Interfacing Inorganic Nanocrystals with Biological Systems Using a - PowerPoint PPT Presentation

Interfacing Inorganic Nanocrystals with Biological Systems Using a Coordinating Polymer Coating A. Kapur, W. Wang, G. Palui, N. Zhan, H. Mattoussi Florida State University Chemistry and Biochemistry Email: mattoussi@chem.fsu.edu Website: http://www.chem.fsu.edu/ S. Medina, J. Schneider NIH/NCI, Frederick, MD The 16th U.S.-Korea Forum on Nanotechnology 1

Potential Applications of Colloidal Nanocrystals Zhang et al., Adv. Mater. 2013, 25: 3869 Nat. Mater 2005,4, 435 2 From Invitrogen

QDs and AuNPs: Lipoic Acid Anchors Hydrophilic - OH OH OH OH OH segment O O n n - OCH 3 O O Anchor O O O O n n O O O O - N 3 O O O O HO HO O O OH OH S S S S n n S S S S O O S S NP S S - NH 2 O O FN O O O O O S S S S n n QD QD S S O O S S n O O - COOH S S S S O O S S S S S S S S n n HO HO O O OH OH - Biotin O O O O n n O O O O O O O O O - Mal O O n n O O n n HO HO HO HO SH SH Susumu et al., J. Am. Chem. Soc. 2007 , 129, 13987; Mei et al., J. Mat. Chem. 2008 , 18, 4949 Zhan, Palui , … Mattoussi J. Am. Chem. Soc. Zhan, Palui , … Mattoussi, J. Am. Chem. 3 2013 , 135 , 13786 -13795 Soc. 2015 , 137, 16084−16097

Lipoic Acid/Histidine-Modified Polymer for QDs Wang, Kapur, Ji, Safi, Palui, Palomo, Dawson, and Mattoussi, J. Am. Chem. Soc. 2015, 137 , 5438 – 5451 Stoichiometric insertion can be achieved Nominal numbers per chain a Experimental numbers per chain b Ligand (molar fractions) LA-PIMA-PEG (x:z = 30:70) LA: 12 PEG: 27 LA: ~ 13 PEG: ~ 27 His-PIMA-PEG (y:z = 30:70) His: 12 PEG: 27 His: ~ 10 PEG: ~ 27 His-PIMA-PEG (y:z = 50:50) His: 20 PEG: 20 His: ~ 17 PEG: ~ 19 LA/His-PIMA-PEG (x:y:z = 20:30:50) LA: 8 His: 12 PEG: 20 LA: ~ 10 His: ~ 12 PEG: ~ 18 4 LA/His-PIMA-PEG-R (x:y:z:z ’ = 20:30:45:5) LA: 8 His: 12 PEG: 18 R: 2 ---

Photoligation of LA-containing Polymer Ligands Photoligation TOP/TOPO TOP/TOPO 1.0 Synergistic Anchors 1.0 LA/His-PIMA-PEG LA/His-PIMA-PEG Absorbance (normalized) QD537 (thiol + imidazole) QD537 QD572 QD572 0.8 0.8 PL (normalized) QD600 QD600 QD633 QD633 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 400 500 600 700 400 500 600 700 800 Wavelength (nm) Wavelength (nm) Hexane DI Water 5

The Zwitterion Motif: an Alternative to PEG Bio-reactive ligands 45 ˚C DMSO Wang, Ji, Kapur, Zhang, ... Mattoussi, J. Am. Chem. Soc. 2015 , 137, 14158-14172 Representative ligands His-PIMA-ZW-NH 2 LA-PIMA-ZW/N 3 His-PIMA-ZW 6 Wang, Kapur, Ji, Zeng, Mishra and Mattoussi, Bioconjugate Chem. 2016 , 27, 2024−2036

His-PIMA-ZW: Compact Coating His-PIMA-ZW => Compact size QDs 1.2 R H = 5.7 nm R H = 5.5 nm Intensity (normalized) 1.0 QD-His-PIMA-ZW 0.8 log D (m 2 /s) 0.6 D = 3.88 × 10 -11 m 2 /s 0.4 D 2 O 0.2 0.0 0.1 1 10 100 1000 1 H δ (ppm) Hydrodynamic Radius (nm) Diffusion Ordered Spectroscopy (DOSY) Dynamic Light Scattering 𝐥 𝐂 𝐔 𝐒 𝐈 𝐟𝐲𝐬𝐛𝐝𝐮𝐟𝐞 𝐯𝐭𝐣𝐨𝐡 𝐓𝐮𝐩𝐥𝐟𝐭 − 𝐅𝐣𝐨𝐭𝐮𝐟𝐣𝐨 𝐅𝐫. ∶ 𝐄 = 𝟕𝛒𝛉𝐒 𝐈 7

1.2 109 nm x 20 nm CTAB-AuNRs LA-PIMA-PEG-AuNRs 1.0 Absorbance (normalized) Functionalization of 0.8 0.6 Au-Nanostructures 0.4 0.2 0.0 400 600 800 1000 1200 Wavelength (nm) Gold nano-structures 1.2 CTAB-AuNRs 45 nm x 19 nm LA-PIMA-PEG-AuNRs 1.0 Absorption (normalized) with a PEGylated (or 0.8 0.6 ZW) polymer coating 0.4 0.2 0.0 400 500 600 700 800 Wavelength (nm) 1.2 Wang, Ji, Du, Mattoussi, J. Phys. 9.8 nm oleylamine - AuNPs LA-PIMA-PEG - AuNPs Absorbance (normalized) 1.0 Chem. C 2017 , 121, 22901- 0.8 22913. 0.6 0.4 0.2 8 0.0 400 500 600 700 800 Wavelength (nm)

