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Fluorescence Anisotropy Based Single Liposome Assay to Measure MoleculeMembrane Interactions Nicky Ehrlich,†,‡,§ Andreas L. Christensen,†,‡,§ and Dimitrios Stamou*,†,‡,§ †
Bio-Nanotechnology Laboratory, Department of Neuroscience and Pharmacology, ‡Nano-Science Center, and §Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, 2100 Copenhagen, Denmark
bS Supporting Information ABSTRACT: Nanometer-scaled liposomes are used frequently for research, therapeutic, and analytical applications as carriers for water-soluble molecules. Recent technical advances allow the monitoring of single liposomes, which provides information on heterogeneous properties that were otherwise hidden due to ensemble averaging. Recent observations demonstrated that the efficiency of entrapping water-soluble molecules increases with decreasing vesicle size. The molecular mechanism behind this observation is not clear, but enhanced moleculemembrane interactions due to the increase of the surface area-to-volume ratio could play an important role. To investigate this hypothesis, we extended our single liposome assay based on confocal fluorescence imaging by implementation of fluorescence anisotropy. This combination has not been widely exploited, and confocal fluorescence anisotropy imaging in particular has seldom been used. We investigated different small dye molecules and were able to determine if these molecules interact or not with the liposome membrane. We confirm the liposome size-dependent entrapment of molecules whereas the moleculemembrane interactions appear to be independent of liposome size. Our fluorescence anisotropy assay can be used as a general method to investigate moleculemembrane interactions or moleculemolecule interactions in a high-throughput manner in nanometer-scaled containers like liposomes.
L
iposomes are self-assembled, spherical vesicles formed by a lipid bilayer that separates aqueous interior and exterior environments and are commonly used as model membrane systems in research.1 Of particular interest are nanometer-scaled liposomes as drug delivery carriers,27 as gene therapeutic agents,8,9 for targeting and treating infection and inflammation,10 or for miniaturized reactor systems.11,12 Recent technical advances allowed the monitoring of single liposomes,11,1318 providing unique information on heterogeneous properties that were otherwise hidden due to ensemble averaging. For example, it was shown in encapsulation experiments that the concentration of encapsulants increase with decreasing liposome size.19,20 The molecular mechanism behind this observation is not clear, but more frequent moleculemembrane interactions due to the increase of area-to-volume ratio as the liposomes become smaller could explain this observation. The goal of this study is to examine if liposome size influences the rate of interactions between entrapped water-soluble molecules and the liposome membrane. Fluorescence anisotropy is a method used to investigate interactions between molecules. It is a powerful and robust method that has been used to address a number of biological and biophysical questions.21 Fluorescence anisotropy is sensitive to the orientation and rotational correlation time of fluorophores and capable of sensing change in the physical properties and local environment of fluorophores. Classically, bulk fluorescence r 2011 American Chemical Society
anisotropy assays are used to quantify proteinprotein interactions and are widespread in clinical and biomedical fields for applications such as binding assays, immunoassays, and highthroughput screening, e.g., for drug discovery.22 The combination of fluorescence anisotropy and imaging has not been widely exploited, and confocal anisotropy imaging in particular has seldom been used,23 although this technique can be implemented in a conventional confocal microscope through relatively straightforward modifications and involve only few additional components.21 Fluorescence anisotropy imaging techniques have been used frequently to investigate proteinprotein interactions21,2428 or changes in membrane properties,29,30 but only few examples exist in which interactions of molecules with membranes were investigated.31,32 In this study, we used confocal fluorescence anisotropy imaging to investigate moleculemembrane interactions of water-soluble molecules encapsulated in small unilamellar liposomes. We isolated individual liposomes by immobilizing them on passivated surfaces at low density.3337 The strategy for immobilization of liposomes in arrays is illustrated in Figure 1. This scheme can also be used to immobilize liposomes in regular patterns.33,38,39 Simultaneous imaging of many single nanoscale Received: July 4, 2011 Accepted: September 16, 2011 Published: October 03, 2011 8169
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Figure 1. Scheme of the single liposome assay. Liposomes with encapsulated water-soluble molecules are immobilized via a neutravidin biotin-complex to a pegylated glass surface. Liposomes and encapsulated molecules are fluorescently labeled, which allow us to determine liposome size, concentration of encapsulant, and moleculemembrane interactions.
