Lipid Raft-Mediated Membrane Tethering and Delivery of Hydrophobic

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Lipid Raft-Mediated Membrane Tethering and Delivery of Hydrophobic Cargos from Liquid Crystal-Based Nanocarriers Okhil K. Nag,† Jawad Naciri,† Eunkeu Oh,‡,§ Christopher M. Spillmann,† and James B. Delehanty*,† †

Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Code 6900, 4555 Overlook Avenue SW, Washington, DC 20375, United States ‡ Optical Sciences Division, Naval Research Laboratory, Code 5600, 4555 Overlook Avenue SW, Washington, DC 20375, United States § Sotera Defense Solutions, Inc., 7230 Lee DeForest Drive, Columbia, Maryland 21046, United States S Supporting Information *

ABSTRACT: A main goal of bionanotechnology and nanoparticle (NP)-mediated drug delivery (NMDD) continues to be the development of novel biomaterials that can controllably modulate the activity of the NP-associated therapeutic cargo. One of the desired subcellular locations for targeted delivery in NMDD is the plasma membrane. However, the controlled delivery of hydrophobic cargos to the membrane bilayer poses significant challenges including cargo precipitation and lack of specificity. Here, we employ a liquid crystal NP (LCNP)-based delivery system for the controlled partitioning of a model dye cargo from within the NP core into the plasma membrane bilayer. During synthesis of the NPs, the water-insoluble model dye cargo, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), was efficiently incorporated into the hydrophobic LCNP core as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiOLCNP-PEG-Chol) facilitated the localization of the dye-loaded NPs to lipid raft microdomains in the plasma membrane in HEK 293T/17 cell. Analysis of DiO cellular internalization kinetics revealed that when delivered as a LCNP-PEG-Chol NP, the halflife of DiO membrane residence time (30 min) was twice that of free DiO (DiOfree) (15 min) delivered from bulk solution. Time-resolved laser scanning confocal microscopy was employed to visualize the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer as confirmed by Förster resonance energy transfer (FRET) imaging. Finally, the delivery of DiO as a LCNP-PEG-Chol complex resulted in the attenuation of its cytotoxicity; the NP form of DiO exhibited ∼30−40% less toxicity compared to DiOfree. Our data demonstrate the utility of the LCNP platform as an efficient vehicle for the combined membrane-targeted delivery and physicochemical modulation of molecular cargos using lipid raft-mediated tethering.

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INTRODUCTION A continuing goal of interfacing of nanomaterials with living cells and tissues is the controlled modulation of the nanoparticle (NP)-associated cargo for the purpose of diagnostics and/or therapeutics. The use of NPs in a therapeutic capacity is broadly referred to as NP-mediated drug delivery (NMDD) where the primary objective is to take advantage of the unique size-dependent properties of the NP vehicle to increase the efficacy of the drug cargo.1,2 Often this is aimed at overcoming common issues associated with the systemic delivery of therapeutic drugs including multiple dose administrations, poor drug solubility, significant off-target drug toxicity, or induced immunogenicity.3,4 The specific properties of the NPs one seeks to take advantage of in this approach include the following: 1, small size that avails enhanced tissue penetration/circulation and clearance kinetics; 2, large surface area:volume ratio for large loading capacity; 3, amenability to bioconjugation to biologicals (e.g., peptides, proteins) for targeting; 4, size-dependent photophysical properties (e.g., the © XXXX American Chemical Society

size-tunable emission of semiconductor nanocrystals) that allows for simultaneous multifunctional imaging, tracking, and reporting during the drug delivery process; and 5, the ability to synthesize large quantities of high-quality NP materials as monodisperse populations.5−9 From the standpoint of NP delivery, one of the chief desired subcellular locations to be targeted for drug delivery in NMDD is the plasma membrane, the sentinel barrier that maintains cell integrity. This is the site of first contact of the NP with the cell and the initiation point for endocytosis.10 The targeted localization of NP-based therapeutics to the membrane can obviate the need for endocytosis-mediated drug uptake and distribution and can determine the therapeutic efficacy of the drug.11 To date, a limited number of known membrane targeting moieties that can achieve persistent NP residence at Received: January 23, 2016 Revised: March 8, 2016

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DOI: 10.1021/acs.bioconjchem.6b00042 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Chemical components used in synthesis of DiO-LCNPs. (A) Chemical structures of the compounds utilized to prepare the DiO-LCNPs: (1) acrylate liquid crystal cross-linking agent (DACTP11), (2) carboxyl-terminated polymerizable surfactant (AC10COONa), and (3) lipophilic dye, DiO. (B) Schematic of the DiO-LCNP and its conjugation to cholesterol-terminated poly(ethylene glycol) (PEG-Chol) via EDC coupling. Addition of PEG-Chol to the DiO-LCNP mediates preferential binding of the NP to the plasma membrane.

the plasma membrane have been described.12 Examples here include lipophilic molecules such as cholesterol and other membrane-derived components.13−17 For example, Carney et al. utilized 14-carbon lipophilic alkyl chains to generate a series of Gd(III)-based magnetic resonance (MR) contrast agents that showed increased membrane labeling efficiency, retention, and MR image contrast compared to nonlipophilic Gd(III) contrast agents.18 A few select peptidyl motifs have been reported to localize NP materials to the plasma membrane. In addition to peptides based on the classical, integrin-binding “RGD” motif,19,20 recently described peptides that present aliphatic adducts covalently attached to the peptide backbone via particular attachment chemistries have been effective at appending NPs to the plasma membrane with long-term residence.21,22 Finally, a number of small molecule drugs (e.g., homotryptamine) have proven effective at appending NPs to the plasma membrane surface.23,24 In addition to relying on long-term membrane residence of the NP carrier, a strategy for the controlled NP-mediated delivery of hydrophobic cargos to the lipophilic membrane bilayer would take advantage of the unique interplay between the physicochemical properties of the NP and the cargo. Key parameters to be understood here include the efficiency of the targeting and localization of the NP-cargo complex to the plasma membrane and how to affect the controlled delivery/ release of the therapeutic cargo. Our goal in this study was to determine the utility of our previously described liquid crystal NP (LCNP) platform25 for addressing these critical issues. We previously showed that these multifunctional NPs can track the endocytic pathway with spatiotemporal resolution when

