Hydrophobic silver nanoparticles trapped in lipid bilayers: Size distribution, bilayer phase behavior, and optical properties
© Bothun; licensee BioMed Central Ltd. 2008
Received: 02 July 2008
Accepted: 12 November 2008
Published: 12 November 2008
Lipid-based dispersion of nanoparticles provides a biologically inspired route to designing therapeutic agents and a means of reducing nanoparticle toxicity. Little is currently known on how the presence of nanoparticles influences lipid vesicle stability and bilayer phase behavior. In this work, the formation of aqueous lipid/nanoparticle assemblies (LNAs) consisting of hydrophobic silver-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, and bilayer fluidity is determined. Concomitantly, the effect of bilayer incorporation on the optical properties of the AgNPs is also examined.
The dispersions were stable at 50°C where the bilayers existed in a liquid crystalline state, but phase separated at 25°C where the bilayers were in a gel state, consistent with vesicle aggregation below the lipid melting temperature. Formation of bilayer-embedded nanoparticles was confirmed by differential scanning calorimetry and fluorescence anisotropy, where increasing nanoparticle concentration suppressed the lipid pretransition temperature, reduced the melting temperature, and disrupted gel phase bilayers. The characteristic surface plasmon resonance (SPR) wavelength of the embedded nanoparticles was independent of the bilayer phase; however, the SPR absorbance was dependent on vesicle aggregation.
These results suggest that lipid bilayers can distort to accommodate large hydrophobic nanoparticles, relative to the thickness of the bilayer, and may provide insight into nanoparticle/biomembrane interactions and the design of multifunctional liposomal carriers.
Hybrid lipid/nanoparticle conjugates provide a biologically inspired means of designing stable agents for biomedical imaging, drug delivery, targeted therapy, and biosensing . An advantage of using lipids as stabilizing or functional ligands is that they mimic the lipidic scaffolding of biological membranes and have well-characterized physicochemical properties and phase behavior. In lipid vesicles, nanoparticle encapsulation can be achieved by trapping particles within the aqueous vesicle core or within the hydrophobic lipid bilayer. Becker et al , Kim et al , and Zhang et al  have shown that iron oxide (Fe3O4), cadmium selenide (CdSe) quantum dots, and gold nanoparticles, respectively, can be trapped within aqueous vesicle cores. To embed nanoparticles within lipid bilayers, the nanoparticle must be small enough to fit within a DPPC bilayer and it must present a hydrophobic surface. Using physisorbed stearylamine, Park et al [5, 6] have stabilized 3–4 nm gold and silver particles in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers. Likewise, Jang et al  embedded 2.5–3.5 nm silicon particles with chemisorbed 1-octanol into bilayer membranes composed of DOXYL-labeled phosphocholine lipids. The resulting vesicles are analogous to liposomal drug delivery systems with an added functional nanoparticle component.
For hydrophobic nanoparticles embedded within lipid bilayers, which is the focus of this work, the presence of nanoparticles can lead to changes in lipid packing and may disrupt lipid-lipid interactions amongst the headgroups and/or acyl tails [5, 6]. Disruption of such inter-lipid interactions can result in changes in lipid bilayer phase behavior, which is related to the degree of lipid ordering and bilayer viscosity. Hence, depending on their size and surface chemistry, embedded nanoparticles may influence the stability and function of hybrid vesicles, as well as the conditions required for preparation.
DPPC/AgNP and DPPC/DPPS/AgNP assembly formation
Lipid assemblies were prepared in PBS at 1 and 30 mM DPPC using the Bangham method . The 1 mM DPPC samples were prepared for fluorescence anisotropy measurements using DPH as a bilayer probe molecule. In these samples, the AgNP concentration was varied from 1 to 1000 mg/L to provide DPPC/AgNP ratios from 734:1 to 1:1 (w/w), respectively. The 30 mM DPPC samples were prepared for differential scanning calorimetry (DSC) and dynamic light scattering (DLS) studies. For these samples, the AgNP concentration was varied from 0.1 to 11.0 g/L to provide DPPC/AgNP ratios from 200:1 to 2:1 (w/w). To form the LNAs, an aliquot of the Ag-decanethiol NP/hexane solution was added to DPPC dissolved in chloroform to yield a transparent, miscible brown phase. For anisotropy measurements, an aliquot of DPH in THF was also added at a DPPC to DPH molar ratio of 500:1. The solvent phase was evaporated under nitrogen and the sample was placed under vacuum for 2 hours, leaving a dry DPPC/AgNP film. Hydration and processing steps were performed at 50°C, which is above the DPPC gel-fluid melting temperature (T m = 42°C). The films were hydrated with PBS, incubated for 1 hour, and sonicated for 2 hours. Portions of each sample were stored at 25°C (gel phase bilayers) and 50°C (fluid phase bilayers) for 15 days without agitation.