liposomes allowed us to perform a variety of experiments with attoliter volumes and a few molecules in an unprecedented ultrahigh-throughput fashion.11 In particular, we wanted to investigate if moleculemembrane interactions are enhanced in smaller liposomes which could explain the observed increased encapsulation of water-soluble molecules in smaller liposomes.19,20
’ EXPERIMENTAL SECTION Liposome Preparation and Characterization. Liposomes were prepared by the rehydration technique.40 Lipids were received in powder form from Avanti Polar Lipids if not stated otherwise. Lipids were dissolved in chloroform and were mixed in a small glass vial in ratios corresponding to the desired lipid composition. Chloroform was evaporated under gentle nitrogen flow, and the resulting lipid film was stored in vacuum for 2 h to remove remaining organic solvent residues. The dry lipid film was rehydrated in PBS buffer to a final lipid concentration of 1 g/L. Freezethaw cycles (10) were applied to the liposome solution by immersion in liquid nitrogen followed by thawing in a water bath (45 C). Afterward, the liposomes were extruded several times through polycarbonate membrane filters (Avanti’s mini extruder, Avanti Polar Lipids Inc.) with defined pore size (50200 nm). The extrusion process generates an upper size limit of the vesicle; hence, the average liposome size but also the size distributions become broader with increasing filter size.17 The standard lipid composition used in this work mainly consist of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 76.577.5 molar %) and of 20 mol % of the negatively charged lipid dioleloyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG), 2 molar % of the membrane dye 3,30 -dioctadecyloxacarbocyanine perchlorat (C18-DiO, Molecular Probes) and 0.5 molar % of the linker 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineN-(cap biotinyl) (DOPE-biotin). Instead of DOPE-biotin, 1 mol % of the linker 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineN-[biotinyl(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-biotin) was used in the DNA encapsulation experiments. In the control experiment, in which the interaction of the molecules with the lipid membrane was enforced, the negatively
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charged lipid DOPG were exchanged by the same amount of the positively charged lipid 1-hexadecanoyl-2-(9Z-octadecenoyl)-snglycero-3-ethylphosphocholine (chloride salt) (POPC+). Instead of changing the liposome charge, the used Atto-dyes were linked to a lipid (DOPE-Atto633 and DOPE-Atto655, AttoTec) and premixed with the other lipids in chloroform in a concentration of 0.5 mol %. To encapsulate water-soluble molecules in liposomes, the molecules were added to the rehydration buffer in concentration of 1001000 μM. The following water-soluble molecules were used: Cy5 hydrazide (Cy5, GE Healthcare), Cy5-labeled DNA (ssDNA-Cy5 and dsDNA-Cy5, Cybergene AB), the Atto-dyes Atto633-NHS (Atto633) and Atto655-maleimide (Atto655) from AttoTec, and Alexa Fluor 633-NHS (Alexa633, Invitrogen). Fluorophores with the highly reactive linker NHS were kept several hours at room temperature before use to deactivate the NHS activity. Surface Preparation and Immobilization. Liposomes were immobilized on functionalized glass surfaces. The surfaces were prepared by plasma etching the coverslip for 2 min in a plasma cleaner (PDC-32G, Harrick Plasma) before incubation with PLL-g-PEG/PLL-g-PEG-biotin solution for 30 min and finally incubation with streptavidin for 10 min. After each incubation step the chamber was washed thoroughly with PBS buffer. Liposomes were immobilized on the streptavidin functionalized surface by incubating the sample in the chamber 10 min followed by washing with PBS buffer. The density of liposomes on the surface was varied by the amount of sample added. In the control experiment, in which positively charged liposomes without encapsulant were used to enforce membrane interaction, the immobilized liposome were incubated for 10 min with a 1 μM solution of the molecules of interest. Samples were washed to remove free molecules in solution before data acquisition. Confocal Microscope Imaging. Solutions of fluorophore or fluorescently labeled molecules as well as immobilized liposomes were imaged with a commercial inverted confocal microscope (Leica TCS SP5, Leica) using an oil immersion objective (HCX PL APO CS 100, NA 1.4, Leica). The microscope stage was placed in a ludin chamber, in which the temperature was monitored and remained constant at 22 ( 1 C. A 488 nm laser (argon laser, Lasos Lasertechnik GmbH) was used to excite the membrane dye DiO. The maximal laser power output of the 488 nm laser was 1 mW measured at the back aperture of the objective. The fluorescence signal of DiO was detected at 500600 nm using a photomultiplier tube (PMT). For the anisotropy measurements, a linear polarized 633 nm laser, with a maximal laser power output of 0.7 