Real-time monitoring of lipid transfer between vesicles and hybrid bilayers on Au nanoshells using surface enhanced Raman scattering (SERS)

Abstract

To investigate the dynamics of exchange/transfer of lipids between membranes, we have studied the interaction of donor-deuterated DMPC vesicles with DMPC hybrid bilayers on Au nanoshells using SERS. Experimental data confirm partial lipid exchange/transfer in the outer leaflet of the hybrid bilayer. The kinetics of the exchange/transfer process follows a first order process with a rate constant of 1.3 × 10⁻⁴ s⁻¹. Changes in lipid phase behavior caused by the exchange/transfer process were characterized using generalized polarization measurements. In situlipid transfer can potentially be utilized for preparation of asymmetric supported lipid bilayers and for incorporation of reporter lipids in biological membranes.

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Recently there has been renewed interest in understanding the dynamic properties of lipids, including lipid exchange/transfer processes, since lipids, an integral constituent of cell membranes, frequently appear to act as messengers that trigger important metabolic events.¹Lipid transfer by nonspecific forces, along with specific lipid transfer proteins, play a vital role in a myriad of biological processes, such as cell signaling, antimicrobial defense, lipid absorption during digestion, and parasitic invasion of erythrocytes.^1–3 With insight into the thermodynamics of lipid mixing and the kinetics of lipid exchange/transfer, these processes can be used for effective tracking of lipid metabolism and for studying membrane organization and biogenesis by incorporating reporter lipids into biological membranes of interest.¹

Processes at cellular membranes usually take place in a very complex environment, therefore it is particularly enlightening to examine the interaction of two simple model lipid membranes to understand the mechanisms and dynamics of membrane interaction and lipid exchange/transfer. Among the plethora of synthetic membranes explored, supported lipid bilayers (SLBs) and lipid vesicles are appropriate mimics for investigating interacting membranes, due to their ease of preparation and study.⁴ Assessing lipid transfer dynamics between vesicles and SLBs can shed light on the subtle aspects of membrane architecture and provide new perspectives for SLB modification and asymmetric SLB formation.^5–9 SLBs such as phospholipid–nanoparticle composites have recently being proposed as nonviral vectors for drug delivery across cell membranes,¹⁰ calling for further study of the interactions between these composite materials and cellular membranes. Recently, we have successfully demonstrated the fabrication and functionality of hybrid bilayers (HBLs) assembled on Au nanoshells as a biomembrane mimic.¹¹ HBLs on nanoshells constitute a special class of SLBs typically consisting of an alkanethiol self-assembled monolayer bound to a noble metal substrate, with an associated outer layer of lipids. Nanoshells, acting as an underlying metal support, are spherical plasmonic nanoparticles consisting of a silica core and a thin, gold shell that can support highly tunable plasmon resonances from visible to mid-IR. The optical excitation of plasmons at desired wavelengths generates a strongly enhanced local electromagnetic field close to the metal surface (within 10 nm) that has been successfully exploited for surface enhanced spectroscopies with high spectral reproducibility.¹² Hence, HBLs are good composite materials that can simultaneously serve as robust biomembrane mimics and strongly enhancing substrates for surface enhanced spectroscopy.

The kinetics of the exchange/transfer of lipids between membranes has been studied using various methods such as fluorescence, calorimetry, and light scattering.^13–16 However, these methods either require special probes (fluorescent tags, labeled lipid) or lack sensitivity; for example, a large lipid fraction (>10%) must be transferred in order to detect exchange/transfer. Hence, there has been great research interest in developing significantly more sensitive techniques to overcome these limitations.^5,8,17Surface enhanced Raman scattering (SERS), a powerful spectroscopic method that permits the identification and detection of chemicals at very low concentrations,¹⁸ is an ideal probe and well suited for studying the exchange/transfer process. In this study, we have investigated the exchange/transfer of lipids between hybrid bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) on nanoshells (acceptor) and small unilamellar vesicles of deuterated DMPC (donor). We probed changes in the chemical structure of the HBL using SERS, addressing the three possible outcomes of interaction of the donor lipid vesicles with acceptor HBL. They are: (I) no lipid exchange/transfer, (II) formation of a new bilayer on top of the existing HBL, and (III) partial and/or complete exchange/transfer of lipid into the outer leaflet of the HBL (Fig. 1). Our experimental observations provide conclusive, real-time, spectroscopic evidence for case (III), i.e., partial and/or complete exchange/transfer of lipids into the outer leaflet of the HBL.

