Article pubs.acs.org/Langmuir
Interactions of Doxorubicin with Organized Interfacial Assemblies. 2. Spectroscopic Characterization Dorota Nieciecka,† Agata Królikowska,† Iwan Setiawan,‡ Pawel Krysinski,*,† and G. J. Blanchard*,‡ †
Faculty of Chemistry, University of Warsaw, 02-093 Warsaw, Pasteur 1, Poland Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States
‡
ABSTRACT: Doxorubicin is an anthracycline that has found wide use as a chemotherapeutic agent, with the primary limitation to its use being cardiotoxicity. Depending on the identity and location of pendent side groups, the anthracyclines exhibit varying degrees of chemotherapeutic activity and toxicity, and a key area of research activity lies in understanding how the structure of the anthracycline influences its interactions with amphiphilic interfaces. We have studied interactions between doxorubicin and interfacial adlayers of octadecylamine (C18NH2), dihexadecylphosphate (DHP), and both monolayers and bilayers of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) on mica using time- and frequencyresolved spectroscopic measurements. We report surfaceenhanced resonance Raman data and fluorescence lifetime and anisotropy imaging data for doxorubicin at these interfaces. For all monolayers, there is a substantial interaction between doxorubicin and the interface. For DMPC bilayers, the extent of the interaction between doxorubicin and the interface depends on how the interface was formed.
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INTRODUCTION The anthracyclines have found wide use as chemotherapeutic agents, with the primary limitation to their use being cardiotoxicity. There are several structurally related anthracyclines in common use, with doxorubicin being used most widely. Doxorubicin is thought to interact with the topoisomerase II-DNA complex,1,2 giving rise to double-stranded breaks of or intercalation into DNA, thereby inhibiting DNA replication and transcription to mRNA.3 In addition to this function, doxorubicin is also known to generate reactive oxygen species catalytically by several routes, and to interfere with iron homeostasis. While the anticancer function of doxorubicin is important, the side effects, up to and including cardiac failure, have led to caution in its use. The structural variety that exists among the anthracyclines has been shown to be related to chemotherapeutic efficacy and toxicity,4,5 and a key area of research activity lies in understanding the structure−property relationship(s) that are operative with this family of compounds.6,7 The manner in which anthracyclines can penetrate plasma membrane structures is, of course, central to their chemotherapeutic action, and the factors important to this process are beginning to emerge. We reported in the preceding paper on the interactions between doxorubicin and biomimetic mono- and bilayer interfaces from an electrochemical perspective.8 In that work we showed that doxorubicin was able to penetrate monolayer structures on a planar Au substrate, but lipid bilayers did not allow for electron transfer between doxorubicin and the electrode surface. These results provided significant insight into © 2013 American Chemical Society
the role of solute hydrophobic character in mediating the interactions between doxorubicin and interfaces. While the electrochemical data provided significant insight into the ability of doxorubicin to interact with biomimetic interfacial adlayer structures, we were ultimately interested in obtaining a clearer understanding of the molecular details of these interactions. To gain more information on the interactions between doxorubicin and interfacial monolayers of octadecylamine (C18NH2), dihexadecylphosphate (DHP), and both mono- and bilayers of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) deposited on mica substrates using Langmuir−Blodgett or Langmuir−Schaeffer deposition,9−11 we have performed both time- and frequency-resolved spectroscopic measurements. We report surface-enhanced resonance Raman scattering (SERRS) data (Ag support) and fluorescence lifetime and anisotropy imaging data for doxorubicin (mica support). For all monolayer structures, there is a substantial interaction between doxorubicin and the interface. For DMPC bilayers, the extent of the interaction between doxorubicin and the interface depends on how the interface was formed. Taken collectively, the data point to interactions between the anthracycline and the interfacial structures where the doxorubicin experiences both the adlayer polar headgroup and the aliphatic nonpolar environments. Received: September 29, 2013 Revised: October 31, 2013 Published: November 1, 2013 14570
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SERS Active Substrate Preparation. SERS-active substrates were prepared employing an electrochemical roughening procedure involving oxidation and reduction cycling (ORC), as described elsewhere.12 The ORC-roughened silver electrodes were milkybrown when exhibiting the highest SERS activity. Roughened silver substrates were rinsed thoroughly with the ultrapure water immediately following the roughening procedure and were then stored in it, prior to further adsorption of the analyte. Fluorescence Lifetime and Anisotropy Imaging Measurements. The instrument used to acquire fluorescence lifetime and anisotropy images is based on an inverted microscope (Nikon Eclipse Ti−U) equipped with a Hg lamp illuminator for steady state fluorescence images. The microscope is equipped with 10x, 20x, 40x, 60x, and 100x objectives. The microscope is equipped with polarized dual channel confocal scanner (Becker & Hickl DCS-120) that is driven by a galvo-drive unit (Becker & Hickl GDA-120) and with two avalanche diode detectors (ID-Quantique ID100) for the acquisition of fluorescence lifetime and anisotropy decay images. The transients used to create these images are acquired using time-correlated single photon counting electronics (Becker & Hickl SPC-152, PHD-400N reference diode) characterized by sub-100 ps time resolution. The TCSPC and confocal scanning instruments are controlled by Becker & Hickl software run on a PC. The light source for this instrument is a synchronously pumped cavity dumped dye laser (Coherent 702) excited by the output of a passively mode locked Nd:YVO4 laser (Spectra Physics Vanguard) that produces 13 ps pulses at 80 MHz repetition rate with 2.5 W average power at 355 and 532 nm. The dye laser is cavity dumped (Gooch and Housego 64389.5-SYN-9.5-1 cavity dumper driver) to control the repetition rate. The output of the dye laser is characterized by 5 ps pulses with a repetition rate that is variable between 80 kHz and 80 MHz. For the measurements reported here, the repetition rate is 4 MHz and the average power at the sample is less than 0.5 mW. The dye laser output can be tuned from 430 to 850 nm depending on the dye and optics used. A wavelength of 480 nm was used for sample excitation in this work.
MATERIALS AND METHODS
Reagents. Reagents used in this work were of the highest purity available commercially and were used as received. Doxorubicin hydrochloride, dihexadecylphosphate (98%), and octadecylamine (98%) were all purchased from Aldrich. DMPC (1,2-dimyristoyl-snglycero-3-phosphocholine, >99% purity) was from Avanti Polar Lipids. Ethanol (99.8% purity) and chloroform were obtained from Chempur (Poland). Aqueous solutions were prepared with Milli-Q water (resistivity 18.2 MΩ-cm) obtained from a Millipore system. Monolayer Preparation. Monolayers of C18NH2, DHP and DMPC were prepared by dissolving each compound in chloroform to obtain a concentration of 2 mg/mL in the monolayer spreading solution. Langmuir monolayers were spread from 15 μL of solution deposited on the aqueous subphase of the Langmuir trough. After solvent evaporation (ca. 10 min.), the monolayer was compressed continuously at a rate of 30 cm2/min to obtain pressure−area isotherms in an isotherm cycling mode set for 2 cycles of compression−decompression. This mode of operation allowed us to obtain at least 2 isotherms and to evaluate the reproducibility and monolayer type. For Langmuir−Blodgett transfer onto the mica, cleaned substrates were immersed into the subphase immediately prior to spreading the monolayer-forming chloroform solution at the air/ water interface. After the monolayer-forming solution was applied, the chloroform was allowed to evaporate, and the monolayer was compressed to a desired target pressure (40 mN/m) that was kept constant (PC-controlled) during the upstroke Langmuir−Blodgett transfer. Bilayer Preparation by Langmuir−Schaeffer transfer (L-S, horizontal touch). This procedure is similar to that used for the Langmuir−Blodgett (L-B) technique. The L-B monolayers were allowed to dry in air for two hours before the next transfer. After the trough was filled, the monolayer was compressed to the required pressure, 40 mN/m. The L-B DMPC monolayer-covered substrate was mounted on the dipper parallel to the water surface with the DMPC layer facing the trough surface. The substrate was lowered until it touched the compressed monolayer, and allowed to equilibrate for ca. 5 min. Then, the horizontal touch transfer was performed at a barrier speed of 30 cm2/min and a dipper speed 5 mm/min (upstroke). Bilayer Preparation by Vesicle Formation and Fusion. Chloroform was evaporated from lipid solution to dryness under nitrogen. Tris buffer solution was added to dry films of DMPC for a final lipid concentration of 1 mg/mL. The lipid solution was processed five times through a freeze−thaw−vortex cycle. Each cycle consisted of immersing the sample in liquid nitrogen (5 min), then heating in a 60 °C water bath (5 min), then vortexing the thawed sample for 2 min. The resulting solution was extruded 11 times through a polycarbonate membrane with a nominal pore diameter of 100 nm to produce 100 nm diameter unilamellar vesicles. For deposition by fusion, 20 μL of the vesicle-containing solution was placed on a planar mica support (ca. 1 cm2) followed by the addition of 60 μL of a pH 7.5 buffer solution of Trizma (10 mM) and NaCl (100 mM), and 7 μL of CaCl2 (2 mM). The resulting vesicle-containing solution was allowed to remain in contact with the mica support for 5 min before rinsing with ∼2 mL of Tris buffer solution. Vesicle fusion was carried out at room temperature. Surface-Enhanced Resonance Raman Scattering (SERRS). SERRS spectra of doxorubicin on the chemically modified solid Ag substrates were collected in a backscattering geometry with a Labram HR800 (Horiba JobinYvon) confocal microscope system, equipped with a Peltier-cooled CCD detector (1024 × 256 pixels). A diode pumped, frequency doubled Nd:YAG laser provided 532 nm excitation radiation, with a total power of less than 0.5mW at the sample. The confocal pinhole size was set to 200 μm and a holographic grating with 600 grooves/mm was used. The calibration of the instrument was performed using the 520 cm−1 Raman band of silicon. SERRS spectra were obtained using a 50x Olympus objective and accumulated from 1 to 2 scans, with 60s or 30s integration time, respectively.
