Introduction

Essential Prevention of Protein Adsorption onto Gold

Nanoclusters in undiluted fetal bovine serum (FBS)

Acknowledgements

Background

Nathan Rebello, Robert Stover, Sai Gourisankar, Gerard Isaac, Thomas Truskett,

Konstantin Sokolov and Keith Johnston

Texas Materials Institute, The University of Texas at Austin, Austin, TX, USA

Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA

NSFSTC (CHE-9876674), NIH (CA 103830), NIH

RO1 EB008821-01, NIH Training Grant #HL07446,

Welch Foundation (F-1319)

Johnston Group

Sokolov Group

Emelianov Group

[1] Tam, J.; Murthy, A. et al. Kinetic Assembly of Near-IR-Active Gold

Nanoclusters Using Weakly Adsorbing Polymers to Control the Size.

Langmuir , 2010, 26, 8988-8999.

[2] Choi, H. et al. Renal Clearance of quantum dots. Nature Biotech., 2007,

25, 1165-1170.

[3] Moyano, Daniel F., Krishnendu Saha, Gyan Prakash, Bo Yan, and Hao

Kong. "Fabrication of Corona-Free Nanoparticles with Tunable

Hydrophobicity." ACS Nano 8.7 (2014): 6748-755. Print.

[4] Murthy, A. et al. Equilibrium Gold Nanoclusters quenched with

biodegradable Polymers. ACS Nano, 2013, 7, 239-251.

[5] Murthy, A. et al. Charged Gold Nanoparticles with Essentially Zero

Serum Protein Adsorption in Undiluted Fetal Bovine Serum. JACS, (In

Review).

References

Exchange of highly negative citrate ligand

with positively-charged lysine or zwitterionic

cysteine to produce mix charged monolayer on

gold surface

Conclusions

Relevant Theory

1.4/1 Lysine/Citrate monolayer 1.6 Cysteine/Citrate monolayer

Ligand/Citrate

(Feed) Ligand/Citrate

(XPS)

Zeta Potential

(mV)

0 0 -58.4 ± 5.3

4.5 Lysine 0.5 -28.9 ± 3.9

9.0 Lysine 1.4 -16.1 ± 2.9

0.3 Cysteine 1.0 -28.8 ± 3.2

0.7 Cysteine 1.6 -21.6 ± 1.7

Lysine and cysteine are longer than citrate, both have zwitterionic tips and variation of charge that interacts

with serum proteins

Minimizes short-ranged local electrostatic interactions, charge-dipole, and H bonding interactions between

nanosphere and charges on proteins

Hydrophilicity

0 1 2 3 4-4 -3 -2 -1

Tryptophan Cysteine LysineGlycine

Nanocluster Formation & Methods Exchange of lysine or cysteine on citrate done with 15 min place exchange reaction at room temperature

Particles incubated in 100% Fetal Bovine Serum (FBS) at 37°C for 48 hours before DLS run on sample

Zwitterions the most effective ligand

type in inhibiting protein adsorption

0.337nm

0.766nm

0.154nm

Ζ = -16.1 ± 2.9 mV Ζ = -21.6 ± 1.7 mV

1.6/1 Cysteine/citrate

monolayer

1.4/1 Lysine/citrate

monolayer

Neutral/Zwitterionic charged NPs show minimal protein adsorption

Less attraction to charged groups on proteins

Zwitterionic tips favor hydration and form a hydration layer on particle that inhibits interaction with proteins

Pure Anionic/Cationic NPs show significant adsorption

High electrostatic attraction to proteins

Charge-dipole interactions with H-donor/acceptor sites

Objective

Problem: Protein adsorption onto primary

nanospheres or nanoclusters in the bloodstream will

prevent efficient renal clearance from the body

Goal: Create mixed-charge ligand surface on Au

nanospheres to reduce surface charge and resist

protein adsorption in 100% FBS by depleting

electrostatic attraction through zwitterions

Nanoparticle Hydro-

dynamic Size

in Water

Hydro-

dynamic

Size in

PBS

Zeta

Potential 5

mM PB ph

7.4 (mV)

Zeta

Potential

Water (mV)

NP+ 9.7 ± 2.8 9.0 ± 2.6 16.9 ± 13.6 45.1 ± 10.0

NP- 6.9 ± 2.1 7.6 ± 1.8 -28.2 ± 4.9 -54.7 ± 13.6

ZMe 6.7 ± 1.1 7.9 ± 2.0 -6.8 ± 5.5 -17.9 ± 5.0

[5]

Sedimentation experiments for the series of

NPs in 55% plasma showing that NP's coated

with zwitterions (ZmeZDiPen) did not

aggregate, in contrast to NP+, NP-, and TEG

that formed pellets.

NP’s aggregate in serum, but those coated

with Zme show little aggregation in serum.

~5 nm

~

Addition of weakly adsorbing polymer reduces

electrostatic repulsion via charge screening;

also provides steric stabilization of resulting

asymmetric clusters

GSH/Cit Lys/CitCys/Cit

2nm gold cores coated with various charged or zwitterionic

end groups (ZmeZDiPen)

Incubated in 55% human serum to test proteins stability

Diluted sample to 1% human serum to allow particles to

overwhelm protein signal

Developed powerful solution for inhibiting protein

adsorption by enveloping Au nanospheres in a

mixed-charged monolayer of zwitterionic molecules

Charges on citrate are “buried” by longer lysine and

cysteine

Zwitterionic tips are exposed to proteins and

create a hydration layer

More hydrophilic molecules exposed

By allowing for efficient renal clearance, this method

of introducing zwitterions revolutionizes the

practicality of gold Au for targeted cancer treatment

and imaging

Gold nanoclusters with high near-IR absorbance

composed of primary nanoparticles have important

applications in the biomedical imaging of tumors

Soft tissues are relatively transparent in the

range of 650-950 nm and thus our goal is to

produce a high near-IR in this region

What makes Au suitable?

Both inert and exhibits surface plasmon

resonance (oscillation of electrons)

FBS & PBS-mimic protein concentration in body

Polymer that coats clusters designed to dissociate

at pH 5 into sub 5nm particles for renal clearance

Our Results

Future Work

Develop a ratio of Cysteine/GSH or Cysteine/Lysine

ligands that are stable at body temperature

Determine a minimum nanocluster size for effective

imaging

Initial 0 hr incubation 24 hr Incubation

100%

FBSGSH

5/1

Cys/GS

H

10/1

Cys/G

SH

100%

FBSGSH 5/1

Cys/GSH

10/1

Cys/GSH

Sedimentation

5nm GSH-Capped primary particlesLigand exchange with zwitterionic cysteine for 24hrs

at 60ºC

Incubated at 100% FBS for 24hrs then centrifuged at 15,000 rcf for 20min

Ran FAAS to determine Au concentration in supernatantSignificant stability with samples added Cysteine/GSH

Dilute FBS more accurate for tracking protein adsorption

Lysine/Citrate and Cysteine/Citrate

monolayers are relatively

hydrophilicgreater hydration, thus

less protein interaction

Glutathione Chemical

Structure