Effects of nanoparticle size and charge on interactions with self-assembled collagen

doi:10.1016/j.jcis.2013.11.019

Journal of Colloid and Interface Science

Volume 417, 1 March 2014, Pages 244-249

https://doi.org/10.1016/j.jcis.2013.11.019 Get rights and content

Highlights

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QCM-D to probe interaction of nanoparticles and a biomimetic collagen substrate.
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All charged nanoparticles likely associate with collagen.
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Association of nanoparticles with collagen is not charge polarity dependent.
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Very small nanoparticles (2 nm) were found to mechanically integrate with collagen.
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Larger nanoparticles particles bound to the surface of collagen.

Abstract

Hypothesis

Insights into bone formation have suggested that the critical first step in the biomineralization process is the integration of small (nanometer dimension) mineral clusters into collagen fibers. Not only is such behavior of interest for understanding biomineralization but also should be important to nanotoxicology because collagen is a major component of structural tissues in the human body and accounts for more than 25% of the whole body protein content. Here, utilizing the current insights from biomineralization, we hypothesize that the binding affinity of nanoparticles to self-assembled collagen fibers is size and surface charge dependent.

Experiments

We developed a self-assembled collagen substrate compatible with Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), which is very sensitive to mechanical changes of the substrate as a consequence of nanoparticle binding. QCM-D experiments were conducted with both positively and negatively charged gold nanoparticles between 2 and 10 nm in size. Complementary ex situ imaging Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) were used to confirm the QCM-D results.

Findings

We find that both positively and negatively charged nanoparticles of all sizes exhibited binding affinity for self-assembled collagen fibers. Furthermore, the smallest particles (2 nm) mechanically integrated with collagen fibers.

Graphical abstract

Introduction

Collagen is the principal protein component of many tissues including bone, connective tissue and skin and provides critical support to their exceptional mechanical properties [1], [2]. Collagen self-assembles to form fibers (hundreds of nanometers in diameter) with 67 nm repeating band structures consisting of less dense gap and more dense overlap regions [3]. Recent results suggest that during the biological mineralization process, the interior of collagen fibers is infiltrated by nanometer sized amorphous calcium phosphate clusters [4], [5], which ultimately crystallize to form the mineral apatite. These calcium phosphate clusters are characterized by (1) their small size, generally less than 10 nm, (2) their negative surface charge, [4] and (3) their fluid like character commonly referred to as the Polymer Induced Liquid Precursor (PILP) [6]. Due to the abundance of collagen in the human body, the nature of nanoparticle interactions with collagen fibrils is of fundamental interest in at least two perspectives: (1) biomimetic materials synthesis: how do CaP nanoparticles bind to and integrate with collagen fibers? And (2) toxicology: how do nanoparticles interact with matrix biomolecules?

Here, we hypothesize that in the first step to biomineralization, the interaction between nanoparticles (NP) with self-assembled collagen fibers is driven by the charge of the NP. We further test NP size dependent association with collagen. We developed a model system with gold nanoparticles (AuNP), which are chemically and colloidally stable to study the nature of collagen–AuNP interaction (amount, spatial distribution and reversibility) as a function of particle size and surface charge polarity. Here, we use AuNP ranging from 2 nm to 10 nm in size and possessing positive or negative charges. To study the nanoparticle/collagen binding process, we used Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) along with Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). QCM-D has been used extensively to quantify binding and subsequent conformation changes of proteins [7], [8], other macromolecules [9], nanoparticles [10] binding to surfaces, as well as mineralization processes at surfaces [11].

The findings of this study have potential applications in nanoparticle toxicology, where understanding the fate of nanoparticles in the body after exposure is important [12]. Because of the prevalence of collagen as a structural protein throughout the body, understanding the role of nanoparticle size and charge governed interaction with collagen is essential.

Section snippets

Preparation of QCM-D sensors

To clean the gold-coated QCM-D sensors (QSX-301, Biolin Scientific) they were exposed to Ultraviolet Ozone (UVO, Jelight) for 10 min; soaked in a solution composed of Deionized Water (DI) water (Milli-Q), hydrogen peroxide (30%, Fisher Scientific) and ammonium hydroxide (Sigma–Aldrich), at a proportion of 5:1:1, respectively, at 70 °C for 5 min, N₂ dried and finally exposed to UVO for 10 min. Afterward, the QSX-301 sensors were made hydrophobic by exposure to 1 mmol/L dodecane thiol (Sigma–Aldrich)

Results and discussion

AFM was used to characterize self-assembled collagen fibers on functionalized gold QCM-D sensors, which exhibited a bimodal distribution of fiber diameters of approximately 15 nm and approximately 100 nm (Fig. S1). The smaller fibers formed a dense mat on the surface of the QCM-D sensor, with larger fibers overlying these structures at a lower areal density, consistent with previous findings [13], [15]. The larger fibers are more characteristic of hierarchically assembled type I collagen fibrils

Conclusions

We have developed a QCM-D based approach for investigating nanoparticle binding to self-assembled collagen fibers, a model for structural proteins. It was demonstrated that nanoparticles of both negative and positive charge polarities have affinity for collagen fibers. The smallest tested 2 nm particles produced a stiffening response in the system that is sudden and particle density dependent, suggesting a percolation threshold. Additionally, imaging data demonstrates that nanoparticle binding

Funding sources

This work is in part supported by an Interagency Agreement Y1-DE-7005-01 between the National Institute of Standards and Technology (NIST) and the National Institute of Dental and Craniofacial Research (NIDCR).

Notes

Official contribution of NIST; not subject to copyrights in USA. Certain commercial materials and equipment are identified in this article to specify the experimental procedure. In no instance does such identification imply recommendation or endorsement by NIST or that the material or equipment identified is necessarily the best available for the purpose.

Acknowledgments

D.W. acknowledges the NIST-NRC Research Associate Program for support. We thank N.J. Lin for helpful discussions and comments.

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Published by Elsevier Inc.

Effects of nanoparticle size and charge on interactions with self-assembled collagen

Highlights

Abstract

Hypothesis

Experiments

Findings

Graphical abstract

Introduction

Section snippets

Preparation of QCM-D sensors

Results and discussion

Conclusions

Funding sources

Notes

Acknowledgments

J. Orthopaed. Res.

Biomaterials

Biomaterials

J. Struct. Biol.

Micron

Biomaterials

Compos. Sci. Technol.

Nat. Mater.

Proc. Natl. Acad. Sci.

Nat. Mater.

Polymers

Anal. Chem.

Langmuir

Environ. Sci. Technol.