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RESULTS AND DISCUSSION
In the preceding paper8 we reported on the electrochemical investigation of interactions between doxorubicin and the analogous mono- and bilayer interfaces supported on Au. That work showed that there was an interaction between doxorubicin and the interfacial monolayers but more detailed information was not available. For this reason we have chosen to investigate the spectroscopic response of doxorubicin within these interfaces. Our findings shed light on the molecular-scale interactions between the adlayer constituents and doxorubicin and, while these interactions are qualitatively similar for all monolayers, they differ in detail. We examine the interactions through a SERRS investigation of doxorubicin and through a fluorescence lifetime and anisotropy imaging study. We consider these data sets individually before drawing larger conclusions from the entire body of information. SERRS Spectra. The interactions of doxorubicin with the C18NH2, DHP, and DMPC monolayers were studied by means of surface enhanced resonance Raman spectroscopy (SERRS). The wavelength of the laser is close to the first singlet electronic transition of doxorubicin, and vibrational modes associated with the chromophoric part of the drug are enhanced selectively.13 SERRS data were collected for the monolayers deposited on roughened Ag and exposed to a 1 × 10−5 M aqueous doxorubicin solution. SERRS spectra were collected at 1 min intervals for 1 h, in order to monitor the penetration/ adsorption of doxorubicin into/onto the biomimetic films. For all of the monolayer structures studied, there was no chemical functionality capable of chemisorption onto the Ag substrate. The binding of the monolayer constituents to the 14571
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wavenumber components, respectively. The spectral differences between the one- and two-component monolayers is consistent with the single component DHP layer interfering with intramolecular hydrogen bonding within the doxorubicin dihydroxyanthraquinone structure. A clear implication of this finding is that doxorubicin in the DHP monolayer is in relatively close proximity to the phosphate headgroup, a finding that is consistent with the chromophore residing in a location where there is a gradient in the dielectric constant over molecular length scales (vide inf ra). The second type of SERRS spectrum seen for doxorubicin in the DHP monolayer (top spectrum in Figure 1a) exhibits the splitting of a single band at ca. 445 cm−1 (seen in the lower spectrum in Figure 1a). This band is associated with CO deformation, while the doublet at 1215 cm−1 and 1245 cm−1 (δ(C−O−H)) exhibits a change in the relative band intensities. These spectral changes cause the recorded spectrum to resemble more closely the resonance Raman spectrum of doxorubicin in solution.14−16 In addition, the position of the ring breathing mode at 995 cm−1 is better matched with that found for solution phase doxorubicin. In the top spectrum, a shoulder near 1460 cm−1 appears in addition to the two other components in this region (around 1415 cm−1 and 1440 cm−1), ascribed to CC and C−C stretching modes of the aromatic carbons. The differences seen for these spectra suggest that the structure of doxorubicin is distorted relatively little compared to its native solution phase conformation, but the band associated with the stretching of carbonyl groups engaged in hydrogen bonding at 1635 cm−1 is still not visible. The SERRS spectrum of doxorubicin in the C18NH2 monolayer on Ag is shown in Figure 1b. For this interfacial system, only one SERRS spectrum is seen, over a variety of locations on a given SERS substrate and for the interface assembled on different roughened silver supports taken from different experimental batches. The spectrum shown for doxorubicin in the C18NH2 interface is very similar to the spectrum seen for the second type of DHP monolayer (Figure 1a), and we make the same spectral assignments and interpretations, accordingly. We show in Figure 1c the SERRS spectra of doxorubicin in a DMPC monolayer deposited on a silver support. Similar to the analogous data for the doxorubicin/DHP system, two characteristic types of SERRS spectra are seen. These two spectra are seen either for different locations on a given Ag substrate or for different doxorubicin/DMPC interfacial assemblies. Qualitatively, these two spectral types are the same as those seen for the doxorubicin/DHP interface, with the only difference being for the bottom spectrum in Figure 1c, where the band indicative of (OH···OC) stretching at 1635 cm−1 is slightly pronounced. A common thread of the SERRS data for all three monolayer systems studied here is that the structure of doxorubicin appears to be less disrupted, compared to that in the solution phase, than was seen when this compound was integrated into mixed, thiol-containing monolayers.12 One possible reason for this finding may be the existence of greater spatial freedom in the single component layers examined here, and we examine this issue with fluorescence anisotropy imaging measurements (vide inf ra). It is useful to keep in mind that for the monolayers examined in this work, there is no chemical bonding to the support, as was the case for the thiol-containing mixed monolayers. We note that more than one conformation of doxorubicin was seen in the SERRS data for both DMPC and DHP monolayers, and only one conformation found for
metallic silver support was by means of interactions between the metal surface and the amphiphile polar headgroup, initiated during the monolayer transfer by Langmuir−Blodgett deposition. The resulting interfaces, after the deposition of doxorubicin, produced Raman spectral data that were specific to each interface. Interpretation of the SERRS data was performed in the context of existing vibrational mode assignments.14−17 Two spectral profiles were seen for doxorubicin contained in the DHP monolayer (Figure 1a). The two characteristic
Figure 1. Surface-enhanced resonance Raman scattering spectra (after reaching stable intensity) of (a) DHP, (b) C18−NH2, and (c) a monolayer of DMPC formed by L-B deposition on SERS-active Ag substrates.
spectral types are seen for different locations on a given Ag substrate as well as for different doxorubicin/DHP interfacial assemblies. The lower spectrum resembles the analogous data acquired for doxorubicin interacting with a mixed monolayer composed of C18−SH and DHP, which has been reported previously.12 The differences between the spectrum reported here and that for the mixed monolayer are the absence of a band at 1635 cm−1 attributed to CO stretching of hydrogenbonded carbonyl groups (OH···OC) and the splitting of a broad band at 1325 cm−1, seen for the mixed monolayer, into two components, at 1310 cm−1 and 1340 cm−1. The presence of the two bands can be ascribed to the coupling of the ν(C− O) and ν(C−C) (ring stretching) modes for the low and high 14572
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Figure 2. Doxorubicin in a C18−NH2 monolayer on mica. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a three-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 200 ± 50 ps.
