Understanding the Dynamic Behavior of an Anticancer Drug

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Understanding the Dynamic Behavior of an Anti-Cancer Drug - Doxorubicin on a Lipid Membrane Using Multiple Spectroscopic Techniques Yi Hou, Shun-Li Chen, Wei Gan, Xing Ma, and Qunhui Yuan J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01941 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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The Journal of Physical Chemistry

Understanding the Dynamic Behavior of an Anticancer Drug - Doxorubicin on a Lipid Membrane Using Multiple Spectroscopic Techniques Yi Houa,†, Shun-Li Chena,†, Wei Gana, *, Xing Mab,*, Qunhui Yuanb,* a

State Key Laboratory of Advanced Welding and Joining, and School of Science, Harbin

Institute of Technology (Shenzhen), University Town, Shenzhen 518055, Guangdong, China b

State Key Laboratory of Advanced Welding and Joining, and School of Materials Science and

Engineering, Harbin Institute of Technology (Shenzhen), University Town, Shenzhen 518055, Guangdong, China †

These two authors contributed to this work equally.

Corresponding Author * Email: [email protected], [email protected], [email protected]

ABSTRACT: The interaction, including the adsorption and embedding, of a widely applied anticancer drug, doxorubicin, with a lipid membrane was investigated. Second harmonic generation and two photon fluorescence were used as a powerful combination capable in revealing this dynamic process at the interface. The adsorption, association, de-association and

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embedding of doxorubicin on the lipid membrane were clearly identified based on the consistency in the dynamic parameters revealed by the time dependent second harmonic generation and two-photon fluorescence measurements. This work also presents a new approach for in situ measurement of the adsorption density of doxorubicin on lipid membrane, benefiting from the two-photon fluorescence signal of doxorubicin being significantly altered by its chemical environment. The analysis of the location and molecular density based on the fluorescent efficiency of the chromophores makes the fluorescence measurement a “surface sensitive” technique as well. The analytical procedures used in this work are expected to aid in understanding the interaction between fluorescent molecules and lipid membranes in general. Introduction: Doxorubicin (DOX) is a widely used anticancer drug effective against many types of cancer cells.1-2 The interaction of DOX with lipid membranes has been intensively investigated because it not only influences the delivery of DOX into cancer cells in medical treatments but also affects the loading/release of DOX in/from liposomes in drug preparations.3-4 The interaction of DOX and other anthracycline antibiotics at the surface of lipid membranes under various chemical environments, such as under different pH levels, ionic strength or cholesterol concentration, has been compared.4-11 To determine the interaction of DOX with a lipid membrane, researchers applied techniques including fluorescence, circular dichroism, UV-vis adsorption, X-ray diffraction and NMR. Many of these methods need to measure the bulk concentration of DOX molecules and estimate its interfacial behavior. For example, in the studies of the interaction between drug molecules and phospholipid membranes, it is necessary to use probing molecules such as DNA to interact with the drug molecules and detect the changed fluorescence intensity from the drug molecules in the bulk phase, or from the probing molecules in the lipid layers.12


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The studies on the interaction between anthracycline drugs with polymer or lipid membranes based on the changed intensity or shape of the fluorescence spectra have presented some structural information

on the lipid interface.13-14 However, molecular level structure and

dynamics associated with these interfacial dynamic processes are still highly desired. Second harmonic generation (SHG) has been demonstrated as a powerful technique in the analysis of molecular density and orientation at interfaces, benefiting from its interface selectively and sub-monolayer sensitivity.15-27 Based on this advantage, SHG has been used to study molecular transportation through the liposome membrane and living cell membrane in situ and in real time.28-33 For example, Prof. Eisenthal’s group was first to detect the transportation of dye molecules across the bilayer of a phospholipid liposome with the help of the SHG technique.28 The influence of cholesterol on the delivery rate of an organic cation across the dioleolyphosphatidylglycerol liposome surface has also been analyzed.34 The binding of DOX with DNA on a SiO2 particle surface and the energetics during this dynamic process was also investigated using SHG measurements.35 Later, Prof. Dai’s group was first to demonstrate that SHG can be used to monitor the adsorption and transportation of molecules through membranes of living bacteria, e.g., Escherichia coli cells.29 Based on SHG analysis, understanding of the molecular mechanism for Gram’s staining protocol was clarified.36 The changes in the adsorption and transportation of dye molecules on bacterial membranes induced by an antimicrobial compound, azithromycin, were also studied.37 Obviously, SHG measurements are based on the idea that the measured SHG field is proportional to the density of adsorbed species at the interfaces.27,