LNAs were also prepared with a lipid mixture of DPPC and DPPS at a molar ratio of 85:15, and a lipid/AgNP weight ratio of 100:1. In this case DPPS was dissolved in a 1:2 chloroform to methanol mixture, and added to the DPPC/chloroform + AgNP/hexane solution. The melting temperature of the mixed DPPC/DPPS bilayer without AgNPs was 43.4°C (measured by DSC).
Colloidal stability and size distribution: Dynamic light scattering
The hydrodynamic diameter and stability of the assemblies were analyzed at the storage temperatures (25 or 50°C) using a Brookhaven light scattering system consisting of a BI-200SM goniometer, a Lexel 95-2 argon laser, and a BI-9000AT Digital Correlator. DLS samples were analyzed at 0.4 mM DPPC. Size distributions were obtained using a continuous non-negative least squares (NNLS) fit of the autocorrelation function (RMS < 3.6 × 10-3).
Bilayer phase behavior: Differential scanning calorimetry
The pretransition temperatures associated with gel to rippled-gel lipid bilayer transitions, and the main transition or melting temperatures associated with rippled-gel to fluid transitions, were analyzed by differential scanning calorimetry (DSC, TA Instruments Q10) at 30 mM DPPC. Heat/cool scans were conducted from 25 to 50°C at 1°C/min.
Bilayer melting and fluidity: Fluorescence anisotropy
Bilayer melting temperatures and fluidity were also examined by fluorescence anisotropy (Perkin Elmer LS 55) of the hydrophobic bilayer probe diphenylhexatriene (DPH) at 1 μM DPPC from 30 to 50°C at a rate of 1°C/min under continuous mixing. Steady-state DPH anisotropy within the DPPC bilayer was determined at λ ex = 350 nm and λ em = 452 nm using the expression <r> = (I VV - I VH )/(I VV + GI VH ) where I represents the fluorescence emission intensity, V and H represent the vertical and horizontal orientation of the excitation and emission polarizers, and G = I HV /I HH accounts for the sensitivity of the instrument towards vertically and horizontally polarized light .
Optical properties: Ultraviolet-visible (UV-vis) spectroscopy
The optical absorbance properties of DPPC/AgNP vesicles were examined by UV-vis spectroscopy (Varian Cary 50) at 0.6 mM DPPC from 25 to 55°C under mixing. For varying DPPC/AgNP ratios, the absorbance data presented was normalized against the absorbance at 300 nm (A/A300) to account for differences in turbidity amongst the samples. Raw absorbance data is presented for fixed DPPC/AgNP and DPPC/DPPS/AgNP ratios.
Results and discussion
Synthesis and stability of hybrid DPPC/AgNP assemblies
Phase behavior and fluidity of DPPC/AgNP bilayers
Phase transition temperatures of DPPC/AgNP assemblies determined by DSC.
Melting temperature and bilayer fluidity determined by fluorescence anisotropy of diphenylhexatriene (DPH).
DSC and fluorescence anisotropy results indicate that the hydrophobic nanoparticles were interacting with the bilayer in a concentration-dependent manner. Given the hydrophobicity of the nanoparticles and their preference to partition into a hydrophobic environment, it is likely that a portion or all of the nanoparticles were embedded within the bilayer acyl region (Figure 1) and suppressed the pretransition and melting temperatures via bilayer disruption. The pretransition involves the transformation of a tilted-gel phase to a more disordered rippled-gel phase. While the rippled-gel phase is not completely understood, it has been described as being a gel phase that contains liquid crystalline domains . Mismatches between the bilayer thickness of neighboring gel and liquid crystalline phases produce periodic ripples. The absence of a pretransition with increased AgNP loading suggests that the presence of the nanoparticles inhibited ripple formation. Bilayer melting describes the transition from a rippled-gel to liquid crystalline phase, or fluid phase, due to melting of the lipid acyl tails. The highest nanoparticle loadings (2:1 and 1:1) suggest that the bilayer was appreciably disrupted by the presence of the nanoparticles.
