Exploiting Protein Corona around Gold Nanoparticles Conjugated to

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Exploiting Protein Corona around Gold Nanoparticles Conjugated to p53 Activating Peptide to Increase the Level of Stable p53 Proteins in Cells Kian Ping Chan, Sheng-Hao Chao, and James Chen Yong Kah Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00032 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 18, 2019

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Bioconjugate Chemistry

Exploiting Protein Corona around Gold Nanoparticles Conjugated to p53 Activating Peptide to Increase the Level of Stable p53 Proteins in Cells Kian Ping Chan1,2,3, Sheng-Hao Chao2,4*, James Chen Yong Kah1,5*

1NUS

Graduate School for Integrative Sciences and Engineering, Centre for Life

Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456

2Bioprocessing

Technology Institute, Agency for Science, Technology and Research,

Singapore, 20 Biopolis Way, #06-01 Centros, Singapore 138668

3Present

Address: NanoBio Lab, 31 Biopolis Way, #09-01 The Nanos, Singapore

138669

4Department

of Microbiology and Immunology, National University of Singapore, 5

Science Drive 2, Blk MD4, Level 3, Singapore 117597

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

5Department

of Biomedical Engineering, National University of Singapore, 4

Engineering Drive 3, Blk E4, #04-08, Singapore 117583

ABSTRACT

Therapeutic peptides suffer from major drawbacks such as peptide degradation in vivo due to proteolysis. Gold nanoparticles (AuNPs) are an effective carrier for therapeutic peptides that improve their stability in vivo, while also enabling non-specific adsorption of complementary proteins to enhance their effectiveness. Using p53 peptide as a model known to disrupt the intracellular MDM2-p53 protein-protein interaction which tags the endogenous p53 proteins for degradation, we conjugated p53 peptides to AuNPs (AuNP-p53) and examined the functionality of AuNP-p53 to release the endogenous p53 proteins from being tagged for degradation, thereby increasing the level of stable p53 proteins in acute myeloid leukemia 2 (AML2) cells. We found that


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AuNPs did not just protect conjugated p53 peptides from trypsin degradation, but also helped to recruit 56.5% and 26.4% of total MDM2 and p53 proteins in the cells to form a protein corona around AuNP-p53. The proximity of MDM2/p53 complexes and p53 peptide on the surface of AuNP-p53 facilitated the action of p53 peptide to cause a sustained elevation of p53 level in AML2 cells up to 6 hours, which was not possible with free p53 peptide alone at the same concentration. Even a 20-fold higher concentration of free p53 peptide caused only a short-lived elevated p53 level of 1 hour. The outcome of this study highlights the utility of combining conjugated ligands and complementary protein adsorption on nanoparticles to improve the biological functionality of the therapeutic ligands.

INTRODUCTION



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Protein-protein interactions are crucial for many biological processes in cells, including degradation of proteins such as p53 to maintain cellular homeostasis. Mouse double minute 2 homolog (MDM2) is an important protein to maintain cell homeostasis by interacting with p53 protein, a tumor suppressor

1, 2,

and tagging it for proteasome-

mediated degradation 3. MDM2 functions as an E3 ubiquitin ligase, with its activity mainly found in the evolutionarily conserved C-terminus RING finger domain to transfer ubiquitin tags onto six lysine residues found in the C-terminus of p53 4. MDM2 is often up-regulated in cancer cells, which binds wild type p53 and targets them for degradation 5, 6

to facilitate uncontrolled cellular proliferation.

Disassociation of MDM2/p53 complexes by disrupting MDM2-p53 protein-protein interaction could be achieved with small therapeutic molecules

7-12

or peptides

13-17,

which stabilize and activate the p53 proteins for downstream anti-tumor signaling cascade. Specifically, a p53 activating peptide, corresponding to residues 361 to 382 in the p53 C terminus region and containing the six lysine residues ubiquitinated by MDM2, could interact with MDM2 and disrupt its interaction with wild type p53 proteins,


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leading to the disassociation of MDM2/p53 complexes and hence prevent degradation of p53 13, 18. However, free peptides are susceptible to proteolytic degradation and hence limiting their bioavailability at target site

19, 20.

Gold nanoparticles (AuNPs) have been shown to

protect conjugated peptides and preserve their functions against proteolysis by trypsin 21.