conjugated to the iron transport protein, transferrin. We further showed the amenability of the LCNP to be loaded postsynthesis with the amphiphilic anticancer drug doxorubicin (Dox) to affect a ∼ 40-fold improvement in the IC50 of Dox relative to free Dox in HEK 293T/17 cells.25 We show here the extended utility of the LCNP platform as a drug/cargo delivery vehicle by demonstrating its ability to controllably modulate the membrane partitioning, cellular uptake kinetics, and cytotoxicity of a model dye cargo, 3,3′dioctadecyloxacarbocyanine perchlorate (DiO). DiO is a waterinsoluble, potentiometric membrane labeling dye that has been used for anterograde and retrograde tracing in living and fixed neurons, membrane potential measurements, and for general membrane labeling.26−30 DiO is typically added directly to cell monolayers or tissue slices as a crystalline form31 or incubated in solution at high concentrations (typically 1−20 μM) after dilution from a concentrated stock solution in organic solvent (e.g., DMSO).32,33 However, such high DiO concentrations delivered from bulk solution can result in dye precipitation, internalization of the dye to the cellular cytosol (and loss of signal resolution due to high background staining), and concomitant cytotoxicity.34−37 Similar to DiO, these challenges are common to other membrane probes (e.g., other carbocyanine dyes, voltage-sensitive dyes, and water-insoluble drugs). Hence, DiO serves as an excellent model compound for NP-assisted membrane partitioning. Thus, our primary goal here was to use the LCNP platform, surface-functionalized with the lipid raft-directing moiety, cholesterol, to realize a DiO delivery system that more efficiently partitions the dye to the membrane bilayer. DiO was incorporated into the LCNP core B


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Figure 2. Physicochemical characterization of the DiO-LCNPs. Shown are the size distribution data for the NPs: (A) PER104, (B) DiO-LCNP, and (C) DiO-LCNP-PEG-Chol. Size data were obtained by DLS. Error bars represent standard deviation of 5 different measurements. (D) Gel electrophoresis analysis of NPs. The horizontal bar represents location of sample loading wells. Unconjugated PER104 (lane C) migrate toward the cathode (+), DiO-LCNPs (lane 1) exhibited minimal electrophoretic mobility while DiO-LCNP-PEG-Chol conjugates (lane 2) moved toward anode (−). Particles were analyzed by 1% agarose gel electrophoresis.

Table 1. Physicochemical and Spectral Properties of DiOfree and DiO-LCNPs PER104 DiO-LCNP DiO-LCNP in 50% DMSO DiO-LCNP-PEG-Chol DiO-LCNP-PEG-Chol in 50% DMSO DiO in water DiO in 50% DMSO

H-DiaAVGa (nm)

PDIb

ζc (mV)

DiO λabsd(nm)

DiO λemie (nm)

66.2 88.8 nd 167.3 nd nd nd

0.18 0.22 nd 0.2 nd nd nd

−27.2 ± 2.6 −27.6 ± 1.9 nd −8.6 ± 0.5 nd nd nd

nd 491 493 493 496 462.0, 488.5g 471

nd 510 511 512 512 544.0, 577.0g 539.0, 577.0g

DiOϕf (%) nd 11.9 ± 14.7 ± 13.1 ± 14.0 ± 1.6 ± 1.4 ±

3.8 3.5 3.3 3.6 0.4 0.3

a H-DiaAVG, average hydrodynamic diameter. bPDI, polydispersity index. cζ, zeta potential ± SEM. dAbsorption maxima. eEmission maxima. fϕ, relative quantum yield versus fluorescein standard ± SEM. gAbsorption/emission maxima accompanied by distinct shoulder peaks; nd, not determined.

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during synthesis and the as-synthesized DiO-LCNPs were bioconjugated with a PEGylated cholesterol derivative (DiOLCNP-PEG-Chol) to promote efficient localization to lipid raft domains, confirming the functional integrity of the cholesterol moiety on the surface of the LCNP. Once tethered to the plasma membrane of HEK 293T/17 cells, the overall membrane residence time of the LCNP form of DiO was observed to be twice that (30 min) of the free form of DiO (DiOfree). Along with the enhanced membrane persistence and residence time, the delivery of DiO as a NP formulation resulted in a ∼40% attenuation of the cytotoxicity of the DiOfree. Cumulatively, the data presented herein demonstrate a new functionality for the LCNP platform and points to lipid raft-mediated NP tethering as a new pathway for the delivery/ modulation of NP-associated cargos.

RESULTS

Synthesis and Characterization of DiO-LCNP and DiOLCNP-PEG-Chol. We synthesized LCNPs wherein the NP core was loaded with the membrane-labeling probe, DiO (DiOLCNPs). DiO is an amphiphilic molecule composed of a positively charged carbocyanine fluorophore appended with a pair of 18 carbon alkyl chains to facilitate plasma membrane binding and insertion. DiO-LCNPs were synthesized using a two-phase mini-emulsion technique38 with the chemical components DACTP11, AC10COONa, and DiO as shown in Figure 1A. In this NP system, the polymeric network of the crystalline cross-linking agent DACTP11 provides a hydrophobic core where the DiO is incorporated during NP synthesis. The negatively charged carboxylate headgroup of the surfactant AC10COONa on the NP surface stabilizes the C


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Figure 3. Spectral properties of DiO in solvents and in LCNPs. (A) Normalized absorption (Abs, dashed line) and emission (Emi, solid line) spectra of DiO in water, 50% DMSO, and LCNPs. DiO in LCNPs exhibited discrete absorption and emission profiles expected for the dye in hydrophobic environment. DiO in water and 50% DMSO showed eximeric spectra indicative of dye aggregation. (B) Absorption spectra of DiO-LCNPs in water, 2× dilution in water, and in 50% DMSO showed the same absorption maxima and spectral pattern. (C) Normalized absorption (Abs) and emission (Emi) spectra of two different preparations of LCNPs loaded with 10 μM and 25 μM DiO showed minimal changes in spectral pattern and maxima. (D) Fluorescence quantum yield of DiO in water and in LCNPs as measured relative (%) to fluorescein standard in water at pH 11 and expressed as an average (n = 4, ± SEM). Statistically differences between DiO and DiO-LCNPs (p < 0.01; *) or DiO-LCNP-PEG-Chol (p < 0.001; **) were noted.