doxorubicin in the C18NH2 monolayer. The interaction between the amine headgroup of C18NH2 and the Ag support is expected to be stronger than phosphate interactions with Ag.18 The electrochemical data presented in the preceding paper show multiple redox peaks for doxorubicin in the C18NH2 and DHP interfaces and a single, reversible peak for doxorubicin in DMPC. The electrochemical and SERRS data thus indicate collectively that the interfaces exhibit subtle structural diversity, but the two techniques are sensitive to different aspects of the doxorubicin local environment. The prominent SERRS signal seen for doxorubicin for all of the monolayer structures examined is confirmation that the compound is in sufficiently close proximity to the Ag substrate to experience some level of surface enhancement. Doxorubicin is thus in intimate contact with the aliphatic chain region and likely is located near the polar headgroups of the monolayers. We note that the SERRS signal from doxorubicin dominates the SERS signal associated with the monolayer species due to the effect of resonance enhancement. The SERRS signal for doxorubicin was not diminished greatly by rinsing of the monolayers with water, indicating that this compound has a significant affinity for the nonpolar aliphatic region of the monolayers examined. The inability of doxorubicin to penetrate the DMPC bilayer structure, as seen in the electrochemical data, is consistent with the SERRS measurements reported here, where there was no detectable SERRS response for doxorubicin. We interpret the absence of SERRS signal for this
system as indicating the inability of doxorubicin to locate in sufficiently close spatial proximity to the Ag support to exhibit a large surface enhancement effect. Neither the electrochemical or SERRS data for the doxorubicin/DMPC bilayer system should be taken as an indication that doxorubicin is not interacting with the bilayer, but that whatever interaction is occurring does not place the compound in sufficiently close proximity to the support to allow either Raman surface enhancement or facile electron transfer. In fact, fluorescence lifetime and anisotropy imaging data (vide inf ra) indicate the presence of doxorubicin in/on the bilayer system. Fluorescence Lifetime and Anisotropy Imaging. As noted in the previous section, it appears that doxorubicin is in relatively intimate contact with the nonpolar and headgroup regions of the monolayer structures we have examined. The information provided by electrochemical and SERRS data is not sufficient, by itself, to provide further insight into the manner in which the monolayers interact with doxorubicin. For this reason, we have undertaken a series of fluorescence lifetime and anisotropy imaging measurements, with the goal of revealing complementary information on the nature of the dielectric environment (fluorescence lifetime) and the confinement (fluorescence anisotropy) experienced by doxorubicin. The instrument we use to obtain these data is a confocal scanning system where the monolayer sample is excited by a polarized light pulse and polarized fluorescence transients are acquired pixel-by-pixel across the sample. For each pixel (the images 14573
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Figure 3. Doxorubicin in a DHP monolayer on mica. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a three-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 93 ± 32 ps.
shown in Figures 2−6 are 256 × 256 pixels), the polarized fluorescence transients, I||(t) and I⊥(t), are combined according to eqs 1 to produce fluorescence lifetime and fluorescence anisotropy decay transients, respectively.
quantitative information from these imaging data, we take blocks of data, sum the data contained in these regions, and then analyze the resulting transients using software that is known to produce accurate results. Thus the imaging data in the left-most panels of Figures 2−6 provide color images of the spatial heterogeneity of the lifetime (top) and anisotropy decay (bottom) time constants. The distributions shown in the center panels should be taken as instructive in a qualitative sense only. The quantitative results are shown in the right panels of each figure and in Table 1. For all of the monolayer systems studied here, we observe a complex fluorescence lifetime decay functionality that requires three exponential components to fit. These data are significantly different than the fluorescence lifetime of doxorubicin in solution, where a single exponential decay with a time constant of 1.1 ns is seen.19 There are two possible explanations for the complexity of the fluorescence decay functionality. The first is that doxorubicin is distributed in these monolayer films in such a manner that, for a given monolayer, there are three (or more) distinct environments in which the chromophore resides, and these environments do not exchange population on a time scale that is comparable to the fluorescence lifetime. Such an interpretation implies a system where neither translational nor rotational dynamics occur rapidly, and this is not the case for a monolayer that is physisorbed to a support. We note that the lifetime distribution
Ifl(t ) = I + 2I⊥(t ) R (t ) =
I (t ) − I⊥(t ) Ifl(t )
(1)
The information contained in the fluorescence lifetime data is of use in determining the local dielectric environment of the chromophore, while the anisotropy (R(t)) data provide information on the rotational motion of the molecule. Both types of information are complementary to the electrochemical and SERRS data on these interfaces. Before examining the imaging results shown in Figures 2−6, a few caveats are in order. The functional form of the timedomain transients that comprise these images is sufficiently complex that the commercial software does not provide quantitatively accurate values for either the fluorescence lifetime or anisotropy decay time constants. This issue is exacerbated by the limited signal-to-noise ratio of the data contained in individual pixels. Despite these limitations, the results from the imaging software are useful in providing the functional form of the distributions of lifetimes and anisotropy decay times for these images because they apply the same mathematical algorithm to all of the data present. To obtain 14574
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Figure 4. Doxorubicin in a DMPC monolayer on mica. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a three-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 124 ± 32 ps.
Anisotropy decay data provide information on molecular rotational motion and, as such, afford insight into the nature and extent of confinement the chromophore experiences within the monolayer structure. There is a well-established body of theory that is used to interpret anisotropy decay data.26−29 In this instance, the chromophore is not bound to the monolayer constituents and is nominally free to rotate. Accordingly, we model the motion of doxorubicin in these monolayers in the context of the free rotor model. Chuang and Eisenthal have developed a general theory that relates anisotropy decay data, R(t), to the Cartesian components of the chromophore rotational diffusion constant (D = 1/3(Dx + Dy + Dz)) and the orientations of the absorbing and emitting transition dipole moments.26 There are two limiting cases of interest. These are when the absorbing and emitting transition dipole moments are nominally parallel, and two of the three Cartesian axes are assumed to be equal. The third, unique axis can either be coincident with the transition moments or normal to them. For the former case, the volume swept out by the rotating chromophore is modeled as a prolate ellipsoid and for the latter case the rotating chromophore is modeled as an oblate ellipsoid (eqs 2 and 3).30
data (Figures 2−6 center panels) does not show any evidence for isolated, nonoverlapped lifetime distributions. The second explanation for a multiple component exponential decay in a fluorescence lifetime measurement is that the chromophore resides in an environment where there is a significant variation in the dielectric response of the medium on the length scale of the emitting moiety.20−25 For the monolayer systems we have constructed, a dielectric gradient could exist normal to the plane of the support and in close proximity to the support surface. In the region where the amphiphilic monolayer is sitting on top of the support, likely with a cushion of several Å of water, the dielectric gradient normal to the surface can be significant owing to the presence of the surface, water, the amphiphile headgroup and then the amphiphile aliphatic chain region. The lifetime data we show in Figures 2−5 is consistent with doxorubicin residing in the monolayer in a region characterized by a large dielectric gradient, i.e. near the amphiphile headgroup, and this finding is in agreement with the SERRS data. We note that the regressed lifetime components are similar for our interfaces, but we do not attempt to extract any information of chemical significance from these fitted values because there is no theoretical framework in which such quantities can be related to system properties. The information contained in these data is that doxorubicin is in a region of the monolayer characterized by a substantial dielectric gradient normal to the surface plane.
R(t ) = 0.4 exp( −6Dz t )
(prolate)
(2)
R(t ) = 0.1 exp( −(2Dx + 4Dz )t ) + 0.3 exp( −6Dx t ) (oblate) 14575
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Figure 5. Doxorubicin in a DMPC bilayer on mica, deposited by the Langmuir−Blodgett/Langmuir−Schaefer method. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a three-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 98 ± 12 ps.
image individually, but it is important to note that these monolayer interfaces exhibit varying degrees of heterogeneity, depending on the specific interface. The extent of this spatial heterogeneity is evaluated in greater detail for these systems through signal averaging of the transients contained in the white, numbered boxes. In addition to revealing the spatial heterogeneity of dynamics within the layers, these data also provide valuable comparative information on the extent and nature of chromophore confinement for the different monolayer structures. As noted above, the anisotropy decay images and distributions (Figures 2−6, bottom row, left and center panels) provide valuable qualitative information, but quantitation requires more detailed analysis. Obtaining meaningful anisotropy decay time constants required data with a higher signalto-noise ratio than that available from a single pixel. For this reason, we summed the polarized transients in the areas that are boxed in Figures 2−6, and from these summed pixels we generated R(t) decay functions. For all samples, we obtained anisotropy decays characterized by single exponential decay components (Table 1). This finding is consistent with the chromophore residing in a single chemical environment that is characterized by a dielectric gradient. To ensure that the utilization of data within boxed regions did not distort our results, we selected several different locations on each interfacial assembly to analyze (see Figures 2−6) and evaluated several
For all of the data we report here, doxorubicin is seen to produce anisotropy decays characterized by a single exponential functionality (eq 2). The transition dipole moments lie along the doxorubicin long, in-plane axis, which we designate as x, and the only motional component we sense is that perpendicular to the x-axis. Because we recover only one of the Cartesian components of D, we are left to assume that Dz ∼ D, and with an estimate of D we can compare the local environments formed by the monolayer. The anisotropy decay time constant is 6D−1, which is related to the local viscosity sensed by the chromophore, its hydrodynamic volume, and the thermal energy in the system through the modified Debye− Stokes−Einstein (DSE) equation,27−29 τOR = 6D−1 =
ηVf kBTS
(4)
where η is the bulk viscosity of the immediate environment of the chromophore, V is the hydrodynamic volume on doxorubicin (V = 456 Å3),31 f is a frictional term to describe the interactions between doxorubicin and the surrounding monolayer,28 S is a shape factor to account for the nonspherical shape of doxorubicin29 and kBT is the Boltzmann thermal energy term. The images presented in Figures 2−6 are maps of the anisotropy decay time constant, τOR, as a function of position. We will consider the information content of each 14576
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Figure 6. Doxorubicin in a DMPC bilayer on mica, deposited by vesicle fusion. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a one-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 260 ± 30 ps.
