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Open AcceResearchHydrophobic silver nanoparticles trapped in lipid bilayers: Size distribution, bilayer phase behavior, and optical propertiesGeoffrey D Bothun

Address: Department of Chemical Engineering, University of Rhode Island, Kingston, RI, 02881, USA

Email: Geoffrey D Bothun - bothun@egr.uri.edu

AbstractBackground: Lipid-based dispersion of nanoparticles provides a biologically inspired route todesigning therapeutic agents and a means of reducing nanoparticle toxicity. Little is currently knownon how the presence of nanoparticles influences lipid vesicle stability and bilayer phase behavior. Inthis work, the formation of aqueous lipid/nanoparticle assemblies (LNAs) consisting of hydrophobicsilver-decanethiol particles (5.7 1.8 nm) embedded within 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers is demonstrated as a function of the DPPC/Ag nanoparticle(AgNP) ratio. The effect of nanoparticle loading on the size distribution, bilayer phase behavior, andbilayer fluidity is determined. Concomitantly, the effect of bilayer incorporation on the opticalproperties of the AgNPs is also examined.

Results: The dispersions were stable at 50C where the bilayers existed in a liquid crystalline state,but phase separated at 25C where the bilayers were in a gel state, consistent with vesicleaggregation below the lipid melting temperature. Formation of bilayer-embedded nanoparticles wasconfirmed by differential scanning calorimetry and fluorescence anisotropy, where increasingnanoparticle concentration suppressed the lipid pretransition temperature, reduced the meltingtemperature, and disrupted gel phase bilayers. The characteristic surface plasmon resonance (SPR)wavelength of the embedded nanoparticles was independent of the bilayer phase; however, the SPRabsorbance was dependent on vesicle aggregation.

Conclusion: These results suggest that lipid bilayers can distort to accommodate largehydrophobic nanoparticles, relative to the thickness of the bilayer, and may provide insight intonanoparticle/biomembrane interactions and the design of multifunctional liposomal carriers.

BackgroundHybrid lipid/nanoparticle conjugates provide a biologi-cally inspired means of designing stable agents for bio-medical imaging, drug delivery, targeted therapy, andbiosensing [1]. An advantage of using lipids as stabilizingor functional ligands is that they mimic the lipidic scaf-folding of biological membranes and have well-character-ized physicochemical properties and phase behavior. Inlipid vesicles, nanoparticle encapsulation can be achieved

by trapping particles within the aqueous vesicle core orwithin the hydrophobic lipid bilayer. Becker et al [2], Kimet al [3], and Zhang et al [4] have shown that iron oxide(Fe3O4), cadmium selenide (CdSe) quantum dots, andgold nanoparticles, respectively, can be trapped withinaqueous vesicle cores. To embed nanoparticles withinlipid bilayers, the nanoparticle must be small enough tofit within a DPPC bilayer and it must present a hydropho-bic surface. Using physisorbed stearylamine, Park et al

Published: 12 November 2008

Journal of Nanobiotechnology 2008, 6:13 doi:10.1186/1477-3155-6-13

Received: 2 July 2008Accepted: 12 November 2008

This article is available from: http://www.jnanobiotechnology.com/content/6/1/13

2008 Bothun; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Nanobiotechnology 2008, 6:13 http://www.jnanobiotechnology.com/content/6/1/13

[5,6] have stabilized 34 nm gold and silver particles in1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)bilayers. Likewise, Jang et al [7] embedded 2.53.5 nm sil-icon particles with chemisorbed 1-octanol into bilayermembranes composed of DOXYL-labeled phospho-choline lipids. The resulting vesicles are analogous to lipo-somal drug delivery systems with an added functionalnanoparticle component.

For hydrophobic nanoparticles embedded within lipidbilayers, which is the focus of this work, the presence ofnanoparticles can lead to changes in lipid packing andmay disrupt lipid-lipid interactions amongst the head-groups and/or acyl tails [5,6]. Disruption of such inter-lipid interactions can result in changes in lipid bilayerphase behavior, which is related to the degree of lipidordering and bilayer viscosity. Hence, depending on theirsize and surface chemistry, embedded nanoparticles mayinfluence the stability and function of hybrid vesicles, aswell as the conditions required for preparation.