EDC coupling. These modifications in surface charge properties of the NPs were further confirmed by the NPs’ movement as observed in gel electrophoresis (Figure 2D). As anticipated, PER104 NPs showed clear and strong mobility toward the cathode. Interestingly, DiO-LCNPs showed minimal mobility even though their ζ is comparable to the PER104 NPs. We attribute this to the presence of the positively charged DiO molecules inside the particle core which are attracted to the anode resulting in minimal overall mobility of the NPs in the agarose matrix. However, after functionalization with PEG-Chol (and the resulting consumption of free carboxyl groups) the DiO-LCNP-PEG-Chol particles exhibited modest migration toward the anode, suggesting that the decrease in negative surface charges coupled with the presence of positively charged DiO molecules imparted a net positive charge on the ensemble NP that drove migration toward the negative terminal. Cumulatively, these data provide strong evidence for the successful loading of DiO into the NP core and the conjugation of PEG-Chol onto the NP surface. Photophysical characterization of the DiO-LCNPs provided further evidence of the successful incorporation of DiO into the NP core. This was characterized by a spectral red-shift in the UV−vis absorbance of DiO in the LCNP compared to DiOfree in water or 50% DMSO (Figure 3A, Table 1). The absorbance and emission spectra of DiOfree in water showed two distinct peaks indicative of molecular aggregation of the dye and the resulting eximeric excited state of the dye in aqueous solvent. In 50% DMSO/water, the absorption peak resolved to a single, broad peak due to the increased solubility of DiOfree in the more hydrophobic 50% DMSO. These spectra were also observed to be concentration-dependent (Figure S1). In the

NP in aqueous media (Figure 1B). In contrast to other surfactant-based surface modifications that physically adsorb the surfactant molecules onto the NP surface, which can result in loss of NP colloidal and steric stability,39−42 the polymerizable surfactant used herein covalently binds to the liquid crystal polymeric network, providing added stability. The carboxylate moiety also presents a functional handle for attachment of cell-targeting ligands. For targeting of the NPs to the plasma membrane, further surface modification of the DiO-LCNPs was performed by conjugation of cholesterolterminated PEG (PEG-Chol) via EDC coupling (Figure 1B). A similar LCNP (PER104), loaded with the hydrophobic dye perylene, was synthesized as a control. The average hydrodynamic diameter (H-DiaAVG) of the DiO-LCNP (89 nm) and PER104 (66 nm) NPs were comparable to one another and both exhibited a narrow polydispersity index (Figure 2 A,B and Table 1). Conjugation of PEG-Chol to the DiO-LCNP (to yield DiO-LCNP-PEG-Chol) increased the H-DiaAVG of the NPs by ∼2-fold (Figure 2C and Table 1), strong evidence of the addition of the PEG-Chol layer to the NP surface. We also characterized the surface charge properties of the NPs using zeta potential (ζ) measurements (Table 1). As anticipated, both PER104 and DiO-LCNP displayed strong negative surface charge (−27 mV) due to the presence of the negatively charged carboxylate termini on the NP surface. Loading of the positively charged DiO into the LCNP core did not alter the surface charge of the particle. However, conjugation of the PEG-Chol significantly reduced the negative charge of the DiO-LCNP surface from −27.6 mV to −8.6 mV, consistent with the consumption of surface carboxylate moieties while forming the neutral amide linkage through D


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Bioconjugate Chemistry context of the LCNP, however, the DiO absorbance and emission spectra appeared as narrow peaks that were redshifted ∼20 nm red and blue-shifted ∼28 nm, respectively, relative to DiO in 50% DMSO. This spectral behavior of DiO in the LCNP is indicative of the increased solubility of the DiO within the hydrophobic microenvironment of the LCNPs. Further, we also noted no significant change in the absorption spectra of the DiO-LCNPs upon dilution in water or in 50% DMSO (Figure 3B), indicating the minimal leakage of the DiO from the LCNPs into the surrounding hydrophilic media. Next, we assessed any changes in the spectral behavior and measured the quantum yield of DiO in the DiO-LCNPs. Here, we prepared LCNPs loaded with different amounts of DiO (10 μM or 25 μM) and recorded their spectral properties. The spectral properties (Figure 3C) and quantum yields of these two preparations were very similar. These results provide strong evidence of the encapsulation of DiO in the hydrophobic core of the LCNPs without major aggregation of the dye. PEGylation of the DiO-LCNPs with PEG-Chol did not alter the optical properties of the DiO in the NP system (Table 1). Finally, we measured an ∼8-fold increase in the fluorescence quantum yield of DiO in the LCNPs (∼11%) compared to DiO in water (Figure 3D, Table 1). Taken together, these results support the partitioning of the DiO into the hydrophobic LCNP core, an environment that destabilizes the emitting states and reduces quenching, resulting in a blueshift in the DiO emission spectra (compared to DiO in aqueous) and concomitant increased quantum yield. Cellular Uptake of DiOfree. DiO is not directly soluble in aqueous media and, therefore, it must first be solubilized in toxic organic solvent prior to dilution into aqueous media for use in plasma membrane labeling. For cellular labeling with DiOfree, a concentrated stock solution of DiO (2 mM in DMSO) was further diluted into DMEM-HEPES prior to application to HEK 293T/17 cell monolayers. Initial experiments revealed that when DiOfree was applied in this manner, the resulting efficiency of membrane labeling was heavily dependent on both the DiOfree concentration and the time of incubation. For example, no significant labeling at DiOfree concentrations below 1.0 μM DiOfree (Figure S2) was observed while higher concentrations (e.g., 6.0 μM) robustly stained the plasma membrane with high efficiency upon 15 min incubation with the cells. However, significant cellular internalization of DiOfree was observed upon only a modest increase in the incubation time to 30 min (Figure S2). The extent of DiOfree internalization determined by time-resolved colocalization experiments where cells were first incubated with 6.0 μM DiOfree (30 min) followed by removal of the DiOfree-containing incubation medium and subsequent culture of the cells for 1, 2, or 4 h (Figure 4A). The plasma membrane of the cells was then counterstained with a phosphoethanolamine membrane lipid conjugated to Rhodamine B (PE-Rhoda). As shown in Figure 4A, upon 15 min of incubation almost 100% (Pearson’s colocalization coefficient (PCC) = 0.99 ± 0.01) of DiOfree was colocalized with the PE-Rhoda marker. However, upon 30 min of incubation ∼80% of DiOfree remained bound to the membrane (∼20% internalized). The degree of DiO free internalization increased steadily as the incubation time was extended to as long as 4 h and this was reflected in the concomitant decrease in the PCC between the DiO and RhodaPE (Table S1). A fit of the data to a one-phase exponential decay equation revealed that the DiOfree internalization reached a maximum of ∼50% at 1 h with an internalization rate (k =

