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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:

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SLIDE 1
  • 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

Interfacing Inorganic Nanocrystals with Biological Systems Using a Coordinating Polymer Coating

The 16th U.S.-Korea Forum on Nanotechnology

1

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SLIDE 2
  • Nat. Mater 2005,4, 435

From Invitrogen 2

Potential Applications of Colloidal Nanocrystals

Zhang et al., Adv. Mater. 2013, 25: 3869

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SLIDE 3

QDs and AuNPs: Lipoic Acid Anchors

S S O O O OH

n

S S O O O OH

n

S S O O O OH

n

S S O O O HO

n

S S O O O HO

n

S S O O O HO

n

S S O O O HO

n

S S O O O OH

n

QD

S S O O O OH

n

S S O O O OH

n

S S O O O OH

n

S S O O O HO

n

S S O O O HO

n

S S O O O HO

n

S S O O O HO

n

S S O O O OH

n

QD

  • OH
  • OCH3
  • N3
  • NH2
  • COOH
  • Biotin
  • Mal

Anchor

FN

O O O

NP

n Hydrophilic segment

SH SH O

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. 2013, 135, 13786 -13795 Zhan, Palui, … Mattoussi, J. Am. Chem.

  • Soc. 2015, 137, 16084−16097

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SLIDE 4

Lipoic Acid/Histidine-Modified Polymer for QDs

Wang, Kapur, Ji, Safi, Palui, Palomo, Dawson, and Mattoussi,

  • J. Am. Chem. Soc. 2015, 137, 5438–5451

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Stoichiometric insertion can be achieved

Ligand (molar fractions) Nominal numbers per chaina Experimental numbers per chainb

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 LA/His-PIMA-PEG-R (x:y:z:z’ = 20:30:45:5) LA: 8 His: 12 PEG: 18 R: 2

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SLIDE 5

Synergistic Anchors (thiol + imidazole) Photoligation

Photoligation of LA-containing Polymer Ligands

400 500 600 700 0.0 0.2 0.4 0.6 0.8 1.0

Absorbance (normalized) Wavelength (nm)

TOP/TOPO LA/His-PIMA-PEG QD537 QD572 QD600 QD633

400 500 600 700 800 0.0 0.2 0.4 0.6 0.8 1.0 TOP/TOPO LA/His-PIMA-PEG QD537 QD572 QD600 QD633

PL (normalized) Wavelength (nm)

Hexane DI Water

5

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SLIDE 6

DMSO 45 ˚C His-PIMA-ZW His-PIMA-ZW-NH2

Representative ligands Bio-reactive ligands

Wang, Ji, Kapur, Zhang, ... Mattoussi, J. Am. Chem. Soc. 2015, 137, 14158-14172

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The Zwitterion Motif: an Alternative to PEG

Wang, Kapur, Ji, Zeng, Mishra and Mattoussi, Bioconjugate Chem. 2016, 27, 2024−2036

LA-PIMA-ZW/N3

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SLIDE 7

His-PIMA-ZW: Compact Coating

0.1 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Intensity (normalized) Hydrodynamic Radius (nm)

1H δ (ppm)

log D (m2/s)

D2O

QD-His-PIMA-ZW

Diffusion Ordered Spectroscopy (DOSY) Dynamic Light Scattering RH = 5.5 nm RH = 5.7 nm

D = 3.88 × 10-11 m2/s

𝐒𝐈 𝐟𝐲𝐬𝐛𝐝𝐮𝐟𝐞 𝐯𝐭𝐣𝐨𝐡 𝐓𝐮𝐩𝐥𝐟𝐭 − 𝐅𝐣𝐨𝐭𝐮𝐟𝐣𝐨 𝐅𝐫. ∶ 𝐄 = 𝐥𝐂𝐔 𝟕𝛒𝛉𝐒𝐈

His-PIMA-ZW => Compact size QDs

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SLIDE 8

400 500 600 700 800 0.0 0.2 0.4 0.6 0.8 1.0 1.2 CTAB-AuNRs LA-PIMA-PEG-AuNRs

Absorption (normalized) Wavelength (nm)

400 600 800 1000 1200 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Absorbance (normalized) Wavelength (nm)

CTAB-AuNRs LA-PIMA-PEG-AuNRs

109 nm x 20 nm

400 500 600 700 800 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Absorbance (normalized) Wavelength (nm)

  • leylamine-AuNPs

LA-PIMA-PEG-AuNPs

9.8 nm 45 nm x 19 nm

Gold nano-structures with a PEGylated (or ZW) polymer coating

Wang, Ji, Du, Mattoussi, J. Phys.

  • Chem. C 2017, 121, 22901-

22913.

Functionalization of

Au-Nanostructures

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SLIDE 9

300 400 500 600 700 800 0.0 0.1 0.2 0.3 0.4

Wavelength (nm) Absorbance (a.u.)

624 nm

300 400 500 600 700 800 0.0 0.1 0.2 0.3 0.4

Absorbance (a.u.) Wavelength (nm)

LA-PIMA-PEG-AuNRs

DTT Stability Test: Polymer vs Monomer PEG-Coating

LA-PEG-AuNRs

Au Nanorods

Similar results were collected for AuNPs. Similar differences between polymer and monomer were found with the zwitterion motif.