Fig. 1 Schematic diagram illustrating the possible outcomes of lipid exchange when DMPC HBL on Au nanoshells are exposed to a solution of deuterated DMPC vesicles: (I) no lipid transfer, (II) formation of a second bilayer on top of the existing HBL, and (III) partial and/or complete transfer of lipid into the outer leaflet.

For the present study, small unilamellar vesicles (SUV) of deuterated DMPC (D-DMPC), typically measuring 85–100 nm, were fabricated using a published protocol.⁸ SERS-active Au nanoshells, fabricated by a previously published approach,¹⁹ have inner and outer radii of [r₁, r₂] = [63, 85] nm as confirmed by the optical extinction spectra and SEM analysis (Fig. S1A, ESI† ). A HBL of alkanethiol-DMPC lipids was fabricated on nanoshells following a recently reported method.¹¹ The fabricated hybrid bilayer-encased nanoshells, deposited on quartz, were then incubated (for times ranging from 10 to 400 min.) with vesicles of D-DMPC. SERS measurements were performed at room temperature (∼23 °C) after washing off the free vesicles. SERS spectra of the three different systems investigated were acquired: (a) hybrid bilayers formed with DMPC (HBL DMPC), (b) hybrid bilayers formed with DMPC that have undergone exposure to D-DMPC vesicles (HBL DMPC + D-DMPC) for 2 h, and (c) hybrid bilayers formed with D-DMPC lipid (HBL D-DMPC) (Fig. 2). SERS spectra collected on these systems are highly reproducible (Fig. S1 B–D, ESI† ), a prerequisite for performing any quantitative spectral analysis. Systems (a) and (c) are effectively two control systems that provide a reference (“starting and end points”) for comparison to the interacting system (b). There are important and readily observable spectral differences that appear in the SERS spectra of (b) when compared to (a) and (c). This clearly implies that the acceptor HBL and the donor vesicles interact with each other, ruling out case (I). Closer examination of the SERS spectra of system (a) reveals the absence of the C–D stretching mode in the 2000–2200 cm⁻¹ region since system (a) lacks any deuterated lipids while system (c), having deuterated HBL, has a clearly observable C–D stretching peak (Fig. 2). Notably, the interacting system (b) also reveals the presence of the C–D stretching mode. For the interacting system (b), these observations clearly indicate a close association of the deuterated lipids from the vesicles with the HBL, suggesting the state of this system is likely (II) and/or (III). In order to distinguish between scenarios (II) and (III), we examined the change of the CH content of the HBLs by monitoring the intensity of the C–H stretch mode (2850 cm⁻¹) normalized to the C–S stretch mode (710 cm⁻¹). It is important to note that for case (II) the presence of an additional bilayer of deuterated lipids on top of the existing HBL of DMPC will not result in a net decrease of the CH content. For (III), the insertion of deuterated lipids in the outer leaflet of the HBL that may occur upon DMPC replacement would result in a net decrease of CH content. The normalized C–H intensities calculated for the three systems from the SERS spectra are plotted (Fig. 2, inset). The decrease in CH content values clearly rules out (II) and favors (III), where there is partial and/or complete exchange/transfer of lipids. The CH content for system (b) lies between those of systems (a) and (c), indicating a partial exchange of lipids. This would also be consistent with the fact that the HBL was exposed to the vesicles for a time period of only 2 h duration.

Fig. 2 SERS spectra of (a) HBL formed with DMPC, (b) HBL DMPC incubated (2 h) with deuterated DMPC vesicles, and (c) HBL formed with D-DMPC. The decrease in I_CH/I_CS (2850 and 710 cm⁻¹, respectively) for the three different systems, as shown in the inset, clearly demonstrates exchange/transfer.