Figure 4. Time resolved cellular uptake of DiOfree in HEK293T/17 cells. (A) DiOfree (6.0 μM) was incubated on cell monolayers for 15 or 30 min as indicated. For the 1, 2, and 4 h time points, DiOfree was incubated on cells for 30 min, removed, and the cells cultured for the indicated time. Cells were subsequently stained with PE-Rhoda (2.0 μM), fixed, and the nuclei stained with DAPI. (B) Time-resolved plot of the percent of membrane-bound DiOfree signal as a function of increasing incubation time. The data was obtained from the Pearson’s colocalization coefficient (PCC, n = 3 ± standard deviation) of the green and red channels, and is expressed as a percentage after normalization to the PCC corresponding to 15 min incubation.

0.045 min−1) and half-life (15 min) that reflected the rapid and efficient cellular uptake of DiOfree. These data demonstrate the rapid concentration- and time-dependent transition of DiOfree from the plasma membrane to the cytosol where it remained largely excluded from the nucleus over the 4 h time period examined (Figure 4B). Further, these observations provided motivation for the development of NP-mediated controlled modulation of the dye’s membrane-binding and internalization kinetics. Cellular Uptake of DiO-LCNP and DiO-LCNP-PEGChol. Given the uncontrolled cellular uptake of DiOfree after initial membrane binding as evidenced by our initial experiments, we sought to determine the ability to modulate this behavior by delivering DiO as a LCNP formulation. To pursue this, DiO-loaded LCNPs were first tested to assess their degree of nonspecific binding to the cell surface. Upon incubation of DiO-LCNP (6.0 μM DiO; ∼50 nM LNCP) for 30 min on HEK 293T/17 monolayers, we observed negligible nonspecific binding of the NPs to the cell surface (Figure S3). These results are in keeping with our previously reported results where we E


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contiguous (Figure 4A), the staining pattern of the DiO-LCNPPEG-Chol after 15 min incubation was more punctate and not nearly as uniform in nature (Figure 5A), a first indication that the DiO-LCNP-PEG-Chol NPs were collecting in discrete regions within the plasma membrane. Time-resolved tracking of the DiO staining pattern further revealed a more persistent membrane retention of the DiO-LCNP-PEG-Chol NPs as compared to DiOfree. Increasing the incubation time of the DiO-LCNP-PEG-Chol conjugates to 30 min resulted in membrane retention of ∼94% of the DiO signal (compared to ∼80% for DiOfree at this same time point) (Figure 5B). This trend became more pronounced as the cells were cultured with the DiO-LCNP-PEG-Chol NPs for increasingly longer times after the initial 30 min incubation. For example, after culture for 1 h after the initial incubation nearly 80% of the DiO-LCNPPEG-Chol NP signal remained membranous and colocalized with the PE-Rhoda marker (compared to ∼55% for DiOfree). Notably, cellular internalization of the DiO-LCNP-PEG-Chol NPs reached a maximum of 30% at 2 h (70% membrane retention), which corresponded to a cellular uptake rate (k = 0.024 min−1; half-life 29 min) that was exactly one-half that observed for the internalization of DiOfree. These results clearly demonstrate the ability to attenuate the cellular uptake of DiO and promote its membrane residence by delivering it as a LCNP formulation rather than from bulk solution. DiO-LCNP-PEG-Chol Localize with Lipid Raft Microdomains. To ascertain the precise microdomain location of the DiO-LCNP-PEG-Chol NPs within the plasma membrane, we performed colocalization experiments where the lipid raft domains were labeled and tracked with the Vybrant Lipid Raft Labeling Kit. Here, a dye conjugate of cholera toxin B binds to the pentasaccharide chain of plasma membrane ganglioside GM1 which selectively localizes to lipid rafts.45,46 When the DiOLCNP-PEG-Chol membrane location was probed with the lipid raft marker, an extremely high degree of colocalization with the marker was noted (Figure 6A). The corresponding PCCs for the localization of the LCNPs with lipid raft marker (and vice versa) were 0.86 and 0.88, respectively, demonstrating the significant degree of colocalization (Figure 6B). These data confirm the role of the cholesterol moiety in localizing the DiO-LCNP-PEG-Chol NPs to lipid raft microdomains in the plasma membrane and show the retention of its biological activity upon bioconjugation to the LCNP surface. More generally, these results demonstrate the utility of the cholesterol ligand in directing the collection of NP materials to which it is appended to these regions that are rich in cholesterol content.47,48 Importantly for our purposes here, the localization of the DiO-LCNP-PEG-Chol NPs to lipid raft domains persisted for hours and allowed us to assess the degree of passive efflux and transition of the DiO cargo from the NP core into the lipophilic region of the plasma membrane bilayer (vide infra). Time-Resolved Release of DiO from DiO-LCNP-PEGChol. Having demonstrated the membrane tethering and subsequent localization of the DiO-LCNP-PEG-Chol NPs to lipid raft microdomains, we next sought to interrogate whether the NP-embedded DiO cargo could efficiently efflux out of the NP core and enter the lipophilic environment of the plasma membrane bilayer in a controlled and time-dependent manner. To address this, we devised a FRET-based strategy wherein the DiO serves as a FRET donor engaged in energy transfer to the acceptor dye, DiI. In this configuration, efflux of the NPcontained DiO cargo into the membrane bilayer would be

noted low nonspecific binding of perylene-containing LCNPs (PER104) to HEK 293T/17 cell monolayers.25 To facilitate plasma membrane association of the DiOLCNPs, amine-functionalized PEG-Chol was conjugated to the carboxyl termini of the stabilizing surfactant, producing NPs referred to herein as DiO-LCNP-PEG-Chol. Cholesterol has been shown previously to mediate the efficient targeting and localization of NP constructs to the plasma membrane.43,44 As evidenced by the fluorescence micrographs in Figure 5A, the