mW, was used to excite the water-soluble molecules. The laser power was optimized for each fluorophore to obtain the best signalto-noise ratio but also to prevent photobleaching. The two orthogonal polarization components of the fluorescence were separated in the wavelength range between 650 and 690 nm with a polarization beam splitter (AHF Analysetechnik, Germany) and focused on avalanche photodiodes (APD, SPCM-AQR-13FC, Perkin-Elmer). From this data it was possible to calculate the fluorescence anisotropy. Micrographs from the PMTs and APDs were acquired sequentially to avoid cross excitation. Images had a resolution of 1024 1024, with a physical pixel area of 50.5 50.5 nm2 and a bit depth of 16. Specimens were scanned bidirectional with a speed of 400 Hz. This corresponds to an exposure time for each pixel of 2.4 μs. To improve the signal-to-noise ratio, each image line was recorded three times 8170
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and the average value of the measured intensity values was used. Four images were taken for anisotropy measurements to check the measured fluorescence stability. The count rates measured by the APDs were compensated for the dead time (td) by multiplying the measured count rate (CR) with a correction factor of 1/(1 - tdCR) (Figure S-1 in the Supporting Information) as described in the literature.41 Image Analysis and Size Calibration. A custom made software routine in Igor Pro 6 (Wavemetrics) was used for imaging analysis. The software routine localized and gave out the fluorescence signal of the membrane dye for each single liposome. Thresholding, minimum particle area, and ellipticity were used as tools to faithfully localize particles. The localization data of the liposomes were used to extract the fluorescence signal in both APD images for calculating the anisotropy value of the water-soluble molecules associated with the liposomes. Conversion from liposome intensity to size was achieved according to the procedure of Kunding et al.17 Briefly, a combination of confocal fluorescence microscopy and dynamic light scattering measurements has been used. The integrated fluorescence intensity (I) is proportional to the number of dye molecules in the membrane and hence the surface area. Thus, the liposome diameter is equal to the square root of I multiplied by a constant (Ccal). The Ccal was determined by size calibration to reference liposomes. Reference liposomes were extruded through a very small pore size of 50 nm to obtain liposome samples with a very narrow size distribution. The mean size of the reference samples were determined by dynamic light scattering (DLS) using an ALV-5000 Correlator (ALV, Langen, Germany) connected to a 633 nm laser line. Scattering data were collected at 21 C at 50110 with 20 intervals, and the autocorrelation curve was fitted in the ALV software to yield a mean radius of the liposomes. Recently, we showed that the lipid composition of individual nanoscale liposomes can be rather heterogeneous.42 This lipid variation is constant with liposome sizes and therefore is not introducing any systematic bias in the determination of the liposome size but an increased uncertainty of e15% for 70% of the liposome population. Intensity calibration of encapsulants were done in solution at a concentration ranging from 10 to 400 nM with the same settings used for imaging immobilized liposomes. The data were fitted by a standard linear fit, and the slopes were used to calculate the number of encapsulants within single liposomes. Steady-State Anisotropy Measurements. For anisotropy measurement, linearly polarized light was used to excite fluorophores of interests. The fluorescence emission was measured by a two channel (T-format) configuration, where the parallel and perpendicular emission components (I|| and I^) are detected simultaneously. This gave us the required anisotropy: r ¼
III GI^ III þ 2GI^
ð1Þ
Here the G factor corrects for the different detection efficiencies of the parallel and perpendicular emission pathways. To calculate the G factor, we performed a daily calibration by imaging an aqueous solution of a reference dye with known anisotropy (e.g., Atto633 in PBS with rref = 0.015) as described in the literature.21,24,28,43 The fast rotation of the fluorophore is resulting in an emission that is completely depolarized. Reference anisotropy measurements were done on a standard spectrofluorometer (Fluoromax-4, Horiba Jobin Yvon). Fluorescent anisotropy
Figure 2. Line profiles of fluorescence anisotropy images of molecules free in solution. Anisotropy values of water-soluble molecules were measured as reference to the encapsulation experiments. The solutions were imaged in proximity to the coverslip surface in the xz-direction, and the anisotropy values in each pixel across the coverslip surface were calculated according to eq 1. Because of the higher molecular weight of BSA, the anisotropy values are shifted to higher values compared to the pure Atto655 dye.