DTT Stability Test: Polymer vs Monomer PEG-Coating Au Nanorods 0.4 0.4 LA-PIMA-PEG-AuNRs LA-PEG-AuNRs Absorbance (a.u.) 0.3 0.3 Absorbance (a.u.) 0.2 0.2 0.1 0.1 624 nm 0.0 0.0 300 400 500 600 700 800 300 400 500 600 700 800 Wavelength (nm) Wavelength (nm) Wang, Ji, Du, Mattoussi, J. Phys. Chem. C 2017 , 121, 22901-22913. Similar results were collected for AuNPs. Similar differences between polymer and monomer were found with the zwitterion motif. 9

Cellular Uptake of Nanoparticles: The Endocytosis Problem 10 Zhao & Zhao et al. Small 2011, 7, 1322

Uptake Promoted by SVS-1 Peptide  Cationic , amphiphilic peptide with anti-cancer activity  Peptide: “ CGG-KVKVKVKVDPPTKVKVKVK-NH 2 ”  Preferentially folds at the negatively charged surface of cells, adopting an amphiphilic β -hairpin structure capable of disrupting cell membranes . Sinthuvanich et al. J. Am. Chem. Soc. 2012 , 134, 6210 Medina et al., J. Controlled Release 2015 , 209 317 – 326 11

Cell Uptake Studies: Case of AuNR/NP-SVS-1 Conj. 9.8 nm Proof of Imidazole-to-AuNC coordination Aldeek, Safi, Zhan, Palui, AuNP Mattoussi, ACS Nano 2013, 11, 10197 109 nm x 20 nm or AuNR S. Medina et al., J. Controlled Release 2015 , 209 317 – 326 R 2 = R 1 = (SVS-1) (TXR) 100 nm 1.NHS-Texas Red/NHS-maleimide SVS-1: CGG-KVKVKVKV D PPTKVKVKVK-NH 2 12 2.SVS-1 peptide (thiol-maleimide)

Merge DIC DAPI Texas-Red Control • SVS-1-mediated delivery of AuNR-TXR AuNR  conjugates into HeLa [0.3 nM] cells. • Here, Texas-Red dye coupled to AuNR-SVS-1 AuNR- conjugates allowed [0.1 nM] visualization of the AuNR-SVS-1 distribution inside cells. • Scale bar ~ 10 μm . AuNR-SVS-1 [0.2 nM] Kapur, Medina, Wang , … , Mattoussi, ACS Omega 2018 , 3, 12754−12762 AuNR-SVS-1 [0.3 nM] 13

Merge DIC DAPI Texas-Red Control AuNP-TXR [5 nM] SVS-1-mediated delivery of AuNP  conjugates into HeLa cells. AuNP-SVS-1 [2 nM] 10-nm NPs were used. Scale bar = 10 μm . AuNP-SVS-1 [4 nM] Kapur, Medina, Wang , … , Mattoussi, AuNP-SVS-1 ACS Omega 2018 , 3, 12754−12762 [5 nM] 14

Intracellular Uptake of QDs 75 nM 50 nM 15

Confocal Images Three color QD-peptide conjugates have been tested 0.38 μ m 1.14 μ m 1.52 μ m 1.90 μ m 2.28 μ m 3.04 μ m 3.42 μ m 0.37 μ m 1.12 μ m 1.86 μ m 2.24 μ m 2.61 μ m 3.35 μ m 3.73 μ m 0.34 μ m 1.72 μ m 2.06 μ m 2.41 μ m 2.75 μ m 3.35 μ m 4.13 μ m Kapur, Medina, Wang, Palui … ACS Omega 2018 , 3, 17164−17172 16

Testing Uptake in the Presence of Inhibitors 1. Incubation at 4°C 2. ATP depletion using NaN 3 3. Clathrin mediated endocytosis inhibition using Sucrose Kapur, Medina, Wang, Palui … ACS Omega 2018 , 3, 17164−17172 C [QD-SVS1] 50 nM, 1hr, 4°C 17

Acknowledgements Group Members: Students Collaborations with: Xin Ji (Ph.D.) Prof. Mikhail Zamkov (BGSU Physics) Naiqian Zhan (Ph.D.) Prof C. Donega (U Utrecht) Wentao Wang (Ph.D.) Prof Sam Grant (NHMFL) Anshika Kapur (Ph.D.) Dr. Joel Schneider (NCI-NIH) Dinesh Mishra (Ph.D.) Prof. Phil Dawson (Scripps Inst) Zhicheng Ji Prof J. Johnson (Scripps Research Inst.) Liang Du Prof. Igor Alabugin (FSU Chem.) Woody Perng Prof. Ken. Knappenberger (FSU Chem.) Chengqi Zhang Prof. Debi Fadool (FSU Bio.) Juan Hernandez Prof. Daniel Hallinan (FSU Eng.) Sisi wang Dr. David Gray (Pfizer) Dr. Goutam Palui Prof. Birong Zeng Dr. Victor Klimov at LANL Dr. Fadi Aldeek Jan-Philip Merkl Dr. Malak Safi Laura Alfonso Dr. Hyon Bin Na AFOSR Dr. Yuya Sugiyama Asahi-Kasei 18