Table 1. Fluorescence Lifetime and Anisotropy Decay Time Constants adlayer on mica C18NH2 monolayer DHP monolayer DMPC monolayer DMPC bilayer (L-B/L-S) DMPC bilayer (vesicle fusion)
τfl(1) (ps) 68 51 37 67
± ± ± ±
1 (79%) 1 (85%) 1 (86%) 1 (80%) --
τfl(2) (ps)
τfl(3) (ps)
⟨τfl⟩ (ps)
τOR (ps)
351 ± 25 (16%) 248 ± 9 (12%) 178 ± 3 (11%) 255 ± 8 (17%) --
1436 ± 111 (5%) 1193 ± 47 (3%) 1099 ± 13 (3%) 1230 ± 43 (3%) 1173 ± 1
177 106 81 135 1173
200 ± 50 93 ± 32 124 ± 32 98 ± 13 260 ± 30
reorientation times of ca. 100 ps (Table 1). Interpreting these results in the context of eq 4, where V = 456 Å3, and we take S = 129 and f = 128 in the absence of information that could cause these values to deviate slightly, we estimate the local viscosity of the interface sensed by doxorubicin to be ca. 0.9 cP for the DHP and DMPC monolayers and to be ca. 1.8 cP for the C18NH2 monolayer. We recognize that these are estimates of the local viscosity and are limited by our assumptions of values for S and f, but the data point to the doxorubicin being in a less constrained environment for the DHP and DMPC monolayers than it is in the C18NH2 monolayer, and this makes sense in the context of the strength of amphiphile-headgroup interactions. We note that this finding is consistent with our SERRS data. In all cases, the recovered viscosity estimates indicate that the chromophore resides in an environment of limited restriction,
smaller subsections of the boxed regions to determine whether the average values for the decay constants were sample size invariant, and they were (data not shown). The physical and chemical significance of these anisotropy decay data lies in how their values vary, or not, depending on the identity of the interfacial layer. There can be two contributions to the anisotropy decay: orientational relaxation of individual chromophores and energy transfer between identical chromophores. The efficiency of this latter effect will scale inversely with the average distance between chromophores. For our bilayers, that average distance is ca. 80 Å, more than the critical radius. Thus the R(t) decay time constants we report are associated with molecular motion. The reorientation data for these interfaces falls into two broad categories, with the C18NH2 interface producing doxorubicin reorientation times of ca. 200 ps and the DHP and DMPC monolayers producing 14577
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*E-mail: [email protected]; tel.: (+4822) 8220211 x389; Fax: (+4822) 8225996.
quite likely with the opportunity for exposure to the aqueous solution overlayer. Examining the analogous data for DMPC bilayer structures likewise provides useful insights. We have formed DMPC bilayers according to two methods, Langmuir−Blodgett/ Langmuir−Schaeffer deposition and vesicle fusion, and the results for bilayers formed by these two methods differ markedly. The reorientation time constant recovered for the L-B/L-S DMPC bilayer is the same to within the experimental uncertainty as that measured for the DMPC monolayer, ca. 100 ps, indicating a similar local environment for the two structures. For the bilayer formed by vesicle fusion, we recover a reorientation time constant of 260 ps, substantially longer than that of the L-B/L-S bilayer. This finding indicates that the bilayer formed by vesicle fusion is more organized (and thus more confining) than the L-B/L-S monolayer. We note that the electrochemical data for doxorubicin in the L-B/L-S DMPC bilayer shows the absence of electron transfer across this interface, and the fluorescence lifetime data for this interface is of the same multiexponential functionality as was seen for the monolayers, demonstrating that the doxorubicin is within the bilayer and senses the lipid headgroups (vide supra). These findings suggest collectively that the L-S overlayer plays an important role in isolating the doxorubicin from the solution overlayer and precluding access to protons. The dynamics and lifetime information for bilayers formed by fusion on mica using vesicles that were extruded (ca. 100 nm diameter) in the presence of doxorubicin reveal significantly different information. The reorientation time for DMPC bilayers formed by vesicle fusion is more than twice as long as that of the L-B/L-S bilayers. We assert that because this reorientation time is much longer than that expected for aqueous solution and that we obtained data with a high S/N ratio (Figure 6), the doxorubicin is indeed contained in the bilayer structure. The fluorescence lifetime for doxorubicin in the bilayer formed by vesicle fusion exhibits a single exponential decay functionality, in sharp contrast to that seen for the other interfaces reported here. We understand this finding in the context of the doxorubicin being sequestered within the nonpolar region of the bilayer and that the bilayer structure is sufficiently well organized to minimize the access of doxorubicin to the lipid headgroup region.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a Project PSPB-079/2010 Grant from Switzerland through the Swiss Contribution to the enlarged European Union. G.B. gratefully acknowledges support by the Donors of the ACS Petroleum Research Fund. The fluorescence lifetime and anisotropy imaging instrument was purchased using funds from National Science Foundation Grant CHE 1048548.
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CONCLUSIONS The time- and frequency-domain spectroscopic data we have reported here point collectively to the extent of organization of the interfaces we have formed. Raman spectroscopic data show the interactions, or not, of doxorubicin with the interfacial adlayers, while the time-domain lifetime and anisotropy decay data reflect the extent to which the chromophore is constrained by its immediate environment and what access it has to the headgroup region of the interface. These findings are fully consistent with the electrochemical data in the preceding paper and, taken as a whole, demonstrate that doxorubicin has a substantial affinity for hydrophobic environments, a factor that may play a role in the transport of anthracyclines across bilayer membranes.