To investigate the extent of the exchange/transfer process and gain insight into the dynamics of the interacting system, kinetic studies were performed by varying the incubation time of the HBL with the donor vesicle. Plotting the intensity of the normalized C–H stretching mode versus incubation time yields the kinetics of the exchange/transfer process (Fig. 3 (A)). The data support a first order exponential fit to the kinetic data points, yielding a rate constant for loading of the outer leaflet with deuterated lipids of K = 1.3 × 10⁻⁴ s⁻¹. Previous studies have reported biexponential kinetics for the exchange/transfer process of lipids between DMPC vesicles and D-DMPC SLBs on a plate of Si ATR crystal.⁷ This observed difference is likely due to the lack of a large disparity in the sizes of the interacting systems used in this study (average size of HBL is 170 nm, compared to 85–100 nm for vesicles). Moreover, the HBLs here have an inner leaflet of covalently attached alkanethiols, which differs from bilayers on a planar surface attached by electrostatic forces. In fact, it has been reported that lipid exchange between small sonicated vesicles of DMPC and D-DMPC that are very similar in size and chemical composition follows first order kinetics.^1,17 Theoretical modeling, along with further experiments to quantify the activation energy for the exchange process, are required to clarify the mechanisms of lipid exchange/transfer between HBLs and vesicles of comparable dimensions. The extent of exchange/transfer was calculated to be ∼76% at the end of 7 h, using the CH contents of systems (a) and (c) (obtainable from Fig. 2 inset) and at the time point where progress of the exchange/transfer process saturates, i.e., the kinetic data point at 400 min (Fig. 3 A). A reasonable scheme of events that may occur during the kinetics study of exchange/transfer is shown in Fig. 3, which is consistent with the observed decrease of CH content, implying a partial exchange/transfer of lipids.

Fig. 3 (A) Kinetics of the transfer of deuterated lipids from vesicles to HBLs as obtained by monitoring the change in I_CH/I_CS. The line is a first order exponential fit to the data points. Accompanied is a schematic of the plausible changes in HBL composition. (B) The excitation generalized polarization (GP_ex) spectra of (a) HBL DMPC, (b) HBL DMPC incubated (6 h) with deuterated DMPC vesicles, and (c) HBL D-DMPC.

We also characterized the molecular structure of the three systems using Laurdan as a phase-sensitive fluorescent dye, since the phase in which the lipids reside is critically important for the exchange/transfer process. The excitation generalized polarization spectra (GP_ex) as defined by

GP_ex = (I₄₄₄ − I₄₈₄/I₄₄₄ + I₄₈₄)

where I₄₄₄ and I₄₈₄ are fluorescence intensities at 444 and 484 nm, respectively, can be utilized to indicate phase behavior below, near, and above the main phase transition temperature.²⁰ The GP_ex spectra for systems (a) HBL DMPC, (b) HBL DMPC incubated (6 h) with deuterated vesicles, and (c) HBL D-DMPC are presented in Fig. 3 B. For HBL DMPC (a), the GP_exspectrum show no appreciable tilt, indicating that the system has gel phase characteristics at room temperature. For HBL D-DMPC (c), the observed slight downward tilt of the GP_exspectrum indicates that the system resides in a liquid phase. These observations agree with the fact that a deuterated lipid has a ∼4–5 degree decrease in its main phase transition temperature.^11,21 Interestingly, the GP_exspectrum of the interacting system (b) is seen to be very similar to that of system (c). The observed similarity indicates that HBL DMPC incubated with deuterated vesicles exists in a liquid phase and is a result of a change in the chemical composition of HBLs due to the exchange/transfer process.

In summary, we have utilized DMPC hybrid bilayers on Au nanoshells and small unilamellar vesicles of deuterated DMPC to examine the interactions and exchange/transfer of lipids using SERS. The exchange/transfer process kinetics, studied in real time, is determined to be first order and provides conclusive evidence for partial exchange/transfer of lipids. The kinetics data provide a quantitative estimate of the extent of the exchange/transfer process. Generalized polarization spectra suggest that exchange/transfer of lipids cause changes in the phase transition behavior. The presented hybrid bilayer system with inbuilt SERS sensing capability and the experimentally observed kinetic findings can be explored further both for mechanistic insight into membrane interactions and for modification of SLBs. Theoretical modeling, along with future experiments, are required to clarify the mechanisms of lipid exchange/transfer between these interacting membrane systems.

Acknowledgements

We thank Dr Rebekah Drezek for allowing us to use her fluorolog, Dr Vengadesan Nammalvar, and Dr Robert M. Raphael for insightful discussions. This work was supported by the Robert A. Welch Foundation (C-1220) and the Multidisciplinary University Research Initiative (MURI) Grant of the Department of Defense W911NF-04-01-0203.

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Footnote

† Electronic supplementary information (ESI) available: Nanoshell extinction spectra and SEM, SERS spectral reproducibility, and details of experimental procedures. See DOI: 10.1039/b9nr00063a

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