9

Wang, Ji, Du, Mattoussi, J. Phys.

  • Chem. C 2017, 121, 22901-22913.
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SLIDE 10

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Cellular Uptake of Nanoparticles: The Endocytosis Problem

Zhao & Zhao et al. Small 2011, 7, 1322

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SLIDE 11
  • Cationic, amphiphilic peptide with anti-cancer activity
  • Peptide: “CGG-KVKVKVKVDPPTKVKVKVK-NH2”
  • Preferentially folds at the negatively charged surface of cells, adopting an

amphiphilic β-hairpin structure capable of disrupting cell membranes.

11

Uptake Promoted by SVS-1 Peptide

Sinthuvanich et al. J. Am. Chem. Soc. 2012, 134, 6210 Medina et al., J. Controlled Release 2015, 209 317– 326

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SLIDE 12

Cell Uptake Studies: Case of AuNR/NP-SVS-1 Conj.

SVS-1: CGG-KVKVKVKVDPPTKVKVKVK-NH2

1.NHS-Texas Red/NHS-maleimide 2.SVS-1 peptide (thiol-maleimide)

100 nm

109 nm x 20 nm

9.8 nm R1 = R2 =

(TXR) (SVS-1)

AuNP

12

  • r AuNR
  • S. Medina et al., J. Controlled

Release 2015, 209 317–326 Proof of Imidazole-to-AuNC coordination Aldeek, Safi, Zhan, Palui, Mattoussi, ACS Nano 2013, 11, 10197

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SLIDE 13
  • SVS-1-mediated delivery of

AuNR conjugates into HeLa cells.

  • Here, Texas-Red dye coupled to

AuNR- conjugates allowed visualization of the AuNR-SVS-1 distribution inside cells.

  • Scale bar ~ 10 μm.

13 AuNR-TXR [0.3 nM] AuNR-SVS-1 [0.1 nM] AuNR-SVS-1 [0.2 nM] AuNR-SVS-1 [0.3 nM]

DIC DAPI Texas-Red Merge Control

Kapur, Medina, Wang, … , Mattoussi, ACS Omega 2018, 3, 12754−12762

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SLIDE 14

SVS-1-mediated delivery of AuNPconjugates into HeLa cells. 10-nm NPs were used. Scale bar = 10 μm.

AuNP-TXR [5 nM] AuNP-SVS-1 [2 nM] AuNP-SVS-1 [4 nM] AuNP-SVS-1 [5 nM]

DIC DAPI Texas-Red Merge Control

14

Kapur, Medina, Wang, … , Mattoussi, ACS Omega 2018, 3, 12754−12762

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SLIDE 15

50 nM 75 nM

15

Intracellular Uptake of QDs

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SLIDE 16

0.38 μm 1.14 μm 1.52 μm 1.90 μm 2.28 μm 3.04 μm 3.42 μm 0.37 μm 1.12 μm 1.86 μm 2.24 μm 2.61 μm 3.35 μm 3.73 μm 0.34 μm 1.72 μm 2.06 μm 2.41 μm 2.75 μm 3.35 μm 4.13 μm

Three color QD-peptide conjugates have been tested

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Kapur, Medina, Wang, Palui … ACS Omega 2018, 3, 17164−17172

Confocal Images

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SLIDE 17

C

  • 1. Incubation at 4°C
  • 2. ATP depletion using NaN3
  • 3. Clathrin mediated endocytosis inhibition using Sucrose

[QD-SVS1] 50 nM, 1hr, 4°C

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Testing Uptake in the Presence of Inhibitors

Kapur, Medina, Wang, Palui … ACS Omega 2018, 3, 17164−17172

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SLIDE 18

Acknowledgements

Group Members: Students Xin Ji (Ph.D.) Naiqian Zhan (Ph.D.) Wentao Wang (Ph.D.) Anshika Kapur (Ph.D.) Dinesh Mishra (Ph.D.) Zhicheng Ji Liang Du Woody Perng Chengqi Zhang Juan Hernandez Sisi wang

  • Dr. Goutam Palui Prof. Birong Zeng
  • Dr. Fadi Aldeek

Jan-Philip Merkl

  • Dr. Malak Safi

Laura Alfonso

  • Dr. Hyon Bin Na
  • Dr. Yuya Sugiyama

Collaborations with:

  • Prof. Mikhail Zamkov (BGSU Physics)

Prof C. Donega (U Utrecht) Prof Sam Grant (NHMFL)

  • Dr. Joel Schneider (NCI-NIH)
  • Prof. Phil Dawson (Scripps Inst)

Prof J. Johnson (Scripps Research Inst.)

  • Prof. Igor Alabugin (FSU Chem.)
  • Prof. Ken. Knappenberger (FSU Chem.)
  • Prof. Debi Fadool (FSU Bio.)
  • Prof. Daniel Hallinan (FSU Eng.)
  • Dr. David Gray (Pfizer)
  • Dr. Victor Klimov at LANL

AFOSR Asahi-Kasei

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