Figure 3. Effect of high numerical aperture of the objective in the confocal microscope on anisotropy measurements. Comparison of anisotropy values of five different samples measured with the confocal microscope and a fluorometer. Shown uncertainties are the standard deviation of the Gaussian distribution of the anisotropy histograms as shown in Figure 2. The black line indicates the trend if the microscope and the fluorometer values would be the same. However, lower anisotropy values are determined with the microscope due to the depolarization effect of the high numerical aperture of the objective. The red fit line was derived by fitting the data to rconfocal = (rfluorometer 2brfluorometer)/(3a + 2rfluorometer 2brfluorometer).44,47 Samples are from the low to high anisotropy value Atto633 (9), Alexa633 (b), Atto655 (2), BSA-Atto655 ([), and BSA-Alexa633 (1).
images were recorded four times to determine the mean anisotropy value and standard deviation of each single liposome.
’ RESULTS AND DISCUSSION Principle of Confocal Anisotropy Measurements. The water-soluble molecules of interest were imaged as a reference free in solution in the xz-direction. The fluorescence signal close to and across the coverslip surface were determined for each pixel in both detectors to verify the accuracy of the anisotropy in both on- and off-axis across the entire field of view. The pixel values were averaged over several pixels to reduce the fundamental uncertainty in fluorescence imaging due to Poisson statistics.44 The anisotropy values for each pixel were calculated according to eq 1. As proof of principle, the anisotropy values of Atto655 and Atto655 attached to bovine serum albumin (BSA) are shown in Figure 2. Fluorescently labeled BSA shows a higher anisotropy value compared to the free fluorophore due to the higher molecular weight of BSA and therefore longer rotational time. 8171
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Figure 4. Determination of the anisotropy value of encapsulated water-soluble molecules within nanometer scaled liposomes. (a) The fluorescence signal of the membrane dye are used to determine the position and size of single liposomes. (b) The two orthogonal polarization components of the fluorescence signal from the encapsulated molecules are separately collected by two detectors. This allows us to determine the anisotropy values of encapsulated molecules within single liposomes as well as the size of the corresponding liposome (c).
The mean anisotropy value and standard deviation were calculated by fitting the anisotropy distribution with a Gaussian. We used in our study an objective with a large numerical aperture. It is already known that the use of this kind of objective leads to a significant depolarization of the fluorescence anisotropy.45,46 For regular imaging conditions this is largely negligible but plays a significant role in high-resolution microscopy, spectroscopy, and single molecule studies.46 This depolarization effect and therefore the resolution of anisotropy can be reduced by using objectives with lower numerical aperture, which however leads also to a reduction in optical resolution and light collection efficiency. It is possible to correct for the depolarization effect of the objectives by, for example, comparing samples measured with the microscope and with a spectrofluorometer. This is shown in Figure 3 for five different samples, namely, Atto633, Alexa633, Atto655, BSA-Atto655, and BSA-Alexa633, which display different anisotropy values due to differences in their fluorescence lifetimes or rotational times. Anisotropy values measured with the microscope are systematically smaller compared to the fluorometer measurement, a phenomenon that is especially pronounced at higher anisotropy values. Aperture correction parameters can be calculated by fitting the data to rconfocal = (rfluorometer 2brfluorometer)/(3a + 2rfluorometer 2brfluorometer),44,47 which yields the values a = 1.20 and b = 1.02. In the following, we present the uncorrected microscope anisotropy values in this work, since the relative difference in anisotropy between molecules free in solution and encapsulated within liposomes and not the absolute values are important in this study. To investigate moleculemembrane interactions of encapsulated molecules, liposomes were loaded by rehydrating a lipid film in buffer containing the water-soluble fluorescent molecules. Loaded liposomes with membrane-associated fluorophores were immobilized at dilute densities on a functionalized coverslip surface through the interaction between membrane tethered biotin and immobilized streptavidin.11,33 This allowed us to measure several hundreds of liposomes at the same time and to perform single liposome analysis.17 Liposomes immobilized in