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REFERENCES
(1) Capanico, G.; Zunino, F. Molecular Basis of Specificty in Nucleic Acid−Drug Interactions; Pullman, B., Jortner, J., Eds. ; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1990, p 167−176. (2) Duguet, M.; Lavenot, C.; Harper, F.; Mirambeau, G.; DeRecondo, A.-M. DNA topoisomerases from rat liver: Physiological variations. Nucleic Acids Res. 1983, 11, 1059−1075. (3) D’Arpa, P.; Liu, L. F. Topoisomerase-targeting antitumor drugs. Biochim. Biophys. Acta 1989, 989, 163−177. (4) Cortes-Funes, H.; Coronado, C. Role of anthracyclines in the era of targeted therapy. Cardiovasc. Toxicol. 2007, 7, 56−60. (5) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: Molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004, 56, 185−229. (6) Nawara, K.; McCracken, J. L.; Krysinski, P.; Blanchard, G. J. Structure dependent complexation of Fe3+ by anthracyclines. 1. The importance of pendent hydroxyl functionality. J. Phys. Chem. B 2013, 117, 6859−6867. (7) Nawara, K.; Beeckman, H.; Krysinski, P.; Blanchard, G. J. Structure dependent complexation of Fe3+ by anthracyclines. 2. The roles of methoxy and daunosamine functionalities. J. Phys. Chem. B 2013, 117, 6868−6873. (8) Nieciecka, D.; Joniec, A.; Setiawan, I.; Blanchard, G. J.; Krysinski, P. Interactions of doxorubicin with organized interfacial assemblies. 1. Electrochemical characterization. Langmuir 2013, DOI: 10.1021/ la403765w. (9) Langmuir, I. The adsorption of gases on plane surfaces of glass, mica and platinum. J. Am. Chem. Soc. 1918, 40, 1361−1403. (10) Blodgett, K. B. Films built by depositing successive monomolecular layers on a solid surface. J. Am. Chem. Soc. 1935, 57, 1007−1022. (11) Langmuir, I.; Schaefer, V. J. Rates of evaporation of water through compressed monolayers on water. J. Franklin Inst. 1943, 235, 119−162. (12) Nieciecka, D.; Krolikowska, A.; Joniec, A.; Krysinski, P. Partitioning of doxorubicin into Langmuir and Langmuir−Blodgett biomimetic mixed monolayers: Electrochemical and spectroscopic studies. J. Electroanal. Chem. 2013, DOI: 10.1016/j.jelechem.2013.03.004. (13) Manfait, M.; Bernard, L.; Theophanides, T. Resonance and preresonance Raman spectra of the antitumor drugs adriamycin and daunomycin. J. Raman Spectrosc. 1981, 11, 68−74. (14) Angeloni, L.; Smulevich, G.; Marzocchi, M. P. Absorption, fluorescence and resonance Raman spectra of adriamycin and its complex with DNA. Spectrochimica Acta A 1982, 38, 213−217. (15) Smulevich, G.; Feis, A. Surface-enhanced resonance Raman spectra of adriamycin, 1-deoxycarminomycin, their model chromophores, and their complexes with DNA. J. Phys. Chem. 1986, 90, 6388−6392. (16) Nonaka, Y.; Tsuboi, M.; Nakamoto, K. Comparative study of aclacinomycin vs. adriamycin by means of resonance spectroscopy. J. Raman Spectrosc. 1990, 21, 133−141.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]; tel.: (+011) 517-3559715 x224. 14578
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Interactions of Doxorubicin with Organized Interfacial Assemblies. 2. Spectroscopic Characterization Dorota Nieciecka,† Agata Królikowska,† Iwan Setiawan,‡ Pawel Krysinski,*,† and G. J. Blanchard*,‡ †
Faculty of Chemistry, University of Warsaw, 02-093 Warsaw, Pasteur 1, Poland Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States
‡
ABSTRACT: Doxorubicin is an anthracycline that has found wide use as a chemotherapeutic agent, with the primary limitation to its use being cardiotoxicity. Depending on the identity and location of pendent side groups, the anthracyclines exhibit varying degrees of chemotherapeutic activity and toxicity, and a key area of research activity lies in understanding how the structure of the anthracycline influences its interactions with amphiphilic interfaces. We have studied interactions between doxorubicin and interfacial adlayers of octadecylamine (C18NH2), dihexadecylphosphate (DHP), and both monolayers and bilayers of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) on mica using time- and frequencyresolved spectroscopic measurements. We report surfaceenhanced resonance Raman data and fluorescence lifetime and anisotropy imaging data for doxorubicin at these interfaces. For all monolayers, there is a substantial interaction between doxorubicin and the interface. For DMPC bilayers, the extent of the interaction between doxorubicin and the interface depends on how the interface was formed.
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INTRODUCTION The anthracyclines have found wide use as chemotherapeutic agents, with the primary limitation to their use being cardiotoxicity. There are several structurally related anthracyclines in common use, with doxorubicin being used most widely. Doxorubicin is thought to interact with the topoisomerase II-DNA complex,1,2 giving rise to double-stranded breaks of or intercalation into DNA, thereby inhibiting DNA replication and transcription to mRNA.3 In addition to this function, doxorubicin is also known to generate reactive oxygen species catalytically by several routes, and to interfere with iron homeostasis. While the anticancer function of doxorubicin is important, the side effects, up to and including cardiac failure, have led to caution in its use. The structural variety that exists among the anthracyclines has been shown to be related to chemotherapeutic efficacy and toxicity,4,5 and a key area of research activity lies in understanding the structure−property relationship(s) that are operative with this family of compounds.6,7 The manner in which anthracyclines can penetrate plasma membrane structures is, of course, central to their chemotherapeutic action, and the factors important to this process are beginning to emerge. We reported in the preceding paper on the interactions between doxorubicin and biomimetic mono- and bilayer interfaces from an electrochemical perspective.8 In that work we showed that doxorubicin was able to penetrate monolayer structures on a planar Au substrate, but lipid bilayers did not allow for electron transfer between doxorubicin and the electrode surface. These results provided significant insight into © 2013 American Chemical Society
the role of solute hydrophobic character in mediating the interactions between doxorubicin and interfaces. While the electrochemical data provided significant insight into the ability of doxorubicin to interact with biomimetic interfacial adlayer structures, we were ultimately interested in obtaining a clearer understanding of the molecular details of these interactions. To gain more information on the interactions between doxorubicin and interfacial monolayers of octadecylamine (C18NH2), dihexadecylphosphate (DHP), and both mono- and bilayers of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) deposited on mica substrates using Langmuir−Blodgett or Langmuir−Schaeffer deposition,9−11 we have performed both time- and frequency-resolved spectroscopic measurements. We report surface-enhanced resonance Raman scattering (SERRS) data (Ag support) and fluorescence lifetime and anisotropy imaging data for doxorubicin (mica support). For all monolayer structures, there is a substantial interaction between doxorubicin and the interface. For DMPC bilayers, the extent of the interaction between doxorubicin and the interface depends on how the interface was formed. Taken collectively, the data point to interactions between the anthracycline and the interfacial structures where the doxorubicin experiences both the adlayer polar headgroup and the aliphatic nonpolar environments. Received: September 29, 2013 Revised: October 31, 2013 Published: November 1, 2013 14570
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SERS Active Substrate Preparation. SERS-active substrates were prepared employing an electrochemical roughening procedure involving oxidation and reduction cycling (ORC), as described elsewhere.12 The ORC-roughened silver electrodes were milkybrown when exhibiting the highest SERS activity. Roughened silver substrates were rinsed thoroughly with the ultrapure water immediately following the roughening procedure and were then stored in it, prior to further adsorption of the analyte. Fluorescence Lifetime and Anisotropy Imaging Measurements. The instrument used to acquire fluorescence lifetime and anisotropy images is based on an inverted microscope (Nikon Eclipse Ti−U) equipped with a Hg lamp illuminator for steady state fluorescence images. The microscope is equipped with 10x, 20x, 40x, 60x, and 100x objectives. The microscope is equipped with polarized dual channel confocal scanner (Becker & Hickl DCS-120) that is driven by a galvo-drive unit (Becker & Hickl GDA-120) and with two avalanche diode detectors (ID-Quantique ID100) for the acquisition of fluorescence lifetime and anisotropy decay images. The transients used to create these images are acquired using time-correlated single photon counting electronics (Becker & Hickl SPC-152, PHD-400N reference diode) characterized by sub-100 ps time resolution. The TCSPC and confocal scanning instruments are controlled by Becker & Hickl software run on a PC. The light source for this instrument is a synchronously pumped cavity dumped dye laser (Coherent 702) excited by the output of a passively mode locked Nd:YVO4 laser (Spectra Physics Vanguard) that produces 13 ps pulses at 80 MHz repetition rate with 2.5 W average power at 355 and 532 nm. The dye laser is cavity dumped (Gooch and Housego 64389.5-SYN-9.5-1 cavity dumper driver) to control the repetition rate. The output of the dye laser is characterized by 5 ps pulses with a repetition rate that is variable between 80 kHz and 80 MHz. For the measurements reported here, the repetition rate is 4 MHz and the average power at the sample is less than 0.5 mW. The dye laser output can be tuned from 430 to 850 nm depending on the dye and optics used. A wavelength of 480 nm was used for sample excitation in this work.