Figure 5. Time resolved cellular uptake of DiO-LCNP-PEG-Chol in HEK293T/17 cells. Cells were stained with DAPI (2 μg/mL), DiOLCNP-PEG-Chol ([DiO] = 6.0 μM), and PE-Rhoda (2.0 μM). (A) Shown are DIC and CLSM images showing nucleus stained with DAPI (blue), membrane stained with PE-Rhoda (red), DiO-LCNPPEG-Chol (green) stained and merged fluorescence channels. The images were acquired after 15 min, 30 min, 1 h, 2 h, and 4 h of incubation. For the later time points, DiO-LCNP-PEG-Chol suspension was removed from the cells after 30 min and the incubation was continued for the predetermine time. (B) Changes in the membrane-bound DiO-LCNP-PEG-Chol signal on increasing incubation time. The data was obtained from the colocalization coefficient (n = 3, ±standard deviation) of green and red channels, normalized to 15 min incubation and expressed as a percentage.

addition of Chol-PEG to the surface of the DiO-LCNPs altered the resulting time-dependent cell-labeling behavior of the DiO compared to when DiOfree was delivered from bulk solution. Initially, upon 15 min incubation of the DiO-LCNP-PEG-Chol with the cells, nearly 100% of the DiO signal was located at the plasma membrane where it was colocalized with the PE-Rhoda membrane marker, a result that was comparable to that observed for DiOfree. We did note, however, that while the DiOfree labeling of the membrane was quite uniform and F


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(Figure S4).49,50 This FRET pair has been used elsewhere to track the efflux kinetics of materials from the hydrophobic core of micelles to the plasma membrane and cytosol in KB tumor cells.50 To test the feasibility of this FRET approach, we first tested the FRET efficiency of this DiO−DiI donor−acceptor system when both dyes were delivered to the plasma membrane as free dye from bulk solution. As expected, a clear concentrationdependent FRET sensitization of the DiI acceptor was observed when the plasma membrane was colabeled with DiOfree and DiIfree (Figure S5). Cells were then labeled with DiO-LCNPPEG-Chol (6.0 μM DiO) and DiIfree (6.0 μM) and monitored over time for the transition of the DiO from the NP to the plasma membrane as evidenced by an observed increase in the FRET-sensitized emission intensity of the DiI acceptor. As shown in Figure 7A, direct excitation imaging confirmed the robust labeling of the donor−acceptor pair to the membrane. Time-resolved FRET imaging of the system (excitation at 488 nm) showed that, as expected, upon initial labeling (t = 0 min) the emission signal was dominated by the DiO donor contained within the membrane-tethered NPs (Figure 7B). FRET imaging of this same field 4 h later (t = 240 min), however, now revealed the significant increase in the emission signal of the DiI acceptor, providing strong evidence of the transition of the DiO donor from the LCNP core into the plasma membrane bilayer (Figure 7C). Examination of the time-resolved nature of this transition showed a steady decrease in DiO donor emission coupled with a corresponding increase in DiI acceptor emission over the 4 h experimental window (Figure 7D). Notably, this transition reached its maximum at ∼180 min post-initial labeling, suggesting that DiO donor had reached its saturation point in terms of its efflux from the NP and partition into the membrane (Figures 7E and S6). Further experiments confirmed the release of DiO from the LCNPs where, upon 15 min incubation of DiO-LCNP-PEG-Chol on the cells followed by 4 h culture, a significant amount (∼98%) of the DiO signal was colocalized with a marker for the endoplasmic reticulum (Figure S7). These data provided strong evidence of the time-resolved partitioning of the DiO from the NP to the

Figure 6. Colocalization of DiO-LCNP-PEG-Chol within lipid raft microdomains. Cells were stained with DiO-LCNP-PEG-Chol (DiO] = 6.0 μM) and lipid raft marker. (A) Shown are DIC, nucleus stained with DAPI (blue), DiO-LCNP-PEG-Chol (green), lipid raft marker (red), and the merged images of the fluorescence channels. (B) Pearson’s colocalization coefficient (n = 3, ±standard deviation) of the green (DiO) and red (lipid raft) channels showing the degree of overlap of the two channels.

observed as a concomitant increase in the FRET-sensitized emission of the DiI acceptor. DiI is a lipophilic membrane dye with a similar structure to DiO and whose red-shifted spectral properties make it a suitable FRET acceptor to the DiO donor

Figure 7. Time-resolved FRET confirms efflux of DiO from DiO-LCNP-PEG-Chol to plasma membrane. HEK293T/17 cells were co-stained with DiO-LCNP-PEG-Chol and DiI, where DiO and DiI act as FRET donor and acceptor, respectively. (A) DIC and fluorescence images of the cells stained with DiO-LCNP-PEG-Chol ([DiO] = 6.0 μM, green), DiI (6.0 μM, red), and merged fluorescence channels. DiO and DiI were excited in direct excitation mode (see Experimental Procedures). (B,C) FRET imaging of the same focal plane shown in (A) at t = 0 min (B) and t = 240 (C) min. (D) Normalized, time-resolved emission intensity of DiO donor and DiI acceptor from cells stained with DiO-LCNP-PEG-Chol and DiI and imaged in FRET excitation mode. (E) Time-resolved FRET ratio (DiIemi/DiOemi) for cells labeled with DiO-LCNP-PEG-Chol and DiI and imaged in FRET configuration. The raw spectral data are shown in Figure S6. G


