Environmentally compatible bioconjugated gold nanoparticles as efficient contrast agents for colorectal cancer cell imaging

https://doi.org/10.1016/j.snb.2014.02.008Get rights and content

Abstract

In this study we show, for the first time, that gold nanoparticles (AuNPs) synthesized by a simple, inexpensive, and environmentally-correct method can be easily conjugated with the antibodies anti-β-catenin and anti-E-cadherin to specifically target colorectal carcinoma cells. The antibody/AuNPs conjugates were then successfully applied for imaging cancerous cells with fluorescence confocal microscopy. The AuNPs as well as the conjugates were very stable in high-salinity medium, a pre-requisite for application in physiological-like environments. Fluorescence results suggest that conjugation was achieved by direct adsorption of antibodies on the AuNPs surface. Finally, compared with a standard method of cell staining, our method is less laborious and the preparation time (from immobilization of cells onto glass cover slips until observation by confocal microscopy) decreased from 27 h to about 1 h, which makes the method eligible for colorectal cancer diagnostic.

Introduction

Current cancer diagnostic methodologies include optical imaging techniques such as coherence tomography and fluorescence confocal microscopy. The image is generated by the backscattered light from the endogenous chromophores present in the tissue. The weak optical signal results in poor intracellular contrast and subtle spectral differences between malignant and benign cells hamper a proper diagnostic [1]. In order to overcome these problems, exogenous chemicals (mainly organic dyes) that function as optical contrast agent have been used to enhance the visibility of both precancerous and cancerous cells. However, organic dyes are subjected to rapid photobleaching [2], therefore becoming inefficient as the analysis proceeds. Other disadvantages of conventional dyes include poor hydrophilicity, low quantum yield, and low detection sensitivity in biological environment [2].

Recent advances of nanotechnology have allowed the development of new functional nanomaterials with enhanced specificity for molecular imaging. In particular, colloidal Au nanoparticles (AuNPs) are extremely advantageous as contrast agents in biomedicine [1], [3], [4], [5], [6] as they can be easily combined with a recognition agent. First, AuNPs present a size-tunable surface plasmon resonance (SPR) that leads to strong absorption and scattering in the visible-to-near-infrared region, a characteristic which makes them considerably superior to the conventional dyes applied in biomedical imaging. For instance, the plasmon resonance absorption of spherical gold nanoparticles has an absorption coefficient orders of magnitude larger than those of regular dyes. As an example the absorption coefficient (ɛ) for 40-nm gold nanoparticles [7] at a wavelength of 530 nm was calculated to be ∼7.7 × 109 M−1 cm−1, while the ɛ of rhodoamine 6G is 1.16 × 105 M−1 cm−1 [8]. Second, AuNPs do not suffer photobleaching and seem to be biocompatible [9]. Third, the chemistry of gold is quite rich. The AuNPs surface binds strongly with amines, thiols and disulfides, allowing the surface bioconjugation with a great variety of peptides, DNA and proteins, enabling selective targeting of cancer cells. Furthermore, in opposition to dyes and organic molecules, several functional groups can be attached onto the same nanoparticle, making them also eligible for drug delivery [10].

In this paper we synthesized AuNPs bioconjugated with β-catenin antibody (anti-β-catenin) and E-cadherin antibory (anti-E-cadherin) and applied them as novel contrast agents for the detection of colorectal cancerous cells with fluorescence confocal microscopy. E-cadherin is localized on the surface of epithelial cells in regions of cell–cell contact known as adherens junctions and is implicated in cell–cell adhesion in epithelial tissues [11], [12], [13]. β-Catenin interacts with cadherins through its cytoplasmic domain. α-catenin connects the E-cadherin and β-catenin complex to actin filaments. The dissociation of E-cadherin-catenin complex from cell membrane is important in malignant progression. In many epithelial cancers, membranous E-cadherin is lost and β-catenin dissociates in the cytoplasm and accumulates in the nucleus as a transcription factor, concomitantly with tumor progression [12]. Down-regulation of membranous E-cadherin and β-catenin, and cytoplasmic/nuclear accumulation of β-catenin have been previously reported in several cancers and hold promise as prognostic markers [14]. In humans β-catenin and E-cadherin are encoded by the adenomatous polyposis coli (APC) and CDH1 genes, respectively, and their mutation may result in colorectal cancer leading to an overexpression of β-catenin and E-cadherin by the cell [11], [12], [13], [14], [15], [16]. The strategy was therefore to combine the specificities of anti-β-catenin and anti-E-cadherin with the fluorescent properties of the AuNPs to produce a selective contrast agent for targeting cancer. In this system, the AuNPs function as the signaling part and the anti-β-catenin and anti-E-cadherin act as recognition units for specific binding with the overexpressed β-catenin and E-cadherin in the cell. The confocal microscopy revealed that the antibody-conjugated AuNPs were very efficient in marking cancerous cells. On the other hand, no specific interaction between normal cells and conjugated AuNPs was observed. Another interesting feature of the present work is the use of an environmentally friendly route to produce the AuNPs based on the reduction of gold ions by glycerol in alkaline medium. Glycerol's non-toxicity and biodegradability make it an excellent alternative to commonly used reducing agents. The conjugations with anti-β-catenin and anti-E-cadherin are achieved by simple incubation after reducing the pH of the nanoparticle colloidal solution to around 7. In comparison to current methods for production and conjugation of AuNPs, our method is quite simple, low-cost and environmentally compatible, with a potential to be routinely applied in cancer diagnostics.