MATERIALS AND METHODS
Reagents. Reagents used in this work were of the highest purity available commercially and were used as received. Doxorubicin hydrochloride, dihexadecylphosphate (98%), and octadecylamine (98%) were all purchased from Aldrich. DMPC (1,2-dimyristoyl-snglycero-3-phosphocholine, >99% purity) was from Avanti Polar Lipids. Ethanol (99.8% purity) and chloroform were obtained from Chempur (Poland). Aqueous solutions were prepared with Milli-Q water (resistivity 18.2 MΩ-cm) obtained from a Millipore system. Monolayer Preparation. Monolayers of C18NH2, DHP and DMPC were prepared by dissolving each compound in chloroform to obtain a concentration of 2 mg/mL in the monolayer spreading solution. Langmuir monolayers were spread from 15 μL of solution deposited on the aqueous subphase of the Langmuir trough. After solvent evaporation (ca. 10 min.), the monolayer was compressed continuously at a rate of 30 cm2/min to obtain pressure−area isotherms in an isotherm cycling mode set for 2 cycles of compression−decompression. This mode of operation allowed us to obtain at least 2 isotherms and to evaluate the reproducibility and monolayer type. For Langmuir−Blodgett transfer onto the mica, cleaned substrates were immersed into the subphase immediately prior to spreading the monolayer-forming chloroform solution at the air/ water interface. After the monolayer-forming solution was applied, the chloroform was allowed to evaporate, and the monolayer was compressed to a desired target pressure (40 mN/m) that was kept constant (PC-controlled) during the upstroke Langmuir−Blodgett transfer. Bilayer Preparation by Langmuir−Schaeffer transfer (L-S, horizontal touch). This procedure is similar to that used for the Langmuir−Blodgett (L-B) technique. The L-B monolayers were allowed to dry in air for two hours before the next transfer. After the trough was filled, the monolayer was compressed to the required pressure, 40 mN/m. The L-B DMPC monolayer-covered substrate was mounted on the dipper parallel to the water surface with the DMPC layer facing the trough surface. The substrate was lowered until it touched the compressed monolayer, and allowed to equilibrate for ca. 5 min. Then, the horizontal touch transfer was performed at a barrier speed of 30 cm2/min and a dipper speed 5 mm/min (upstroke). Bilayer Preparation by Vesicle Formation and Fusion. Chloroform was evaporated from lipid solution to dryness under nitrogen. Tris buffer solution was added to dry films of DMPC for a final lipid concentration of 1 mg/mL. The lipid solution was processed five times through a freeze−thaw−vortex cycle. Each cycle consisted of immersing the sample in liquid nitrogen (5 min), then heating in a 60 °C water bath (5 min), then vortexing the thawed sample for 2 min. The resulting solution was extruded 11 times through a polycarbonate membrane with a nominal pore diameter of 100 nm to produce 100 nm diameter unilamellar vesicles. For deposition by fusion, 20 μL of the vesicle-containing solution was placed on a planar mica support (ca. 1 cm2) followed by the addition of 60 μL of a pH 7.5 buffer solution of Trizma (10 mM) and NaCl (100 mM), and 7 μL of CaCl2 (2 mM). The resulting vesicle-containing solution was allowed to remain in contact with the mica support for 5 min before rinsing with ∼2 mL of Tris buffer solution. Vesicle fusion was carried out at room temperature. Surface-Enhanced Resonance Raman Scattering (SERRS). SERRS spectra of doxorubicin on the chemically modified solid Ag substrates were collected in a backscattering geometry with a Labram HR800 (Horiba JobinYvon) confocal microscope system, equipped with a Peltier-cooled CCD detector (1024 × 256 pixels). A diode pumped, frequency doubled Nd:YAG laser provided 532 nm excitation radiation, with a total power of less than 0.5mW at the sample. The confocal pinhole size was set to 200 μm and a holographic grating with 600 grooves/mm was used. The calibration of the instrument was performed using the 520 cm−1 Raman band of silicon. SERRS spectra were obtained using a 50x Olympus objective and accumulated from 1 to 2 scans, with 60s or 30s integration time, respectively.
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RESULTS AND DISCUSSION
In the preceding paper8 we reported on the electrochemical investigation of interactions between doxorubicin and the analogous mono- and bilayer interfaces supported on Au. That work showed that there was an interaction between doxorubicin and the interfacial monolayers but more detailed information was not available. For this reason we have chosen to investigate the spectroscopic response of doxorubicin within these interfaces. Our findings shed light on the molecular-scale interactions between the adlayer constituents and doxorubicin and, while these interactions are qualitatively similar for all monolayers, they differ in detail. We examine the interactions through a SERRS investigation of doxorubicin and through a fluorescence lifetime and anisotropy imaging study. We consider these data sets individually before drawing larger conclusions from the entire body of information. SERRS Spectra. The interactions of doxorubicin with the C18NH2, DHP, and DMPC monolayers were studied by means of surface enhanced resonance Raman spectroscopy (SERRS). The wavelength of the laser is close to the first singlet electronic transition of doxorubicin, and vibrational modes associated with the chromophoric part of the drug are enhanced selectively.13 SERRS data were collected for the monolayers deposited on roughened Ag and exposed to a 1 × 10−5 M aqueous doxorubicin solution. SERRS spectra were collected at 1 min intervals for 1 h, in order to monitor the penetration/ adsorption of doxorubicin into/onto the biomimetic films. For all of the monolayer structures studied, there was no chemical functionality capable of chemisorption onto the Ag substrate. The binding of the monolayer constituents to the 14571
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wavenumber components, respectively. The spectral differences between the one- and two-component monolayers is consistent with the single component DHP layer interfering with intramolecular hydrogen bonding within the doxorubicin dihydroxyanthraquinone structure. A clear implication of this finding is that doxorubicin in the DHP monolayer is in relatively close proximity to the phosphate headgroup, a finding that is consistent with the chromophore residing in a location where there is a gradient in the dielectric constant over molecular length scales (vide inf ra). The second type of SERRS spectrum seen for doxorubicin in the DHP monolayer (top spectrum in Figure 1a) exhibits the splitting of a single band at ca. 445 cm−1 (seen in the lower spectrum in Figure 1a). This band is associated with CO deformation, while the doublet at 1215 cm−1 and 1245 cm−1 (δ(C−O−H)) exhibits a change in the relative band intensities. These spectral changes cause the recorded spectrum to resemble more closely the resonance Raman spectrum of doxorubicin in solution.14−16 In addition, the position of the ring breathing mode at 995 cm−1 is better matched with that found for solution phase doxorubicin. In the top spectrum, a shoulder near 1460 cm−1 appears in addition to the two other components in this region (around 1415 cm−1 and 1440 cm−1), ascribed to CC and C−C stretching modes of the aromatic carbons. The differences seen for these spectra suggest that the structure of doxorubicin is distorted relatively little compared to its native solution phase conformation, but the band associated with the stretching of carbonyl groups engaged in hydrogen bonding at 1635 cm−1 is still not visible. The SERRS spectrum of doxorubicin in the C18NH2 monolayer on Ag is shown in Figure 1b. For this interfacial system, only one SERRS spectrum is seen, over a variety of locations on a given SERS substrate and for the interface assembled on different roughened silver supports taken from different experimental batches. The spectrum shown for doxorubicin in the C18NH2 interface is very similar to the spectrum seen for the second type of DHP monolayer (Figure 1a), and we make the same spectral assignments and interpretations, accordingly. We show in Figure 1c the SERRS spectra of doxorubicin in a DMPC monolayer deposited on a silver support. Similar to the analogous data for the doxorubicin/DHP system, two characteristic types of SERRS spectra are seen. These two spectra are seen either for different locations on a given Ag substrate or for different doxorubicin/DMPC interfacial assemblies. Qualitatively, these two spectral types are the same as those seen for the doxorubicin/DHP interface, with the only difference being for the bottom spectrum in Figure 1c, where the band indicative of (OH···OC) stretching at 1635 cm−1 is slightly pronounced. A common thread of the SERRS data for all three monolayer systems studied here is that the structure of doxorubicin appears to be less disrupted, compared to that in the solution phase, than was seen when this compound was integrated into mixed, thiol-containing monolayers.12 One possible reason for this finding may be the existence of greater spatial freedom in the single component layers examined here, and we examine this issue with fluorescence anisotropy imaging measurements (vide inf ra). It is useful to keep in mind that for the monolayers examined in this work, there is no chemical bonding to the support, as was the case for the thiol-containing mixed monolayers. We note that more than one conformation of doxorubicin was seen in the SERRS data for both DMPC and DHP monolayers, and only one conformation found for
metallic silver support was by means of interactions between the metal surface and the amphiphile polar headgroup, initiated during the monolayer transfer by Langmuir−Blodgett deposition. The resulting interfaces, after the deposition of doxorubicin, produced Raman spectral data that were specific to each interface. Interpretation of the SERRS data was performed in the context of existing vibrational mode assignments.14−17 Two spectral profiles were seen for doxorubicin contained in the DHP monolayer (Figure 1a). The two characteristic
Figure 1. Surface-enhanced resonance Raman scattering spectra (after reaching stable intensity) of (a) DHP, (b) C18−NH2, and (c) a monolayer of DMPC formed by L-B deposition on SERS-active Ag substrates.