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Bioconjugate Chemistry plasma membrane bilayer that reached its maximum ∼3 h post initial labeling with the DiO-LCNP-PEG-Chol NPs. Cumulatively, our data clearly demonstrate the ability of the LCNPPEG-Chol platform to modulate the uptake and delivery kinetics of DiO in a controlled manner that is not attainable with the free dye. Cytotoxicity of DiO and DiO-LCNPs. In addition to the ability to modulate the uptake and distribution kinetics of a dye or drug cargo, another critical parameter that one seeks to control by using NP-facilitated delivery is the concomitant cytotoxicity of the NP-associated cargo. Thus, we performed a comparative cytotoxicity analysis of DiOfree versus DiO-LCNPPEG-Chol. As shown in Figure 8A, when delivered from bulk

mediated drug cargo modulation have shown the ability to modulate with fine control the NP-associated cargo. For example, Seo et al. showed a 30% enhancement of Dox cytotoxicity in HeLa cells by delivering the drug via endocytosis as a complex with gold NP/metal-binding protein hybrids.51 Similarly, the Prosperi group used human apotransferrin as a nanocage to drive the enhanced endocytosis and nuclear uptake of Dox.52 Partlow et al. showed that lipid coated NPs targeting αvβ3-integrin ligands were able to deliver lipophilic substances to target cell membranes with subsequent trafficking via lipid raft-dependent pathways.53 However, demonstrations of the controlled tethering of NP-cargo conjugates to lipid raft domains as a means of controlling cargo delivery parameters (e.g., membrane-specificity, labeling kinetics, cytotoxicity) of the membrane-targeted cargo still remain limited. Lipid raft microdomains play a critical role in biomolecule trafficking and cellular signaling and they are believed to be a unique pathway for cellular uptake of materials that is separate from classical clathrin-mediated endocytosis.44,54 It is within this backdrop that we undertook the study detailed herein where our goal was to use the LCNP platform for the controlled delivery of a model hydrophobic cargo to the plasma membrane bilayer via cholesterol-mediated tethering of the LCNP-cargo conjugates to lipid rafts. In our previous work we demonstrated the multifunctional nature of the LCNP materials as an all-in-one cellular platform for the simultaneous two-color tracking of the endocytic pathway when conjugated to the iron transport protein, transferrin (Tf).25 Further, the LNCP-Tf platform served as an efficient modulator of an anticancer therapeutic Dox where the endosomal uptake of the Dox-loaded LCNPs facilitated the persistent and sustained release of the drug resulting in a ∼40fold improvement of Dox-mediated cell killing when delivered as an LCNP-Tf drug conjugate. Here, we have utilized the same LCNP platform for a different goal: the targeted membrane tethering of the LCNP for the purpose of modulating the membrane labeling efficiency of an LCNP-embedded membrane-labeling dye cargo. To accomplish this, several key modifications to the LCNP system were made. First, the perylene dye was replaced with the lipophilic dye, DiO. Second, the endocytosis-directing transferrin ligand from our previous report was replaced with a PEGylated cholesterol derivative to facilitate the docking of the DiO-loaded LCNP to lipid raft microdomains. DiO is an excellent model hydrophobic dye cargo that has been used for neuronal cell membrane labeling/tracing, membrane potential reporting and for monitoring cell fusion events.26−30,55 However, the conditions used for DiO delivery in these applications lead to nonspecific labeling of nonmembranous areas (cytosol and intracellular membranes) adding to increased background signal and concomitant cytotoxicity. This is a significant challenge for the cellular delivery of hydrophobic cargos in general. Hence, there is a need for alternative delivery systems that can overcome these limitations. The DiO-LCNPPEG-Chol system described in this report addresses many of these shortcomings and demonstrates several novel aspects relative to previous NP systems that have sought to deliver DiO to living cells. In work reported by Chen et al., DiO and DiI were loaded into the hydrophobic core of PEG-polyester micelles and used as a FRET pair to monitor the release of the two cargos and the anticancer drug paclitaxel into the plasma membrane prior to internalization.50 In contrast to our system, no specific membrane-tethering ligand was appended onto the

Figure 8. Quantification of cytotoxicity. DiOfree, LCNP, DiO-LCNPs, or DiO-LCNP-PEG-Chol were incubated on HEK 293T/17 cell monolayers for 15 min and then removed. Cells were washed and cultured in growth medium for 72 h prior to MTS assay. (A) Average (n = 10 ± SEM) cell viability of DiOfree at different concentration from 1.25 to 25 μM. The data is represented as a percentage of control without DiOfree. (B) Comparison of cell viability (n = 5 ± SEM) of DiOfree, LCNP, DiO-LCNP, and DiO-LCNP-PEG-Chol at [DiO] = 3.0 or 6.0 μM. The difference between DiOfree and LCNP, or DiOLCNP, or DiO-LCNP-PEG-Chol are significant (p < 0.001).

solution, we determined the IC50 for the free dye to be ∼6 μM. Interestingly, we chose this same concentration (6 μM) for the cell delivery/labeling experiments as we determined empirically that this was the minimum concentration required to achieve a cell labeling efficiency of at least 75% (Figures S2 and S8). However, this concentration, when used in the incubation regimes described herein, corresponded to a cell viability for DiOfree of ∼50% (Figure 8B). This effect was clearly attributable to the free dye alone as the LCNP and DiOLCNP formulations showed considerably less toxicity (90% and 80% cell viability at 6 μM concentration, respectively). Of critical importance was the observed >90% cell viability for the full ensemble DiO-LCNP-PEG-Chol (at 6 μM DiO concentration) which confirmed the high degree of biocompatibility of the fully assembled DiO delivery vehicle. Results following a similar trend were obtained when the culture time was shortened to 24 h after initial incubation of the material with the cells (Figure S9).

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DISCUSSION A continuing goal of interfacing biocompatible nanomaterials with living cells is the development of a detailed understanding of how new NP formulations can be employed to modulate the physicochemical behavior of NP-associated cargos. To do this with control will ultimately enable enhanced drug/cargo delivery and dosing regimens and improved therapeutic indexes for drugs delivered via NMDD. Recent examples of such NPH