29, 34, 38

With the transportation of molecules through the lipid membrane, the

increased number density of the adsorbed species on another side of the liposomes or cells (the inner surface) causes a decrease in the SHG field.28, 34, 39


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Here, we present a study on the adsorption and embedding of DOX on a lipid membrane using SHG and two-photon fluorescence (TPF) methods. Unlike the SHG method, TPF was generally considered to be a probe to bulk phase because in principle it has no surface selectivity, even though it may reflect structural change on the interface in certain cases.40 However, we found that TPF from DOX varied significantly in different chemical environment. This property can be used to study the dynamic behavior, as well as the density of DOX at the lipid interface. Experimental section Materials 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG, 99.9%) and doxorubicin hydrochloride (95%) were purchased from Sigma-Aldrich (U. S.) and Tokyo Chemical Industry (Japan), respectively. Chloroform (AR) was purchased from Tianjin Damao Chemical Reagent Factory (Tianjin, China). Deionized water (18.2 MΩ·cm) was obtained from a water purification system (WP-UP-UV-20) from Sichuan Water Technology Development Co. Ltd (Chengdu, China). Hepes-buffered saline solutions (HBS) used in part of the experiments contain 20 mM HEPES and 150 mM NaCl, both of which were purchased from Sigma-Aldrich. Vesicle preparation DOPG was dissolved in chloroform (~500 μL) and a thin film was formed by drying the solvent. Then it was dried in vacuum for 2 h, followed by addition of water to a DOPG concentration of 0.5 mM. The solution was sonicated for 25 min. (KQ-300DE, 300 W, 40 kHz, Kunshan Ultrasonic Instrument Co. Ltd, Kunshan, China) at a temperature of 30 ± 2 °C. The formed vesicle solution was cooled to room temperature and stored in a refrigerator at 4 °C. Before the experiment the solution was filtrated two times through a membrane with pore size of 0.8 μm to get rid of the aggregates. By drying the obtained sample and weighing the remaining


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DOPG, the loss of DOPG molecules in the filtration was estimated to be approximately 10%. This loss was taken into account in the data analysis. Vesicle characterization The number mean diameter of the vesicles was characterized by the dynamic light scattering method (90Plus PALS, Brookhaven) with the temperature set as 25 ± 1 °C and the measuring direction as 90°. During the experiments with low DOX addition, the vesicle size remained almost unchanged (90 ± 20 nm). In experiments with a high DOX to lipid mole ratio, the vesicle size increased as described in the discussion.

Second harmonic generation and two-photon fluorescence measurements A Ti: sapphire oscillator (MaiTai-HP, Spectra-Physics) with a pulse width of ~100 fs and repetition rate of 80 MHz was used as the fundamental laser source with the wavelength set at 800 nm. The power of the incident laser used in the experiments was 300 mW. The laser was passed through a high pass filter (FGL9, >720 nm, Thorlabs) that blocks the 400 nm second harmonic light and focused at the center of a cylindric fused quartz cell with 13 mm inner diameter. The SHG (~400 nm) and TPF (~555 nm) scattering signal was collected at a relatively large spatial angle range (~40°, as shown in Figure 1) around the forward direction. The signal was passed through two bandpass filters (FGB37S, 335-610 nm, Thorlabs) that block the residual pumping laser and focused into a monochromator (Omni-λ1509, Zolix Instruments CO., LTD, China). The signal at the desired wavelength was selected and detected with a photomultiplier tube (PMT, Hamamatsu R1527p, -900V). The PMT output was amplified by a factor of 5 with a preamplifier (SR445A, Stanford Research System) and analyzed by a photon counter (SR400, Stanford Research System). The obtained photon counts were then recorded with a computer. The polarization of fundamental laser was set with the direction of the electric


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field in the horizontal plane. During the measurements, the laboratory temperature was kept at 22 ± 1 ºC. Because the fundamental laser, the emitted SHG and the TPF signals were partly scattered/absorbed by the vesicle solution and the DOX molecules, all the SHG and TPF data

were corrected based on the extinction spectra of the vesicle solutions and the DOX solutions. After the correction of the measured SHG intensity, the Hyper-Rayleigh scattering intensity from DOX solutions was subtracted. Then, the electric field of the SHG signal from the surface of the DOPG vesicles (approximately 300 counts/second in intensity) was subtracted from the values obtained in the DOX adsorption experiments.