spectral types are seen for different locations on a given Ag substrate as well as for different doxorubicin/DHP interfacial assemblies. The lower spectrum resembles the analogous data acquired for doxorubicin interacting with a mixed monolayer composed of C18−SH and DHP, which has been reported previously.12 The differences between the spectrum reported here and that for the mixed monolayer are the absence of a band at 1635 cm−1 attributed to CO stretching of hydrogenbonded carbonyl groups (OH···OC) and the splitting of a broad band at 1325 cm−1, seen for the mixed monolayer, into two components, at 1310 cm−1 and 1340 cm−1. The presence of the two bands can be ascribed to the coupling of the ν(C− O) and ν(C−C) (ring stretching) modes for the low and high 14572
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Figure 2. Doxorubicin in a C18−NH2 monolayer on mica. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a three-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 200 ± 50 ps.
doxorubicin in the C18NH2 monolayer. The interaction between the amine headgroup of C18NH2 and the Ag support is expected to be stronger than phosphate interactions with Ag.18 The electrochemical data presented in the preceding paper show multiple redox peaks for doxorubicin in the C18NH2 and DHP interfaces and a single, reversible peak for doxorubicin in DMPC. The electrochemical and SERRS data thus indicate collectively that the interfaces exhibit subtle structural diversity, but the two techniques are sensitive to different aspects of the doxorubicin local environment. The prominent SERRS signal seen for doxorubicin for all of the monolayer structures examined is confirmation that the compound is in sufficiently close proximity to the Ag substrate to experience some level of surface enhancement. Doxorubicin is thus in intimate contact with the aliphatic chain region and likely is located near the polar headgroups of the monolayers. We note that the SERRS signal from doxorubicin dominates the SERS signal associated with the monolayer species due to the effect of resonance enhancement. The SERRS signal for doxorubicin was not diminished greatly by rinsing of the monolayers with water, indicating that this compound has a significant affinity for the nonpolar aliphatic region of the monolayers examined. The inability of doxorubicin to penetrate the DMPC bilayer structure, as seen in the electrochemical data, is consistent with the SERRS measurements reported here, where there was no detectable SERRS response for doxorubicin. We interpret the absence of SERRS signal for this
system as indicating the inability of doxorubicin to locate in sufficiently close spatial proximity to the Ag support to exhibit a large surface enhancement effect. Neither the electrochemical or SERRS data for the doxorubicin/DMPC bilayer system should be taken as an indication that doxorubicin is not interacting with the bilayer, but that whatever interaction is occurring does not place the compound in sufficiently close proximity to the support to allow either Raman surface enhancement or facile electron transfer. In fact, fluorescence lifetime and anisotropy imaging data (vide inf ra) indicate the presence of doxorubicin in/on the bilayer system. Fluorescence Lifetime and Anisotropy Imaging. As noted in the previous section, it appears that doxorubicin is in relatively intimate contact with the nonpolar and headgroup regions of the monolayer structures we have examined. The information provided by electrochemical and SERRS data is not sufficient, by itself, to provide further insight into the manner in which the monolayers interact with doxorubicin. For this reason, we have undertaken a series of fluorescence lifetime and anisotropy imaging measurements, with the goal of revealing complementary information on the nature of the dielectric environment (fluorescence lifetime) and the confinement (fluorescence anisotropy) experienced by doxorubicin. The instrument we use to obtain these data is a confocal scanning system where the monolayer sample is excited by a polarized light pulse and polarized fluorescence transients are acquired pixel-by-pixel across the sample. For each pixel (the images 14573
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Figure 3. Doxorubicin in a DHP monolayer on mica. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a three-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 93 ± 32 ps.
shown in Figures 2−6 are 256 × 256 pixels), the polarized fluorescence transients, I||(t) and I⊥(t), are combined according to eqs 1 to produce fluorescence lifetime and fluorescence anisotropy decay transients, respectively.
quantitative information from these imaging data, we take blocks of data, sum the data contained in these regions, and then analyze the resulting transients using software that is known to produce accurate results. Thus the imaging data in the left-most panels of Figures 2−6 provide color images of the spatial heterogeneity of the lifetime (top) and anisotropy decay (bottom) time constants. The distributions shown in the center panels should be taken as instructive in a qualitative sense only. The quantitative results are shown in the right panels of each figure and in Table 1. For all of the monolayer systems studied here, we observe a complex fluorescence lifetime decay functionality that requires three exponential components to fit. These data are significantly different than the fluorescence lifetime of doxorubicin in solution, where a single exponential decay with a time constant of 1.1 ns is seen.19 There are two possible explanations for the complexity of the fluorescence decay functionality. The first is that doxorubicin is distributed in these monolayer films in such a manner that, for a given monolayer, there are three (or more) distinct environments in which the chromophore resides, and these environments do not exchange population on a time scale that is comparable to the fluorescence lifetime. Such an interpretation implies a system where neither translational nor rotational dynamics occur rapidly, and this is not the case for a monolayer that is physisorbed to a support. We note that the lifetime distribution
Ifl(t ) = I + 2I⊥(t ) R (t ) =
I (t ) − I⊥(t ) Ifl(t )
(1)
The information contained in the fluorescence lifetime data is of use in determining the local dielectric environment of the chromophore, while the anisotropy (R(t)) data provide information on the rotational motion of the molecule. Both types of information are complementary to the electrochemical and SERRS data on these interfaces. Before examining the imaging results shown in Figures 2−6, a few caveats are in order. The functional form of the timedomain transients that comprise these images is sufficiently complex that the commercial software does not provide quantitatively accurate values for either the fluorescence lifetime or anisotropy decay time constants. This issue is exacerbated by the limited signal-to-noise ratio of the data contained in individual pixels. Despite these limitations, the results from the imaging software are useful in providing the functional form of the distributions of lifetimes and anisotropy decay times for these images because they apply the same mathematical algorithm to all of the data present. To obtain 14574
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Figure 4. Doxorubicin in a DMPC monolayer on mica. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a three-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 124 ± 32 ps.
Anisotropy decay data provide information on molecular rotational motion and, as such, afford insight into the nature and extent of confinement the chromophore experiences within the monolayer structure. There is a well-established body of theory that is used to interpret anisotropy decay data.26−29 In this instance, the chromophore is not bound to the monolayer constituents and is nominally free to rotate. Accordingly, we model the motion of doxorubicin in these monolayers in the context of the free rotor model. Chuang and Eisenthal have developed a general theory that relates anisotropy decay data, R(t), to the Cartesian components of the chromophore rotational diffusion constant (D = 1/3(Dx + Dy + Dz)) and the orientations of the absorbing and emitting transition dipole moments.26 There are two limiting cases of interest. These are when the absorbing and emitting transition dipole moments are nominally parallel, and two of the three Cartesian axes are assumed to be equal. The third, unique axis can either be coincident with the transition moments or normal to them. For the former case, the volume swept out by the rotating chromophore is modeled as a prolate ellipsoid and for the latter case the rotating chromophore is modeled as an oblate ellipsoid (eqs 2 and 3).30
data (Figures 2−6 center panels) does not show any evidence for isolated, nonoverlapped lifetime distributions. The second explanation for a multiple component exponential decay in a fluorescence lifetime measurement is that the chromophore resides in an environment where there is a significant variation in the dielectric response of the medium on the length scale of the emitting moiety.20−25 For the monolayer systems we have constructed, a dielectric gradient could exist normal to the plane of the support and in close proximity to the support surface. In the region where the amphiphilic monolayer is sitting on top of the support, likely with a cushion of several Å of water, the dielectric gradient normal to the surface can be significant owing to the presence of the surface, water, the amphiphile headgroup and then the amphiphile aliphatic chain region. The lifetime data we show in Figures 2−5 is consistent with doxorubicin residing in the monolayer in a region characterized by a large dielectric gradient, i.e. near the amphiphile headgroup, and this finding is in agreement with the SERRS data. We note that the regressed lifetime components are similar for our interfaces, but we do not attempt to extract any information of chemical significance from these fitted values because there is no theoretical framework in which such quantities can be related to system properties. The information contained in these data is that doxorubicin is in a region of the monolayer characterized by a substantial dielectric gradient normal to the surface plane.