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by polymerizable surfactant in water. The mixture was then heated to 64 °C to initiate the polymerization of both the crosslinking agent and the surfactant as the chloroform slowly evaporated leaving a DiO-containing NP suspension stabilized by surfactant. Following synthesis, the NP suspension was filtered (3 times) through a 0.2 μm syringe filter to reduce the average particle size and sample polydispersity and any aggregated NPs appearing in the pellet were discarded. The filtered NP solution was stored at 4 °C until further use. During DiO-LCNP synthesis, DiO was included in the reaction mixture at either 4 mol % or 18 mol % relative to DACTP11. The purified NPs were further characterized for their actual content of DiO dye as described below. Conjugation of DiO-LCNPs to Chol-PEG-NH2.HCl. DiOLCNPs were covalently conjugated to Chol-PEG-NH2·HCl via carbodiimide chemistry. A stock solution of Chol-PEG-NH2· HCl (PEG-Chol, 0.9 mM) was prepared in HEPES buffer (pH 7.0, 25 mM). A working solution containing NHSS (40 mM) and EDC (400 mM) was prepared in HEPES buffer from concentrated stock solutions. An aliquot (20 μL) of the freshly prepared working solution of NHSS/EDC was immediately added to 1.0 mL of DiO-LCNP in HEPES buffer and stirred for 5 min. An aliquot (20 μL) of stock solution of Chol-PEG-NH2· HCl was added to this mixture and stirred for 2 h. The reaction mixture was briefly centrifuged and the supernatant was subjected to size exclusion chromatography using a PD-10 column equilibrated with DPBS (0.1×). The colored band was collected and analyzed by agarose (1%) gel electrophoresis. Control samples of DiO-LCNP and perylene-loaded LCNP (PER104) were also prepared as described above without the addition of Chol-PEG-NH2·HCl. Characterization of DiO-LCNPs. The as-synthesized DiOLCNPs were characterized for their particle concentration, size, charge, and the concentration and spectral properties of the incorporated DiO. Particle size and distribution were measured by dynamic light scattering (DLS) on a DiO-LCNP solution (200× dilution) in PBS (pH ∼ 8, 0.1×) using a ZetaSizer NanoSeries equipped with a HeNe laser source (λ = 633 nm) (Malvern Instruments Ltd., Worcestershire, UK) and analyzed using Dispersion Technology Software (DTS, Malvern Instruments Ltd.). Zeta potential was measured on a ZetaSizer NanoSeries equipped with a HeNe laser source (λ = 633 nm) (Malvern Instruments Ltd.) and an avalanche photodiode for detection. For each analysis at least six measurements were performed and the data is represented as average value ± SEM. The concentration of the LCNP dispersion in each preparation was determined to be ∼200 nM using a NanoSight LM10 imaging system (NanoSight Ltd., UK).25 The DiO content of the NPs was determined by measurement of DiO absorbance and comparison to the absorption coefficient (ε) of DiOfree in 50% DMSO. To confirm the inclusion of DiO in the NPs we compared the spectral properties and relative fluorescence quantum yield (ϕ) of DiOfree and DiO-LCNP in water and in 50% DMSO/water. The fluorescence quantum yield of DiO and DiO-LCNPs were measured using the quantum yield of fluorescein at pH 11 as standard, using the equation ϕs = ϕfl (Emis/Abss) (Absfl/Emifl) (ηs/ηfl)2), where ϕs is the fluorescence quantum yield of the DiO-LCNP sample, ϕfl is the quantum yield of the fluorescein standard (ϕfl = 92% at pH ≈ 11)57 and Emis and Emifl are the integrated emission intensities of the samples and the standard, respectively. Abss and Absfl are the absorbance of the samples and the standard at the excitation wavelength (485 nm), respectively, and ηs and ηfl are

micelle surface to drive membrane persistence. This likely accounts for the large degree of NP endocytosis and accumulation within perinuclear areas observed in that study. In our system, the cholesterol moiety facilitates the prolonged membrane residence of the LCNPs where, after 4 h, 75% of the materials remained membrane-associated. This feature is directly responsible for the enhanced membrane partitioning of the DiO cargo in the LCNP-PEG-Chol system compared to DiOfree, a large percentage of which accumulates in the cytosol and intracellular membranes adding to background signal. Cumulatively, the 1, large cargo capacity; 2, enhanced plasma membrane tethering/residence via lipid raft localization; 3, sustained cargo release kinetics; and 4, mitigated cargo cytotoxicity demonstrated by the DiO-LCNP-PEG-Chol system point to its potential utility for the enhanced/improved delivery of a range of other cell-labeling or therapeutic cargos.

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CONCLUSION

In this study, we have presented a new capability of our previously described LCNP system: the tethering of LCNP to lipid rafts coupled with the controlled delivery of the waterinsoluble model cargo, DiO. Tethering of the DiO-loaded NPs to the membrane via a PEGylated cholesterol ligand drives membrane residence of the LNCPs that in turn allows enhanced, sustained DiO efflux, and membrane bilayer insertion of the DiO cargo. Compared to delivery of DiOfree delivery from bulk solution, we have observed a significant improvement in localized DiO loading to the plasma membrane bilayer by tethering the LCNP-DiO conjugate to lipid rafts. The slow, sustained release of the DiO in to the bilayer is coupled with the attenuation of DiO cytotoxicity. We anticipate that these demonstrated improvements will translate to the NPenhanced delivery of other membrane bilayer-directed imaging and diagnostic/therapeutic agents.

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EXPERIMENTAL PROCEDURES Materials. All chemicals were purchased from Sigma (St. Louis, MO) and used as received unless otherwise mentioned. The synthesis of the polymerizable acrylate-functionalized surfactant AC10COONa and the liquid crystal (LC) nematic cross-linking agent DACTP11 is described elsewhere.38 Dulbecco’s phosphate buffered saline (DPBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1 M), Dulbecco’s Modified Eagle Medium (DMEM) containing 25 mM HEPES (DMEM-HEPES), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), hydrochloride, and N-hydroxysulfosuccinimide sodium salt (NHSS) were obtained from Life Technologies (Grand Island, NY). Cholesterol poly(ethylene glycol) amine hydrochloride (Chol-PEG-NH2.HCl, MW 2000) was purchased from Nanocs Inc. (New York, USA). Synthesis of 3,3′-Dioctadecyloxacarbocyanine Perchlorate Liquid Crystal Nanoparticles (DiO-LCNPs). DiO-LCNPs were prepared using a two-phase miniemulsion procedure described previously.25,38,56 Briefly, liquid crystalline diacrylate cross-linking agent (DACTP11, 45 mg), 3,3′dioctadecyloxacarbocyanine perchlorate (DiO, 2 mg), and a free radical initiator (azobis(isobutyronitrile), 1 mg) for polymerization were dissolved in 2 mL chloroform and added to an aqueous solution of acrylate-functionalized surfactant (AC10COONa, 13 mg in 7 mL) (Figure 1A). The mixture was stirred (1 h) and sonicated (5 min) to produce a miniemulsion consisting of small droplets of the organic material surrounded I