Figure 1. The experimental setup for the collecting of the scattered signal at a relatively large spatial angle.

Results and discussion: Figure 2 shows the scattering spectra measured from the equilibrium state after mixing DOPG vesicle solution (in deionized water, with a DOPG molecular concentration of 250 μM) and DOX (10 μM). The spectra of DOX and DOPG vesicle solutions at the same concentrations were also shown. Upon irradiation with an 800 nm femtosecond laser, the DOX molecules at the vesicle surface produce a measurable SHG signal at 400 nm. The two-photon fluorescence signal at around 555 nm is from the DOX molecules at the lipid interface and in the solution.


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Figure 2. The scattered spectra from the DOX solution (10 μM, solid line), DOPG vesicle solution (250 μM, dashed line), and the mixture of them at their respective concentrations (dotted line) when excited by 800 nm laser. Figure 3a shows time-resolved SHG field curves deduced from the measured SHG intensities after addition of DOX at various concentrations into liposome suspensions. Because the SHG field is generally considered in proportion to the density difference of the SHG probing molecules at two sides of the lipid bilayers,31, 41 the monotonic increase trend in the curves and the subsequent leveling off with no decay indicate that the DOX molecules adsorbed at the lipid bilayer surface with no transition across the membrane. Anthracycline drugs may penetrate lipid bilayers at other chemical environment, such as in the presence of salt in the solution42 and in HBS.43-46 We did observe this process in HBS, which was indicated by the decay of the SHG signal after DOX adsorption (Figure 3c). Here, we demonstrate that the adsorption/embedding of DOX at only the outer layer of the DOPG vesicles is somewhat complex. The combination of SHG and TPF methods is powerful in revealing the dynamic behavior and molecular structure at interfaces. As shown in Figure 3a, the SHG field curves recorded with DOX at relatively low concentrations (≤7 μM) can be fitted single-exponentially, indicating typical adsorption kinetics as revealed previously.15-16 However, the curves recorded with DOX at relatively high concentrations (≥9 μM) can only be satisfactorily fitted with a biexponential function, suggesting


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a complex dynamic process rather than typical adsorption. At high DOX concentrations, the interaction between DOX and the lipid bilayer seems to be composed of two steps: a fast process with a time constant at 1-3 seconds (τ1) and a slow process with a time constant at tens of seconds (τ2).

Figure 3. (a) Time-dependent SHG field obtained after the addition of DOX at various concentrations in 250 μM DOPG vesicle solutions. (b) Magnification of the rectangle area marked by dotted lines in (a) (some curves are shifted in the x direction to make the plot clear). (c) SHG field curve measured by mixing DOX and DOPG vesicles in HBS solution. The final concentrations of DOX and DOPG are 20 μM and 250 μM, respectively. (d) The time constants obtained from the fittings plotted as a function of DOX concentration. τ1 represents the rate of the fast SHG change in experiments with DOX at relatively high concentrations (9-25 μM), τ2 indicates the rate of the slow change in all the SHG curves, τ3 reflects the rate of the TPF change as shown in Figure 4. To gain insight into the fast and the slow dynamic processes revealed by the SHG experiments, we recorded TPF signals from DOX after adding DOPG suspension at equal


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volumes to DOX solutions of various concentrations. Figure 4 shows typical TPF curves with the original TPF signal divided by 2 to compensate for the DOX concentration having been reduced by half during DOPG addition. Compared with the original value, the final TPF signal increases at relatively low DOX concentrations ( ≤ 1 μM) while notably decreases at higher DOX

concentrations. In most curves, there is a drastic change in the TPF intensity immediately after DOPG addition followed by a single exponential increase or decrease. In previous studies, it was pointed out that anthracycline drugs interact with the lipid membrane and result in enhanced or diminished single fluorescence intensity, depending on the concentration of the anthracycline drugs.47 It is generally believed that the fluorescence of anthracycline

