Elsevier

Sensors and Actuators B: Chemical

Volume 223, February 2016, Pages 157-165
Sensors and Actuators B: Chemical

Influence of alkane chain lengths and head groups on tethering of liposome–gold nanoparticle on gold surface for electrochemical DNA sensing and gene delivery

https://doi.org/10.1016/j.snb.2015.09.031Get rights and content

Abstract

1,2-Dioleoyltrimethylammoniumpropane (DOTAP) liposome–gold nanoparticle (DOTAP–AuNP) is tethered on different thiol monolayers with different chain lengths (carbon number n = 3 and 10) and head groups (acid and amine) on gold transducer for applications of DNA sensing, gene transfer and antibacterial activities. Gram −ve (Escherichia coli (E. coli)) and +ve (Staphylococcus aureus (S. aureus)) bacterial cells are used as model systems. The structure of planar and/or spherical liposome–AuNP is ascertained electrochemically in presence of transmembrane protein melittin and electroactive K3[Fe(CN)6]/K4[Fe(CN)6] redox couples. Results from transmission electron microscopy (TEM), ultraviolet–visible spectroscopy (UV–vis), dynamic light scattering (DLS) spectroscopic techniques and electrochemical data confirm the presence of more stable spherical structured DOTAP–AuNPs on the alkane thiols with amine head groups than on the acid terminal alkane thiols irrespective of chain length differences. Label free electrochemical DNA sensing in presence of K3[Fe(CN)6]/K4[Fe(CN)6] indicates large discrimination efficiency at concentrations greater than nano mole for the spherical DOTAP–AuNP on the aminoundecane thiol. DNA transfer into E. coli cells and anti-bacterial studies are made using gram −ve and +ve E. coli cells. The DNA transfer and antibacterial activities are confirmed using differential confocal laser scanning microscopy (CLSM).

Introduction

Liposomes are efficient non-immunogenic vesicles used for gene and drug delivery applications. Cationic 1,2-dioleoyltrimethylammoniumpropane (DOTAP) lipid attracts more interest for targeted DNA delivery application due to its ease of forming lipoplex by electrostatic interaction. Critical requirement is to form stable spherical liposome with enhanced circulation life time. The stability of the liposome surface is enhanced in the presence of poly(ethylene glycol) [1], [2] to form ‘stealth liposome’, however, the processes of endocytosis and release of DNA are decreased. This advantage is used in antibacterial drug delivery applications. For other cell types, removal of PEG from the liposome surface is essential for gene and drug delivery applications. Alternatively, biodegradable l-amino acid (peptide), antibodies, folate, nucleic acid and aptamer are utilized for liposome functionalization [3], [4], [5], [6], [7], [8], [9], [10]. Among these, aptamers [11] are used specifically for cell targeted drug delivery by Cell-SELEX method, however, the combination of PEG with aptamer [12], [13] results in complicated and expensive protocols. Recently, loading of silver and gold nanoparticles in dipalmitoylphosphatidylcholine (DPPC) liposome is investigated by spectral techniques and indicate that the association of nanoparticles at the corner edges of liposome. Further studies proved that integration of gold nanoparticles, which are neither toxic nor immunogenic [14], [15] provides an effective way for further functionalization of liposomes [16]. Therefore, gold nanoparticle protected DOTAP is further functionalized using hydrogel [17] for efficient and long term drug delivery in Staphylococcus aureus bacteria. The liposome is labeled with fluorescent dye Rhodamine B (RhB) and variation of fluorescent intensity indicates drug release efficiency. In this, hydrogel function is controlled by pH, similar to stimuli responsive liposome–gold nanoparticle reported by Pornpattananangkul et al. [18]. Other than AuNP [19], PEG and hydrogel polymers, Silica nano particles and DNA [20] are also employed to stabilize liposomes against fusion and to enhance the transfection efficiency, but these are demonstrated only in solution phase experiments. Still efforts continue to develop simple methods to form stable liposomes in solution phase. Recently, fabrication of miniaturized devices at the reduced reagent consumption and cost for multiple applications is of growing interest. In this context, the electronic behavior of liposome/lipid layers tethered on alkane thiols are studied intensively and postulated that the electronic properties are highly dependent on the spacer length of alkane chains [21], [22], [23], [24], [25], [26]; however, this is not investigated for liposome–nanoparticle composites on solid transducers. Recently, behavior of gold nanoparticle liposome has been investigated using short chain mercaptopropionic acid and cysteamine [27], [28], [29], but not studied on the longer chain length alkane thiols. Further, influence of trans membrane proteins on the behavior of the liposome–AuNP complex on different monolayers is also not addressed yet, which is essential to understand the nature of liposome–protein interaction electronically. In order to address all these issues, comparative studies of DOTAP–AuNP complex on both short and long chain length alkane thiols is made in the presence of trans membrane protein melittin. Further, complicated liposome immobilization and detaching protocols reported need to be simplified. Hence, in this work, short chain (number of carbons n = 3, hydrophilic mercaptopropionic acid and cysteamine) and long chain (number of carbons n = 10, hydrophobic mercaptoundecanoic acid and 11-amino-1-undecanethiol (AUT)) alkane thiols are used to tether cationic spherical liposome 1,2-dioleoyltrimethylammoniumpropane (DOTAP) stabilized by AuNP (DOTAP–AuNP) by simple electrostatic interaction. Formation of spherical liposome on these monolayers is ascertained in presence of trans membrane protein melittin and bench-mark redox probe [Fe(CN)6]3−/4−. Melittin interaction with DOTAP and DOTAP–AuNP is examined using the Barrel–Stave and Toroidal models proposed for lipid–protein interaction. The solid surface immobilized DOTAP–AuNP is applied for sensitive target DNA sensing, DNA transfer in gram −ve (Escherichia coli (E. coli)) and +ve (S. aureus) bacteria cells and antimicrobial studies, Scheme 1.