Unfortunately, their high surface energy arising from their nanoscale dimension could

also non-specifically adsorbs other proteins in the cells to form a layer of protein corona which is often deemed undesirable

22-24

as the corona masks the intended functionality

of the peptides on the surface of AuNPs. Our group have demonstrated that protein corona could also be exploited for biological applications including enabling protein quantification uptake

26, 27,

synthesis

enabling drug loading and triggered release

32-34

28-31

25,

improving cellular

and enhancing protein

instead of viewing it as a biological artifact. Here, we extend the concept

of utilizing the protein corona by demonstrating that the conjugation of p53 peptides to AuNPs to form AuNP-p53 does not merely protect p53 peptides from proteolytic degradation, but also simultaneously recruited MDM2/p53 complexes through their non5 ACS Paragon Plus Environment


specific adsorption on the AuNP-p53 to form a part of the protein corona which bring the complexes into close proximity of p53 peptides. The p53 peptides subsequently dissociated p53 proteins from MDM2 more efficiently and consequently resulted in a sustained elevated level of stable p53 proteins in acute myeloid leukemia 2 (AML2) cells (Figure 1), peaking at 6 h post-incubation. Even with a 20-fold higher free peptide concentration that could similarly trigger an elevated level of stable p53 proteins, the elevated p53 level was short-lived, lasting not more than 1 h. Our findings introduce for the first time, the concept of utilizing the non-specific adsorption of protein-protein complexes on peptide-functionalized AuNPs to improve the efficiency and effectiveness of the mode of action of the conjugated peptide on the complex. This is different from other studies where the role of AuNPs as a delivery vehicle for intracellular peptide delivery to enhance biological processes is already widely established

35, 36.

The outcome is significant in its applicability to improve the

efficacy of other therapeutic peptide/protein models with similar mechanism of actions based on peptide-induced dissociation of protein-protein complexes as a treatment target of diseases such as cancers. 6 ACS Paragon Plus Environment

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Figure 1. Schematic showing the use of AuNP to protect p53 peptides and increase the level of stable endogenous p53 proteins in cells. (i) MDM2 interacts with endogenous p53 protein, forming a MDM2/p53 complex, and targets it for (ii) nucleus export into cytoplasm where (iii) MDM2 is capable of ubiquitinating p53 and (iv) allowing proteasomes to recognize and degrade p53 proteins. (v) Free p53 therapeutic peptides are able to interact with N-terminal of MDM2 to release endogenous p53 protein from 7 ACS Paragon Plus Environment


degradation, but (vi) are themselves susceptible to protease degradation. (vii) p53 peptides conjugated on AuNPs are protected from proteolytic degradation while at the same time, the AuNPs recruit MDM2/p53 complexes to their surface during protein corona formation where (viii) conjugated p53 peptides interact with N-terminal of MDM2 to (ix) release the p53 proteins from degradation.

RESULTS AND DISCUSSION

Characterization of peptide conjugated AuNPs The synthesized citrate-capped AuNPs were monodispersed with an average diameter of 12.12 ± 0.13 nm as observed under TEM (Figure 2a, b). Conjugation of p53 peptides to AuNPs was performed at low pH and high peptide to AuNP ratio (see Supporting Information, Figure S1) to yield AuNP-X13 and AuNP-p53. Conjugation of positively-charged peptides (at pH below their isoelectric point) to negatively-charged AuNPs would result in charge compensation and destabilized AuNPs. Thus, using a high peptide to AuNP ratio would ensure that AuNPs remained colloidally stable after peptide conjugation 37. 8 ACS Paragon Plus Environment

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Figure 2. Characterization of synthesized citrate-capped AuNPs and their conjugates with p53 peptides (AuNP-p53) and negative control X13 peptides (AuNP-X13). (a) Citrate-capped AuNPs were mono-dispersed as imaged under TEM and (b) had an average diameter of 12.12 ± 0.13 nm. (c) Absorbance spectra measured using UV-Vis spectroscopy showed a red-shift in peak absorbance from 520 nm (AuNP) to 528 nm (AuNP-X13) and 525 nm (AuNP-p53), an indication of successful conjugation. (d) Hydrodynamic diameter (DH) distribution of AuNPs, AuNP-X13 and AuNP-p53



determined by dynamic light scattering (DLS) showed a monomodal size distribution with size increment and no large aggregates upon conjugation of X13 or p53 peptides. (e) A flip in zeta potential, ζ, indicated a transition from negatively-charged citrate coating to a positively-charged peptide coating (n=3). (f) AuNP-X13 and AuNP-p53 have approximately 960 and 534 peptides per AuNP respectively after conjugation. *One tail t-test, p

Exploiting Protein Corona around Gold Nanoparticles Conjugated to

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