drugs

is

quenched

by

self-association,

but

enhanced

when

the

dihydroanthraquinone part is embedded in the lipid bilayer.13-14, 48 Based on this knowledge, we believe that the quick drop of the TPF signal at relatively high DOX concentrations (≥ 5 μM) upon vesicle addition is due to the quick adsorption/association of DOX molecules at the lipid bilayer surface, while the increase or decrease of the TPF signal that follows reflects the deassociation of the adsorbed DOX molecules and the embedding in the outer leaflet of the lipid bilayer. It is understandable that at relatively low bulk DOX concentrations ( ≤ 1 μM), the

interfacial density of the adsorbed DOX is also relatively low, so the diminishing of TPF is absent while the enhancement of TPF is observable. For experiments with higher DOX concentration (e.g., 3-15 μM), the higher DOX adsorption density leads to a quick diminish of the TPF signal. The following slow increase in the TPF signal then reflects the dynamics in the de-association and embedding of DOX in the lipid membrane. For experiments with even higher DOX concentrations (e.g., 25 μM), the slow decrease of the TPF signal after the quick drop indicates that the embedding of more DOX results in a high interfacial DOX density in the lipid


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bilayer. This high DOX density is not contributing to but weakening the TPF signal, possibly due to the association of DOX molecules embedded in the lipid membrane.

Figure 4. (a), (c) Time-dependent TPF intensity recorded after the addition of DOPG vesicle into DOX solution at various concentrations. (b), (d) Magnifications of the rectangle area marked by dotted lines in (a) and (c), respectively (some curves were shifted in the x direction to make the plot clear). The final concentration of DOPG is 250 μM. It is worth noting that the observed enhancing and quenching effect of the two-photon fluorescence signal from DOX molecules in different chemical environments can also be observed in their one-photon fluorescent scattering. As shown in Figure 5, the one-photon fluorescence peaks at around 560 nm, 595nm and 650 nm from the DOX molecules varied with


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the addition of DOPG vesicles at different DOX:DOPG ratios, while the fluorescence peak at around 685 nm from the DOPG vesicles was not notably affected by DOX addition. Although the two-photon fluorescence spectrum we observed is with no features at wavelengths higher than 600 nm because of the band pass filters (FGB37S) used in the experiments, we choose to mainly analyze the two-photon fluorescence because it can be measured with the same experimental setup used in the SHG measurements. Furthermore, it is very convenient to perform the time dependent two-photon fluorescence measurements needed for the dynamics analysis, as shown in Figure 4 and discussed later. However, because one-photon fluorescence has much higher emission efficiency than its two-photon counterpart, it also delivers valuable information on the structure and chemical environment of the molecules. For example, a recent review summarized the fluorescence sensing approaches based on the changed fluorescence efficiency of chromophores when molecules are assembled in micelles.49


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Figure 5. Single photon fluorescence spectra of DOX solutions before and after the addition of DOPG vesicles (250 μM) with DOX concentration of 0.5 μM (a) and 20 μM (b), respectively. The excitation wavelength is 450 nm. The fluorescence spectrometer used in the experiments is F-7000 purchased from Japan Hitachi high-tech. This physical picture on the interfacial behavior of DOX molecules can also be supported by the SHG data in Figure 3. As shown in Figure 3b, the dynamic process reflected by the SHG curves obtained at relatively high DOX concentrations (≥ 9 μM) is divided into two steps. The fast process is in line with the adsorption of DOX from the aqueous phase to the lipid membrane surface. This step is observable (τ1) in the SHG measurements but too fast to be detected by the TPF experiments, indicating that the adsorption and association of DOX molecules take place first, while the adjustment of the DOX orientation comes later. The slow dynamic step (τ2) observed in the SHG curves then reflects the de-association and embedding of DOX in the lipid membrane. In this step the DOX molecules were aligned in more orderly manner by the DOPG molecules and contributed to a higher SHG signal. Again, the time constant obtained in the SHG experiments (τ2) being relatively larger than those obtained in the TPF experiments (τ3) indicates that the embedding-reorienting of DOX molecules in the lipid bilayers has to come after the dissociation of DOX molecules adsorbed at the vesicle surface. For experiments with low DOX concentrations (< 9 μM), there is no fast rise before the single exponential increase in the SHG field. This is possibly because the adsorbed DOX molecules have relatively low interfacial density, which is not enough to form a structure with an orientational order and radiate a detectable SHG signal, as illustrated in Figure 6a. At relatively high DOX concentrations in the bulk, the adsorbed-associated molecules with higher density formed an oriented structure that led to a fast rise in SHG signal (Figure 3a). A multistep process was also observed during the interaction of the widely applied drug, ibuprofen, with lipid membranes using fluorescence binding assays and vibrational sum frequency spectroscopy experiments.50 The steps of


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electrostatic interaction and the hydrophobic insertion of the drug into the lipid membranes were also revealed in that work.