this way maintain a spherical shape48 and stay intact to concentration gradients of small organic fluorophores19 and ions.33 Liposomes of nanoscale dimensions appeared as diffractionlimited spots and yielded micrographs with discrete intensity spots (Figure 4a). These fluorescence signals were used to determine the position and size of single liposomes by a particle finding procedure and 2D-Gaussian fit in Igor Pro 6.17 The two orthogonal polarization components of the fluorescence signal from the encapsulated molecules were separately collected by two detectors (Figure 4b). Four sequential images were recorded in each APD to calculate the uncertainty in the anisotropy value. With the fluorescence information of the membrane and lumen channel, the anisotropy value of single liposomes can be plotted vs liposome size or as a histogram to determine the distribution of the anisotropy value of the whole liposome sample (Figure 4c). The fluorescence anisotropy resolution is determined by Poisson statistics.44,49 Generally, a typical confocal image is formed by only tens to hundreds of detected photons per pixel and an anisotropy image will therefore generally exhibit a large standard deviation.44 The anisotropy resolution can be improved by averaging over a number of exposures, averaging over neighboring pixels and increasing the total emission intensity.49 The advantage of our liposome assay is that the water-soluble molecules are freely rotating within the liposomes but keep localized due to the immobilization of the liposomes, which allow us to observe the molecules over a long time and thereby gather as many photons as possible. Since the single liposomes and therefore their cargo are clearly separated from each other, all neighboring pixels can be taken into account for the calculation of the anisotropy value. This increase in fluorescence collection helps to improve the signal-to-noise ratio and therefore the accuracy of the anisotropy measurements. Bigelow et al. demonstrated that with a sufficiently high signal-to-noise ratio, relative changes in anisotropy less than 0.4% can be resolved.21 To evaluate how accurate we can determine the anisotropy value, we imaged fluorescently labeled reference beads at different laser powers. The reference beads allow us to image a sample 8172
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Figure 5. Experimental determination of anisotropy accuracy depends on fluorescence signal. The anisotropy resolution was evaluated by imaging the same immobilized fluorescent reference beads at different laser powers. Beads were imaged four times, from which the anisotropy value and its standard deviation for each single bead were calculated. The distribution of both values for all beads were fitted with a Gaussian fit, and the mean values are plotted vs laser power for (a) the standard deviation and (b) the anisotropy value. Error bars represent the standard deviation between the four images, which were acquired for each laser power. The power of the laser is controlled by an acousto-optical beam splitter (AOBS) and is given in percentage (%), which indicates how much of the laser light transmits through the AOBS.
Figure 6. Histogram of fluorescence anisotropy values of several hundred single liposomes. Shown is the anisotropy distribution of Atto655 encapsulated in liposomes (red bar) and incorporated into the membrane as DOPE-Atto655 (blue bar). The anisotropy values of DOPE-Atto655 are shifted to higher values due to restricted rotational freedom. Data were fitted with a Gaussian fit to determine the mean (x0) and the width of the distributions.
with a very narrow distribution of properties, e.g., size, intensity, and anisotropy value. By varying the laser power, we were able to investigate the influence of the fluorescence strength on the accuracy of the anisotropy value determination. Sequential images were recorded, from which the mean anisotropy value and the corresponding standard deviation for each single particle were calculated. The standard deviation decreased with increasing laser power and increasing fluorescence signal, as shown in Figure 5a. A maximal accuracy in the anisotropy value of (0.024 can be achieved for single particles at the maximal laser power. The anisotropy value itself should be independent of fluorescence intensity as shown in Figure 5b. Here the anisotropy value was determined by a Gaussian fit of the distribution of the anisotropy values of single beads and therefore representing the mean anisotropy value of the whole bead population. The variation of this mean anisotropy value is only (0.002 for the different laser powers. However, there are limitations for how much the fluorescence signal can be improved. For instance, too high laser power will result in saturation of the fluorescence output of the dye, which in turn leads to an apparent decrease in measured anisotropy.44 Another effect that leads to a decrease in anisotropy is the occurrence of fluorescence resonance energy transfer (FRET) between identical fluorescent molecules, a phenomena known as