Section snippets

Materials and methods

Potassium ferrocyanide, potassium ferricyanide, sulfuric acid, hydrogen peroxide, sodium chloride, sodium dihydrogen phosphate and potassium chloride of analytical grade were purchased from Himedia, India. 3-Mercaptopropionic acid, cysteamine hydrochloride (Cyst) and mercaptoundecanoic acid, 1,2-dioleoyltrimethylammoniumpropane (DOTAP), Rhodamine B (RhB) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma–Aldrich, USA. Deionized water (DI) was used for preparing all experimental

Characterization of spherical liposome–gold nanoparticle tethered on thiol monolayer modified gold surface by physical and electrochemical methods

Electrochemical behaviors of the DOTAP–AuNP on an un-modified and thiol monolayer modified gold surfaces are evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Although different thiol monolayers are used to tether different lipid or liposome to form bilayers (tBLM) on the gold surface by electrochemical method, no comparative study of electronic behavior of the same lipid or liposome has been made so far. For this, DOTAP and AuNP are assembled by a

Conclusions

In this study, we showed that the use of citrate capped AuNP arrests the spherical structure of DOTAP on thiol monolayers on the solid surface and provides a simple method to tether DOTAP–AuNP on thiols layers having acid and amine functional groups by electrostatic attraction. This reduces complicated protocols used to tether lipids or liposome on thiol monolayer supports and prevents vesicle fusion. The results indicate that the alkane thiols with amine functional groups are better platform

Acknowledgements

Mohanlal Bhuvana acknowledges DST for the DST-INSPIRE fellowship (DST-IF-110327) and Dr. Venkataraman Dharuman is grateful to the Department of Science and Technology (DST, /EMR/2015/000118), New Delhi, Government of India for the project support.

M. Bhuvana received her Master Degree in Bioelectronics and Biosensors from Alagappa University, India in 2010 and currently doing her doctoral program under the guidance of Dr. V. Dharuman, in the Department of Bioelectronics and Biosensors, Alagappa University, India. Her research interests focus on the development of liposome platforms on solid surface for DNA, sensing and drug delivery applications.

References (50)

  • F. Arduini et al.

    Acetylcholinesterase biosensor based on self-assembled monolayer-modified gold-screen printed electrodes for organophosphorus insecticide detection

    Sens. Actuators B

    (2013)
  • J. Takei et al.

    Generalised bilayer perturbation from peptide helix dimerisation at membrane surfaces: vesicle lysis induced by disulphide-dimerised melittin analogues

    FEBS Lett.

    (1999)
  • L. Yang et al.

    Barrel-stave model or toroidal model? A case study on melittin pores

    Biophys. J.

    (2001)
  • M.T.N. Campanha et al.

    Interactions between cationic liposomes and bacteria: the physical-chemistry of the bactericidal action

    J. Lipid Res.

    (1999)
  • M. Stepniewski et al.

    Study of PEGylated lipid layers as a model for PEGylated liposome surfaces: molecular dynamics simulation and langmuir monolayer studies

    Langmuir

    (2011)
  • W. Yan et al.

    Recent advances in liposome-based nanoparticles for antigen delivery

    Polym. Rev.

    (2007)
  • P. Sapra et al.

    Internalizing antibodies are necessary for improved therapeutic efficacy of antibody-targeted liposomal drugs

    Cancer Res.

    (2002)
  • Y. Lu et al.

    Folate targeting of haptens to cancer cell surfaces mediates immunotherapy of syngeneic murine tumors

    Cancer Immunol. Immunother.

    (2002)
  • C.P. Leamon et al.

    Folate–liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo

    Bioconjug. Chem.

    (2003)
  • D. Shangguan et al.

    Aptamers evolved from live cells as effective molecular probes for cancer study

    Proc. Natl. Acad. Sci. U.S.A.

    (2006)
  • H. Kang et al.

    A liposome-based nanostructure for aptamer directed delivery

    Chem. Commun. (Cambridge)

    (2010)
  • E.E. Connor et al.

    Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity

    Small

    (2005)
  • G.F. Paciotti et al.

    Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery

    Drug Deliv.

    (2004)
  • W.K. Rhim et al.

    Lipid–gold-nanoparticle hybrid-based gene delivery

    Small

    (2008)
  • W. Gao et al.

    Hydrogel containing nanoparticle-stabilized liposomes for topical antimicrobial delivery

    ACS Nano

    (2014)
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    M. Bhuvana received her Master Degree in Bioelectronics and Biosensors from Alagappa University, India in 2010 and currently doing her doctoral program under the guidance of Dr. V. Dharuman, in the Department of Bioelectronics and Biosensors, Alagappa University, India. Her research interests focus on the development of liposome platforms on solid surface for DNA, sensing and drug delivery applications.

    Dr. V. Dharuman received his Ph.D degree in electrochemistry from University of Madras, India, in 2002. He worked as a research scientist at the Department of Biotechnical Microsystems, Fraunhofer Institute for Silicon Technology, Germany, from 2001 to 2004. He then worked as a postdoctoral research scientist at the Laboratory for Advanced Biotechnology and Biomedical Micro-Instrumentation, Biotech Centre, Department of Chemistry, Pohang University of Science and Technology, South Korea, from December 2004 to May 2008. He is an Assistant Professor in the Department of Bioelectronics and Biosensors, Alagappa University, Karaikudi, India since 2008. His research interests include development of DNA hybridization label free electrochemical sensing, lipid behaviors on solid surfaces for biosensing and targeted drug delivery, nano-biotechnology and lab-on-a-chips.

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