Figure 6. Schematic illustrations showing the adsorption and embedding of DOX molecules to the outer membrane of DOPG vesicles under relatively low (a) and high (b) DOX concentrations. To acquire the adsorption density of DOX molecules at the liposome surface, we added a relatively small amount (50 μL) of DOPG suspension to 2 mL of DOX solution (30 μM) four times (at points A, B, C, D in Figure 7). As expected, a significantly quenched fluorescence intensity is observed with the adsorption of DOX in the lipid membrane. Because the amount of DOPG vesicles is insufficient at the early stages (e.g., after A, B), the TPF signal is partly quenched. By the ratio of the decreased TPF signal, the DOX molecules adsorbed/embedded to the lipid membrane can be estimated as approximately 1:1.1 (DOX : DOPG. It needs to be noted that in the experiments with such high DOX/DOPG ratios, we observed a fast increase and then decrease in the scattered SHG signal before a slow increase, which implied that some of the DOX molecules penetrated across the lipid bilayer in these experiments. This observation is notably different from the SHG observations in experiments with a low DOX/DOPG ratio shown in Figure 3. The value reported here is calculated considering the distribution of DOX on both sides of the lipid bilayer. Because the DOX density on the outer side of the vesicles were higher than that on the inner side, the obtained value may somewhat overestimate the DOX density on


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the inner side of the vesicles but underestimate that on the outer side.). This value may somewhat underestimate the number of the DOX molecules at the surface because these molecules may still radiate a weak TPF signal. However, this estimation is in line with previous studies that revealed a ratio as 1-2 DOX per anionic phospholipid for the cardiolipin and phosphatidylserine case.51-52 At the late stages (i.e., after D), the TPF signal is almost completely quenched, indicating that the DOX remaining in the aqueous phase of the solution is negligible (less than 4%). It also shows that DOX molecules on the surface of the lipid membrane, both the associated ones on the surface and the embedded ones in the membranes, emitted almost zero TPF signal at a relatively high molecular density. Such a high quenching efficiency in the TPF emission of DOX is much more sensitive than that in its single-photon emission.53-55 This property makes TPF a “surface sensitive” probe of the interfacial molecular density of DOX. According to previous studies, the surface area per lipid molecule in any side of the vesicles or cells is about 0.6 nm2.56-57 This means DOX molecules can be highly enriched at the DOPG membrane with a density of approximately 1.5 molecules/nm2. In this experiment, we did observe a notable increase in the diameter of the DOPG vesicles after point B. The measured diameter can increase up to 180 ± 20 nm based on the dynamic light scattering measurements.


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Figure 7. Time-dependent TPF intensity recorded after addition of DOPG vesicle solutions (50 μL, 0.5 mM) four time to 2 mL DOX solution (30 μM) at points A, B, C, D. Conclusion The dynamics in the adsorption and embedding of DOX on a DOPG lipid membrane was revealed using SHG and TPF methods. It was found that both SHG and TPF were capable of reflecting the molecular densities of DOX at the interface. The adsorption-associationreorientation of DOX on the lipid surface is as fast as 1-3 seconds. The dissociation-embeddingreorientation of DOX in the lipid membrane occurs at a relatively slower rate in the range of 550 s, depending on the bulk concentration of DOX. The maximum adsorption density of DOX at one side of the DOPG vesicle surface was estimated to be approximately 1.5 molecules/nm2. In addition, the tendency of DOX to adsorb at the DOPG lipid membrane can be as high as around 96% in certain concentration range for DOX and DOPG vesicles. Because the altering of the fluorescence efficiency at various chemical environments is a general property of anthracycline drugs, we expect that the analysis in this work may also be used to understand the structure and dynamics of other anthracycline drugs in DOPG or other lipid membranes, including genuine cell membranes.

AUTHOR INFORMATION Notes The authors declare no competing financial interests. ACKNOWLEDGMENT


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This work was supported by the National Natural Science Foundation of China (21673285, 21873024) and the funding from the Shenzhen city (JCYJ20170307150520453).

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Understanding the Dynamic Behavior of an Anticancer Drug

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