This work demonstrates the formation of hybrid lipid/nanoparticle assemblies (LNAs) containing hydrophobicdecanethiol-modified silver nanoparticles (Ag-

decanethiol) and the effect of embedded nanoparticles onbilayer structure. An illustration of a vesicle assembly isshown in Figure 1 (not to scale). DPPC, a zwitterionicphospholipid with dual saturated C16 tails, was chosen forthis study as a model lipid system because of its well-char-acterized phase behavior [8]. Vesicle size, stability, andbilayer phase behavior were examined as a function ofnanoparticle loading and temperature. Ag LNAs were alsoformed with a mixture of DPPC and 1,2-dipalmitoyl-sn-glycero-3- [phospho-L-serine] (DPPS), an anionic phos-pholipid, to investigate the effect of vesicle charge andaggregation on the Ag SPR wavelength.

MethodsChemicalsDPPC and DPPS (>99%) were obtained from Avanti PolarLipids, and chloroform and tetrahydrafuran (THF) fromFisher Scientific (>99.9%). Diphenylhexatriene (DPH)and Ag-decanethiol nanoparticles (AgNPs) dispersed inhexane (0.1 wt%) were obtained from Sigma-Aldrich. Anaverage nanoparticle diameter of 5.7 1.8 nm was meas-ured by transmission electron microscopy (JOEL JEM1200EX) using ImageJ analysis software [9] (Figure 2).Dulbecco's 150 mM phosphate buffered saline (PBS) was

A lipid/nanoparticle assembly (LNA) containing hydrophobic nanoparticles embedded within vesicle bilayersFigure 1A lipid/nanoparticle assembly (LNA) containing hydrophobic nanoparticles embedded within vesicle bilayers. This illustration, which was adapted from Jang et al [7], depicts the incorporation of nanoparticles that have been surface mod-ified with hydrophobic tails (e.g. decanethiol, shown in gray) into a lipid bilayer. Lipid disordering and bilayer disruption will be dependent on the size and surface chemistry of the nanoparticles. The image is not to scale.

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Journal of Nanobiotechnology 2008, 6:13 http://www.jnanobiotechnology.com/content/6/1/13

prepared at pH 7.4 with sterile deionized water from aMillipore Direct-Q3 UV purification system.

DPPC/AgNP and DPPC/DPPS/AgNP assembly formationLipid assemblies were prepared in PBS at 1 and 30 mMDPPC using the Bangham method [10]. The 1 mM DPPCsamples were prepared for fluorescence anisotropy meas-urements using DPH as a bilayer probe molecule. In thesesamples, the AgNP concentration was varied from 1 to1000 mg/L to provide DPPC/AgNP ratios from 734:1 to1:1 (w/w), respectively. The 30 mM DPPC samples wereprepared for differential scanning calorimetry (DSC) anddynamic light scattering (DLS) studies. For these samples,the AgNP concentration was varied from 0.1 to 11.0 g/L toprovide DPPC/AgNP ratios from 200:1 to 2:1 (w/w). Toform the LNAs, an aliquot of the Ag-decanethiol NP/hex-ane solution was added to DPPC dissolved in chloroformto yield a transparent, miscible brown phase. For anisot-ropy measurements, an aliquot of DPH in THF was alsoadded at a DPPC to DPH molar ratio of 500:1. The solventphase was evaporated under nitrogen and the sample wasplaced under vacuum for 2 hours, leaving a dry DPPC/AgNP film. Hydration and processing steps were per-formed at 50C, which is above the DPPC gel-fluid melt-ing temperature (Tm = 42C). The films were hydratedwith PBS, incubated for 1 hour, and sonicated for 2 hours.

Portions of each sample were stored at 25C (gel phasebilayers) and 50C (fluid phase bilayers) for 15 days with-out agitation.

LNAs were also prepared with a lipid mixture of DPPCand DPPS at a molar ratio of 85:15, and a lipid/AgNPweight ratio of 100:1. In this case DPPS was dissolved in a1:2 chloroform to methanol mixture, and added to theDPPC/chloroform + AgNP/hexane solution. The meltingtemperature of the mixed DPPC/DPPS bilayer withoutAgNPs was 43.4C (measured by DSC).

Colloidal stability and size distribution: Dynamic light scatteringThe hydrodynamic diameter and stability of the assem-blies were analyzed at the storage temperatures (25 or50C) using a Brookhaven light scattering system consist-ing of a BI-200SM goniometer, a Lexel 95-2 argon laser,and a BI-9000AT Digital Correlator. DLS samples wereanalyzed at 0.4 mM DPPC. Size distributions wereobtained using a continuous non-negative least squares(NNLS) fit of the autocorrelation function (RMS < 3.6 10-3).

Bilayer phase behavior: Differential scanning calorimetryThe pretransition temperatures associated with gel to rip-pled-gel lipid bilayer transitions, and the main transitionor melting temperatures associated with rippled-gel tofluid transitions, were analyzed by differential scanningcalorimetry (DSC, TA Instruments Q10) at 30 mM DPPC