Bilayer disruption was demonstrated; however, nanoparticle-lipid interaction mechanisms, as well as the structure and morphology of the LNAs are still under investigation. It is likely that the smaller nanoparticles in the size distribution embedded within the bilayers, while the larger particles were capped and dispersed in the aqueous phase by a lipid monolayer with the C16 acyl tails mixing with the decanethiol tails and the headgroups exposed to water. Lipid-capped nanoparticles and possible agglomerates are consistent with the smaller size fractions measured by DLS. Previous experimental studies have been focused on nanoparticle diameters smaller than 5 nm, which is a typical thickness for a lipid bilayer [5, 6, 17]. However, recent computer simulations suggest that it is thermodynamically feasible for 2–8 nm diameter nanoparticles to embed within a lipid bilayer . Based on bilayer phase behavior, it is shown herein that it may be possible to embed nanoparticles that have a diameter in proximity to, or exceeding the thickness of the bilayer, which is consistent with the simulation work .
Optical properties of DPPC/AgNP and DPPC/DPPS/AgNP vesicles
DPPC/DPPS/AgNP assemblies (85:15 DPPC to DPPS) were prepared at 100:1 lipid/AgNP to further investigate the effect of aggregation. DPPS is an anionic lipid that stabilizes vesicles via electrostatic repulsion. With the addition of DPPS, there was no change in the SPR wavelength relative to the native AgNPs in hexane or DPPC/Ag vesicles. DPPC/DPPS/AgNP vesicles remained stable and the absorbance spectra were similar for both the gel and fluid phase (Figure 6b). Results for both the zwitterionic and mixed zwitterionic/anionic lipids suggest that neither AgNP encapsulation within the bilayers or vesicle aggregation affect the SPR wavelength, as AgNP aggregation has been shown to yield a prominent red-shift .
Comparatively, Bhattacharya and Sirvastava  have shown that 2.04 ± 0.4 nm gold nanoparticles containing a hydrophobic surface ligand maintain their characteristic SPR band when embedded within gel phase DPPC bilayers. This work expands upon this observation, and suggests that the SPR of small AgNPs was independent of bilayer phase at the DPPC/AgNP and DPPC/DPPS/AgNP ratios examined.
Aqueous dispersions of hydrophobic Ag-decanethiol nanoparticles were formed using DPPC and DPPC+DPPS as stabilizing components. Our results based on bilayer phase behavior suggest that the DPPC/AgNP assemblies consisted of nanoparticle-embedded bilayer vesicles. The stability of the assemblies was dependent on their storage temperature and, in turn, the state of the bilayer (gel or fluid phase). Given that the nanoparticles had diameters near or exceeding the thickness of a lipid bilayer, this work suggests that DPPC bilayers can distort to accommodate such particles and that this distortion reduces lipid ordering. This result is consistent with the ability for a cell membrane to accommodate large transmembrane proteins . As a therapeutic agent, LNAs may be formed with functional nanoparticles, potentially larger than previously thought, for combined delivery and imaging. With respect to nanoparticle-cell interactions, these results provide further evidence that such hydrophobic nanoparticles could reside within cell membranes. Studies are underway to measure LNA morphology and structure, develop new nanoparticle encapsulation protocols, and explore different lipid compositions.
The author thanks Professor Arijit Bose and Ashish Jha for their assistance with DLS measurements. Alisson Boyko, a high school summer intern, and Sean Marnane, an undergraduate research assistant, assisted with sample preparation and fluorescence anisotropy studies. Steph Aceto, an undergraduate, conducted the UV-vis studies. This material is based in part upon work supported by a National Science Foundation (NSF) Faculty Development Award (Grant No. CHE-0715003), which was made possible by the NSF Discovery Corps Fellowship program, and by RI-INBRE (Grant No. P20RR016457) from the National Center for Research Resources (NCRR), which a component of the National Institutes of Health (NIH). Content is solely the responsibility of the author and does not represent the official views of NSF, NCRR, or NIH.
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