homo-FRET.27,43,50 The magnitude of this interaction depends on the separation of the dyes and is very sensitive in the 210 nm range. Photobleaching alters the anisotropy value in a sample with homo-FRET,44 but it is negligible when fluorescence anisotropy reports only the rotational diffusion of freely rotating molecules. However, photobleaching can affect the anisotropy value of rotational restricted molecules, e.g., if they are bound to the membrane, due to selectively bleaching (Figure S3 in the Supporting Information). Therefore, we used imaging conditions in which the typical photobleaching was minimized to less than 2% per image scan and the effect of photobleaching was negligible. Molecules Encapsulated in Nanometer Scaled Liposomes. Comparison of the anisotropy distributions of Atto655 encapsulated or bound via DOPE to the liposome membrane reveals that fixed DOPE-Atto655 dyes display higher anisotropy values (Figure 6). The width of the anisotropy distribution shown here is broader compared to the anisotropy histogram in Figure 2. This can be explained by the photon noise. The liposome samples are quite heterogeneous in regard to fluorescence intensities, due to heterogeneity in liposomes size and amount of encapsulated dye molecules. Liedke et al. showed that the standard deviation of the anisotropy value is 0.1 or even higher for photon counts below 100, which is not unusual for confocal images.44 Besides photon noise, heterogeneities in lipid composition of single liposomes42 might also broaden the range of anisotropy values of single liposomes. However, Figure 6 demonstrates that we can probe the hindered rotational freedom of molecules bound to the liposome membrane. To determine, if encapsulated water-soluble molecules are completely rotationally unhindered or interact with the liposome membrane, we compared their anisotropy value (rlip) with the reference measurement of the corresponding molecules free in solution (rfree) by taking the ratio of the anisotropy values (rlip/ rfree) (Figure 7). A ratio of 1 implies that the encapsulated molecules experience the same rotational freedom as free in solution, whereas a ratio greater than 1 indicates hindered rotational freedom of the encapsulated molecule. To evaluate ratios above 1, we compared the anisotropy ratio of encapsulated dyes to a situation in which rlip originates from dye molecules associated with the liposome membrane. Thereby, we obtain a reference anisotropy ratio that corresponds to all dyes being associated with the liposome membrane. We enforced the 8173
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interaction between the molecules and the liposome membrane by covalently binding the dye to a lipid molecule (e.g., DOPEAtto655 and DOPE-Atto633) or by electrostatic attraction between the dyes and liposome membrane (e.g., Alexa633 and Cy5). The reader should be aware that the relative values given in Figure 7 do not provide any information about the strength of rotational hindrance and are not suitable for comparison between different dye molecules. The anisotropy scale is restricted from the top and bottom; therefore, the magnitude of the relative values depends on the reference anisotropy value and the free rotating dye in solution, which is different for the different dyes.
Figure 7. Determination of moleculemembrane interactions for a variety of molecules. The figure shows the ratio of anisotropy values from molecules bound to or encapsulated within liposomes (rliposome) and free in solution (rfree) (detailed data can be found in Table 1). Therefore, a ratio of ∼1 indicates that the molecules are freely rotating, whereas values significant above 1 imply rotational restriction of the molecules within the liposome. The degree of rotational restriction can be evaluated by comparing the anisotropy ratio of encapsulated molecules (red bars) to the corresponding bound molecules (blue bars).
Table 1. Fluorescence Anisotropy Values of the Investigated Water-Soluble Moleculesa anisotropy value water-soluble molecules
free in solution
encapsulated
bound to SUVs
Alexa633 Atto633
0.03 ( 0.01 0.015 ( 0.002
0.055 ( 0.009 0.099 ( 0.005
0.07 ( 0.01 0.098 ( 0.002
Atto655
0.040 ( 0.015
0.034 ( 0.006
0.123 ( 0.002
0.11 ( 0.01
0.118 ( 0.003
0.234 ( 0.003
Cy5 a
Molecules free in solution, encapsulated in liposomes, or bound to the membrane of the liposomes are compared. Values are determined by fitting the anisotropy distribution with a Gaussian fit.
