc. Monica Vazzana Mgr. Marketa Vaculovicova, Ph.D. Doc. RNDr. Pavel Kopel, Ph.D. Prof. Ing. Rene Kizek, Ph.D. Investice do rozvoje vzdělávání 11

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