R(t ) = 0.4 exp( −6Dz t )
(prolate)
(2)
R(t ) = 0.1 exp( −(2Dx + 4Dz )t ) + 0.3 exp( −6Dx t ) (oblate) 14575
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Figure 5. Doxorubicin in a DMPC bilayer on mica, deposited by the Langmuir−Blodgett/Langmuir−Schaefer method. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a three-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 98 ± 12 ps.
image individually, but it is important to note that these monolayer interfaces exhibit varying degrees of heterogeneity, depending on the specific interface. The extent of this spatial heterogeneity is evaluated in greater detail for these systems through signal averaging of the transients contained in the white, numbered boxes. In addition to revealing the spatial heterogeneity of dynamics within the layers, these data also provide valuable comparative information on the extent and nature of chromophore confinement for the different monolayer structures. As noted above, the anisotropy decay images and distributions (Figures 2−6, bottom row, left and center panels) provide valuable qualitative information, but quantitation requires more detailed analysis. Obtaining meaningful anisotropy decay time constants required data with a higher signalto-noise ratio than that available from a single pixel. For this reason, we summed the polarized transients in the areas that are boxed in Figures 2−6, and from these summed pixels we generated R(t) decay functions. For all samples, we obtained anisotropy decays characterized by single exponential decay components (Table 1). This finding is consistent with the chromophore residing in a single chemical environment that is characterized by a dielectric gradient. To ensure that the utilization of data within boxed regions did not distort our results, we selected several different locations on each interfacial assembly to analyze (see Figures 2−6) and evaluated several
For all of the data we report here, doxorubicin is seen to produce anisotropy decays characterized by a single exponential functionality (eq 2). The transition dipole moments lie along the doxorubicin long, in-plane axis, which we designate as x, and the only motional component we sense is that perpendicular to the x-axis. Because we recover only one of the Cartesian components of D, we are left to assume that Dz ∼ D, and with an estimate of D we can compare the local environments formed by the monolayer. The anisotropy decay time constant is 6D−1, which is related to the local viscosity sensed by the chromophore, its hydrodynamic volume, and the thermal energy in the system through the modified Debye− Stokes−Einstein (DSE) equation,27−29 τOR = 6D−1 =
ηVf kBTS
(4)
where η is the bulk viscosity of the immediate environment of the chromophore, V is the hydrodynamic volume on doxorubicin (V = 456 Å3),31 f is a frictional term to describe the interactions between doxorubicin and the surrounding monolayer,28 S is a shape factor to account for the nonspherical shape of doxorubicin29 and kBT is the Boltzmann thermal energy term. The images presented in Figures 2−6 are maps of the anisotropy decay time constant, τOR, as a function of position. We will consider the information content of each 14576
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Figure 6. Doxorubicin in a DMPC bilayer on mica, deposited by vesicle fusion. Top, from left to right: Fluorescence lifetime image, distribution of calculated lifetimes, and regression of averaged region providing quantitative information on population decay. Best fit function is a one-component exponential decay. Bottom, from left to right: Anisotropy decay image, distribution of calculated anisotropy decay times, and regression of averaged region providing quantitative information on the anisotropy decay. The anisotropy decay time functionality is single exponential with a time constant of 260 ± 30 ps.
Table 1. Fluorescence Lifetime and Anisotropy Decay Time Constants adlayer on mica C18NH2 monolayer DHP monolayer DMPC monolayer DMPC bilayer (L-B/L-S) DMPC bilayer (vesicle fusion)
τfl(1) (ps) 68 51 37 67
± ± ± ±
1 (79%) 1 (85%) 1 (86%) 1 (80%) --
τfl(2) (ps)
τfl(3) (ps)
⟨τfl⟩ (ps)
τOR (ps)
351 ± 25 (16%) 248 ± 9 (12%) 178 ± 3 (11%) 255 ± 8 (17%) --
1436 ± 111 (5%) 1193 ± 47 (3%) 1099 ± 13 (3%) 1230 ± 43 (3%) 1173 ± 1
177 106 81 135 1173
200 ± 50 93 ± 32 124 ± 32 98 ± 13 260 ± 30
reorientation times of ca. 100 ps (Table 1). Interpreting these results in the context of eq 4, where V = 456 Å3, and we take S = 129 and f = 128 in the absence of information that could cause these values to deviate slightly, we estimate the local viscosity of the interface sensed by doxorubicin to be ca. 0.9 cP for the DHP and DMPC monolayers and to be ca. 1.8 cP for the C18NH2 monolayer. We recognize that these are estimates of the local viscosity and are limited by our assumptions of values for S and f, but the data point to the doxorubicin being in a less constrained environment for the DHP and DMPC monolayers than it is in the C18NH2 monolayer, and this makes sense in the context of the strength of amphiphile-headgroup interactions. We note that this finding is consistent with our SERRS data. In all cases, the recovered viscosity estimates indicate that the chromophore resides in an environment of limited restriction,
smaller subsections of the boxed regions to determine whether the average values for the decay constants were sample size invariant, and they were (data not shown). The physical and chemical significance of these anisotropy decay data lies in how their values vary, or not, depending on the identity of the interfacial layer. There can be two contributions to the anisotropy decay: orientational relaxation of individual chromophores and energy transfer between identical chromophores. The efficiency of this latter effect will scale inversely with the average distance between chromophores. For our bilayers, that average distance is ca. 80 Å, more than the critical radius. Thus the R(t) decay time constants we report are associated with molecular motion. The reorientation data for these interfaces falls into two broad categories, with the C18NH2 interface producing doxorubicin reorientation times of ca. 200 ps and the DHP and DMPC monolayers producing 14577
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quite likely with the opportunity for exposure to the aqueous solution overlayer. Examining the analogous data for DMPC bilayer structures likewise provides useful insights. We have formed DMPC bilayers according to two methods, Langmuir−Blodgett/ Langmuir−Schaeffer deposition and vesicle fusion, and the results for bilayers formed by these two methods differ markedly. The reorientation time constant recovered for the L-B/L-S DMPC bilayer is the same to within the experimental uncertainty as that measured for the DMPC monolayer, ca. 100 ps, indicating a similar local environment for the two structures. For the bilayer formed by vesicle fusion, we recover a reorientation time constant of 260 ps, substantially longer than that of the L-B/L-S bilayer. This finding indicates that the bilayer formed by vesicle fusion is more organized (and thus more confining) than the L-B/L-S monolayer. We note that the electrochemical data for doxorubicin in the L-B/L-S DMPC bilayer shows the absence of electron transfer across this interface, and the fluorescence lifetime data for this interface is of the same multiexponential functionality as was seen for the monolayers, demonstrating that the doxorubicin is within the bilayer and senses the lipid headgroups (vide supra). These findings suggest collectively that the L-S overlayer plays an important role in isolating the doxorubicin from the solution overlayer and precluding access to protons. The dynamics and lifetime information for bilayers formed by fusion on mica using vesicles that were extruded (ca. 100 nm diameter) in the presence of doxorubicin reveal significantly different information. The reorientation time for DMPC bilayers formed by vesicle fusion is more than twice as long as that of the L-B/L-S bilayers. We assert that because this reorientation time is much longer than that expected for aqueous solution and that we obtained data with a high S/N ratio (Figure 6), the doxorubicin is indeed contained in the bilayer structure. The fluorescence lifetime for doxorubicin in the bilayer formed by vesicle fusion exhibits a single exponential decay functionality, in sharp contrast to that seen for the other interfaces reported here. We understand this finding in the context of the doxorubicin being sequestered within the nonpolar region of the bilayer and that the bilayer structure is sufficiently well organized to minimize the access of doxorubicin to the lipid headgroup region.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a Project PSPB-079/2010 Grant from Switzerland through the Swiss Contribution to the enlarged European Union. G.B. gratefully acknowledges support by the Donors of the ACS Petroleum Research Fund. The fluorescence lifetime and anisotropy imaging instrument was purchased using funds from National Science Foundation Grant CHE 1048548.
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CONCLUSIONS The time- and frequency-domain spectroscopic data we have reported here point collectively to the extent of organization of the interfaces we have formed. Raman spectroscopic data show the interactions, or not, of doxorubicin with the interfacial adlayers, while the time-domain lifetime and anisotropy decay data reflect the extent to which the chromophore is constrained by its immediate environment and what access it has to the headgroup region of the interface. These findings are fully consistent with the electrochemical data in the preceding paper and, taken as a whole, demonstrate that doxorubicin has a substantial affinity for hydrophobic environments, a factor that may play a role in the transport of anthracyclines across bilayer membranes.
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