Therefore, we also provide the absolute anisotropy values in Table 1. As seen in Figure 7, Atto655 and Cy5, encapsulated in liposomes, show a ratio of ∼1, indicating that both dyes do not interact with the liposome membrane. The anisotropy ratio of encapsulated Atto633 is comparable to the liposome sample with Atto633 incorporated into the liposome membrane via DOPE. This indicates that this dye is strongly interacting with the liposome membrane and not freely rotating in the lumen of the liposomes. Measurements with giant vesicles, in which the adsorption of the dye to the membrane can be directly seen, support this observation (Figure S-2 in the Supporting Information). The anisotropy ratio of encapsulated Alexa633 is between 1 and the ratio of Alexa633 electrostatically bound to the liposome membrane. This indicates that Alexa633 interacts weakly with the membrane. We extended our study by ss- and ds-DNA olignucleotides since DNA molecules have great potential as biopharmaceuticals.51 The DNA molecules were labeled with the widely used dye Cy5. The anisotropy value of DNA-Cy5 bound to the liposome membrane shows a lower value compared to DNA-Cy5 freely diffusing in solution, and the anisotropy value of encapsulated DNA-Cy5 is even lower (Table 1). If the anisotropy value would indicate a restriction in the rotational freedom of the DNA-Cy5 molecule, one would expect a higher value due to more restricted movement as for Atto633. It has been shown that cyanine dyes covalently attached to the terminus of duplex DNA are mostly stacked at the end of the helix by forming a π-stacked complex with the terminal base pair.52,53 Similar cyanineDNA interactions were also observed for single-stranded oligos.54 These are dynamic interactions, where the dyes are likely to exist in an equilibrium between a bound and unbound state,55 resulting in dramatic variations in dye fluorescence quantum yields and lifetimes.56 This behavior is the consequence of the existence of a photoisomerization process in cyanine dyes that deactivates the first singlet excited state with an efficiency that depends strongly on the local microscopic friction. The binding of the DNA to the liposome membrane probably shifts the equilibrium from the bound to the unbound state. This alters the rotational behavior of the dye around its linker as well as its fluorescence lifetime and thereby decreases the measured anisotropy value. Although steady-state anisotropy can be used for a broad range of fluorescent probes, the DNA data shows that careful controls have to be performed to rule out that the fluorescence properties of the dye is not affected by the molecule membrane interactions. Lohse et al. showed that the small water-soluble fluorophore CoroNa Green is encapsulated in higher concentration in small
Figure 8. Liposome size dependency of concentration and anisotropy value of the encapsulants. The number of encapsulated molecules increases with increasing liposome size (inset in part a). However, the highest concentration of encapsulants can be found within smaller liposomes (a). The fluorescence anisotropy of encapsulated molecules does not show any significant dependency on liposome size (b). 8174
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Analytical Chemistry liposomes than in large ones.19 This observation was later supported by Luisi et al., who used the protein ferritin as an encapsulant,20 pointing toward it being a general property of encapsulation of water-soluble molecules in liposomes. Both these studies showed that the concentration inside single liposomes is inversely proportional to the diameter of the liposomes, which suggests that the mechanism behind this observation could be related to the surface-to-volume ratio of the liposomes and that moleculemembrane interactions could play an important role. Our fluorescence anisotropy imaging assay gives us the tool to investigate these kinds of heterogeneities of moleculemembrane interactions within liposomes of different sizes. While the number of encapsulated Atto655 molecules decreases with decreasing liposome size (Figure 8a, inset), the intraliposomal concentration of the dye molecules increases with decreasing liposome size (Figure 8a) as previously shown with other water-soluble molecules.19,20 To evaluate whether the increased concentration within small liposomes is due to increased moleculemembrane interactions, we plotted the anisotropy value of the encapsulants versus liposome size (Figure 8b). The anisotropy values show a relative broad spread but no systematic dependency on size, suggesting that the moleculemembrane interactions do not change with liposome size. However, it cannot be excluded that these interactions play a role during the formation of the liposomes and thereby effect the final concentration of encapsulants within the liposomes. Besides moleculemembrane interactions, we envision that crowding effects during liposome formation, in which the liposomes act as a crowder, could also play a significant role in up concentration of encapsulants in small liposomes.57,58
’ CONCLUSIONS We have established a fluorescence anisotropy imaging assay based on single liposomes, which allow us to investigate moleculemembrane interactions of molecules encapsulated in liposomes. We used our assay to examine if moleculemembrane interactions could be the reason for the previously reported increase in encapsulation efficiency of water-soluble molecules with decreasing liposome size.19,20 Since we do not see any changes in moleculemembrane interactions of encapsulated molecules within liposomes of different sizes, we conclude that the observed increasing concentration of encapsulants with decreasing liposome size cannot exclusively be explained by moleculemembrane interactions. Nevertheless, our fluorescence anisotropy imaging assay shows great potential for studying moleculemembrane interactions as well as molecule molecule interactions in a high-throughput manner in nanometer-scaled containers. ’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: +4535320479. Fax: +4535320406. E-mail: stamou@ nano.ku.dk.
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