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of DiOfree (0−12.0 μM) and a fixed concentration of 1,1′dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIfree, 6.0 μM). To confirm successful colabeling, the samples were imaged using excitation at 488 nm (DiO) and 561 nm (DiI) with fluorescence detector channels set to the following filters: 500−550 nm (DiO) and 570−620 nm (DiI). FRET imaging was performed by exciting the DiO donor at 488 nm and collecting full emission spectra of both the DiO donor and DiI acceptor from 490 to 700 nm with a 32 channel spectral detector. FRET-sensitized DiI emission spectra was compared with the direct excitation emission spectra as a function of increasing concentration of DiOfree. Similar experiments in live HEK 293T/17 cells were performed at room temperature wherein cells were colabeled with DiO-LCNP-PEG-Chol and DiIfree to study the release kinetics of DiO from the LCNPPEG-Chol and incorporation into the membrane bilayer using time-resolved FRET imaging. Cellular Proliferation Assays. The cytotoxicity of DiOfree and DiO-LCNPs were determined using the CellTiter 96 Aqueous One Solution MTS Cell Proliferation Assay (Promega, Madison, WI). This assay measures the proliferation of cells that have been incubated with a dose range of DiOfree and DiO-LCNPs constructs and is based upon the conversion of a tetrazolium substrate to a soluble formazan product by viable cells following a suitable proliferation period. HEK 293T/17 cells were seeded to the wells of 96-well tissue culture plates (∼5 × 103 cells/well). To each well was added DMEMHEPES containing increasing concentrations of DiOfree or DiOLCNPs and solutions were incubated on the cells for 30 min. After incubation, the materials were replaced with complete growth medium and the cells were cultured for 72 h. After this proliferation period, 20 μL of the tetrazolium substrate was added to each well and color formation proceeded at 37 °C for 4 h. The absorbance of the formazan product was read at 570 nm (absorption minima for the DiO-LCNPs used in this study) and 650 nm (for subtraction of nonspecific background absorbance) using a Tecan Infinite M1000 (Tecan, USA) microtiter plate reader. Absorbance values with the background subtracted were plotted as a function of material concentration and reported as percent of control cell proliferation (degree of proliferation of cells in cell culture media only). Data Analysis. The data were statistically analyzed by the univariate analysis of variance (ANOVA) using GraphPad Prism 6.01 software for Windows (La Jolla, CA). For multiple comparisons, Bonferroni’s post hoc test was applied. All average values were given ± standard error of mean (SEM) unless otherwise mentioned. The acceptable probability for significance was p < 0.05.

the refractive indexes of the corresponding solutions. All absorbance and fluorescence emission spectra were collected on a Tecan Infinite M1000 dual monochromator multifunctional microtiter plate reader (Tecan, U.S.). The DiO loading into the LCNP core, determined as the ratio of DiO:LCNP concentration, was ∼50 or 120 DiO molecules per LCNP, which tracked with the amount of DiOfree included in the reaction mixture during synthesis. For cellular delivery experiments, the NPs containing the higher degree of DiO loading were used. Cell Culture, Cellular Delivery/Staining, and Imaging. HEK 293T/17 cells (ATCC, Manassas, VA) were cultured as described previously25 and all cellular delivery experiments were performed using cells between passages 5 and 15. Cells were seeded to 35 mm Petri dish with 14 mm glass bottom insert (#1.0 cover glass, MatTek Corp., MA, USA) at a density of ∼7 × 104 cells/mL (3 mL/well). Dishes were coated with fibronectin (10−20 μg/mL) in DPBS before adding the cell suspension. Stock solutions of DiOfree (2 mM in DMSO) and DiO-LCNPs were diluted to desired concentrations in DMEMHEPES supplemented with 0.1% FBS and incubated on cell monolayers at 37 °C for 15 or 30 min at 37 °C. The solution was removed and cell monolayers were washed with DMEMHEPES and cultured for various time points. The plasma membrane was counterstained with Lissamine Rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (PERhoda, Life Technologies, Grand Island, NY), triethylammonium salt (1−2 μM in DMEM-HEPES) for 15 min at 37 °C. Lipid rafts were stained with Vybrant Lipid Raft Labeling Kit (Alexa Fluor 555, Life Technologies) following the supplied protocol. The endoplasmic reticulum (ER) was labeled with ER-Tracker Red (BODIPY TR Glibenclamide, Life Technologies) following the supplied protocol. After delivery/counterstaining, the cells were washed with DPBS (pH 7.4) followed by fixation with paraformaldehyde (3.7% in DPBS) for 15 min. Cell nuclei were stained with DAPI (2 μg/mL in DPBS) for 15 min at 25 °C followed by washing with DBPS. The fixed cells were stored in 0.05% NaN3 at 4 °C and imaged within 48 h. The cell samples were imaged by differential interference contrast (DIC) and confocal laser scanning microscopy (CLSM) using a Nikon A1RSi confocal microscope equipped with a 405 nm diode laser, 488 nm argon laser line, and a 543 nm HeNe laser with fluorescence detection channels set to the following filters: 425−475 nm (blue), 500−550 nm (green), and 570−620 nm (red) with dichroic mirrors at 405/488/561/ 640 nm. All images were collected using a Plan Apo 60× objective. Laser power, PMT gain, and threshold for a particular channel were held constant across different samples to allow for quantitative analysis. The confocal images were acquired as a zstack comprising sequential/series optical x−y sections taken at 0.5−1.5 μm z-intervals using NIS-Elements AR 4.3 Software (Nikon Co. Ltd., Tokyo, Japan). The images were processed for publication with Adobe Photoshop CS4 (v 11.0). Colocalization analysis was performed using Pearson’s Correlation equation58 included in the NIS-Elements software. The Pearson’s colocalization coefficients (PCC) for green (DiO) and red (PE-Rhoda) channels describe the contribution of each channel to the overall colocalization. The degree of membrane association for DiO or DiO-LCNP-PEG-Chol was compared with PE-Rhoda and quantified as a percent of total contribution from the green and red channels. Förster Resonance Energy Transfer (FRET) Imaging. HEK 293T/17 cells were costained with varying concentrations

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00042. Additional UV/vis and PL spectra of DiO and DiI, and related CLSM images of cells (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. J


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ACKNOWLEDGMENTS This work was supported by the NRL Base Funding Program (Work Unit MA041-06-41-4943). O.N. is supported by a National Research Council Postdoctoral Research Associateship.

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