Section snippets

Chemicals and reagents

Gold trichloride (30 wt% in HCl), polyvinylpyrrolidone (PVP, MW = 10.000), sodium hydroxide, dialysis tubing cellulose membrane, and glycerol were products of Sigma-Aldrich Chemical Co. Sulphuric acid and hydrogen peroxide were purchased from Vetec. Phosphate-buffered saline (PBS) solution and bovine serum albumine (BSA, 5%) were purchased from Life Technologies Corporation©. β-Catenin antibodies (anti-β-catenin) and E-cadherin antibodies (anti-E-cadherin) were acquired from ABCAM® at the

Results and discussion

The AuNPs used in this work were synthesized by reducing Au3+ with glycerol in alkaline medium. Glycerol is an inexpensive chemical and readily biodegradable under aerobic conditions, therefore a greener option when compared to current reducing chemicals such as formamide, sodium borohydride and hydrazine. Sodium citrate [19] is efficient in generating gold nanoparticles, however they are formed only upon heating the solution (80–100 °C). Fig. 1A and B shows a UV–vis spectrum and a

Conclusions

Anti-β-catenin/AuNPs and anti-E-cadherin/AuNPs conjugates were successfully applied for imaging colorectal cancerous cells. UV–vis results showed that the conjugates are stable in solutions that resemble the physiological medium. Based on fluorescence results, low antibody concentrations are sufficient to achieve maximum adsorption of antibodies onto the surface of the AuNPs. Furthermore, the herein proposed conjugates had similar performance compared with a standard procedure, with the

Acknowledgments

The authors are grateful to CNPq (grant 471794/2012-0), Capes (grant Capes/070/2012), and FAPERN/MCT/CNPq/CT-INFRA 005/2011 for the financial support. We also thank Dr. Celso Sant́Anna (Inmetro) for the help with TEM images.

Kássio M.G. Lima is an assistant professor at the Institute of Chemistry of the Federal University of Rio Grande do Norte since 2009. He received his Ph.D. in sciences (2009) from UNICAMP (Brazil) and he was sandwich-doctorate fellow (09/2007–09/2008) at Universidad Complutense de Madrid in the Optical Fibre Chemical Sensors and Applied Photochemistry Group (supervised by Dr. M.C. Moreno-Bondi) working on simultaneous determination of heavy metals with optical sensors. He is the leader of the

References (26)

  • P.K. Jain et al.

    Nano Today

    (2007)
  • S. Luo et al.

    Biomaterials

    (2011)
  • A.C.A. Silva et al.

    Sens. Actuators, B

    (2014)
  • S. Parveen et al.

    Nanomed. Nanotechnol. Biol. Med.

    (2012)
  • F.H. Brembeck et al.

    Curr. Opin. Genet. Dev.

    (2006)
  • L.H.S. Gasparotto et al.

    J. Power Sources

    (2012)
  • N. Vigneshwaran et al.

    Carbohydr. Res.

    (2006)
  • R.G. Rayavarapu et al.

    Int. J. Biomed. Imaging

    (2007)
  • J. Gao et al.

    Langmuir

    (2012)
  • G.P. Luke et al.

    Nanotechnology

    (2013)
  • P.K. Jain et al.

    J. Phys. Chem. B

    (2006)
  • H. Du et al.

    Photochem. Photobiol.

    (1998)
  • C.J. Murphy et al.

    Acc. Chem. Res.

    (2008)
  • Cited by (0)

    Kássio M.G. Lima is an assistant professor at the Institute of Chemistry of the Federal University of Rio Grande do Norte since 2009. He received his Ph.D. in sciences (2009) from UNICAMP (Brazil) and he was sandwich-doctorate fellow (09/2007–09/2008) at Universidad Complutense de Madrid in the Optical Fibre Chemical Sensors and Applied Photochemistry Group (supervised by Dr. M.C. Moreno-Bondi) working on simultaneous determination of heavy metals with optical sensors. He is the leader of the Research Group of Applied Chemometrics (UFRN, Brazil) since 2011 and his current research interests are on spectroscopy and chemometrics.

    Raimundo F. Araújo Júnior holds a position of assistant professor at the Morphology Department of the Federal University of Rio Grande do Norte, Brazil, since 2006. He got his Ph.D. in Health Sciences in 2005. His interests are molecular biology to oncology. The focus of his work is to observe the apoptosis and proliferation of cancerous cells upon contact with nano engineered and phototherapeutic drugs.

    Aurigena A. Araujo works as assistant professor at the Department of Biophysics and Pharmacology of the Federal University of Rio Grande do Norte, Brazil, since 2004. She got her Ph.D. in Health Sciences in 2005. She was a post-doc fellow at the Universidad Autónoma de San Luís Potosí, Mexico, in 2007. Her research line is the search for new medicines for the treatment of inflammatory process.

    Ana Luiza C. S. L. Oliveira is a Ph.D. student in Pharmaceutical Science/Public Health at Federal University of Rio Grande do Norte, Brazil. The focus of her work is to correlate the expression of the E-cadherin protein with pathological variables in colorectal carcinoma. She is also interested in studying the viability of cancer cells upon treatment with phytotherapeutic extracts.

    Currently Luiz Henrique da Silva Gasparotto holds a position of assistant professor at University of Rio Grande do Norte, Brazil. He obtained his Ph.D. in Sciences in 2009, followed by a post-doc stage at TU Clausthal, Germany, under the supervision of Prof. Frank Endres. Gasparotto's interests comprise the synthesis and application of nanomaterials obtained through chemical and electrochemical routes.

    View full text