Concentration-Induced J-Aggregate Formation Causes a Biphasic

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Concentration-Induced J-Aggregate Formation Causes a Biphasic Change in the Release of trans-Combretastatin A4 Disodium Phosphate from Archaeosomes and the Subsequent Cytotoxicity on Mammary Cancer Cells Varsha P. Daswani, Umme Ayesa, Berenice Venegas, and Parkson Lee-Gau Chong Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00500 • Publication Date (Web): 10 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015

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Molecular Pharmaceutics

Concentration-Induced J-Aggregate Formation Causes a Biphasic Change in the Release of trans-Combretastatin A4 Disodium Phosphate from Archaeosomes and the Subsequent Cytotoxicity on Mammary Cancer Cells

Varsha P. Daswani, Umme Ayesa, Berenice Venegas, and Parkson Lee-Gau Chong*

Department of Medical Genetics and Molecular Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140

*Corresponding author Email: [email protected]; phone: 215-707-4182; fax: 215-707-7536

1 ACS Paragon Plus Environment


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Table of Contents/Abstract Graphic


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Abstract Combretastatin A4 disodium phosphate (CA4P) is a fluorescent, water-soluble prodrug able to induce vascular shutdown within tumors at doses less than one-tenth of the maximum tolerated dose. As a continued effort to develop efficient liposomal CA4P to treat solid tumor, we herein investigate the physical and spectroscopic properties of CA4P in aqueous solution and the mechanism of CA4P release from archaeal tetraether liposomes (archaeosomes). We found that cis-CA4P can be photo-isomerized to trans-CA4P. This photo-isomerization results in an increase in fluorescence intensity. Both cis- and trans-CA4P undergo fluorescence intensity selfquenching after they reach a critical concentration Cq (~0.15-0.25 mM). Moreover, both cis- and trans-CA4P in buffer exhibit a red shift in their excitation spectrum and an increase in excitation spectrum band sharpness with increasing concentration, which can be attributed to the formation of J-aggregates. The onset of the dramatic change in excitation maximum occurs at concentrations close to Cq, suggesting that the self-quenching arises from extensive J-aggregate formation and that, when CA4P concentration exceeds Cq, J-aggregate formation begins to increase sharply. Our data also suggests that the extent of J-aggregate formation plays a critical role in CA4P release from tetraether archaeosomes and in the subsequent cytotoxicity on cultured human breast cancer MCF-7 cells. The drug leakage and cytotoxicity rate constants vary with the initial CA4P concentration entrapped inside archaeosomes in a biphasic manner, reaching a local maximum at 0.25-0.50 mM. A mechanism based on the concept of J-aggregate formation has been proposed to explain the biphasic changes in drug release and cytotoxicity with increasing drug concentration. Tetraether archaeosomes are extraordinarily stable and relatively non-toxic to animals; thus, they are promising nano drug carriers. The results obtained



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from this study pave the way for future development of archaeosomal CA4P to treat solid tumors.

Key words: vascular-disrupting agent, fluorescence, cis-to-trans isomerization, self-quenching, J-aggregates, mechanism of drug release, cytotoxicity, archaeal tetraether liposomes

Abbreviations used: BTL, bipolar tetraether liposomes; CA4, combretastatin A4; CA4P, combretastatin A4 disodium phosphate; GDNT, glycerol dialkylcalditol tetraether; GDGT, glycerol dialkyglycerol tetraether; PLFE, polar lipid fraction E; TDM, n-tetradecyl-β-Dmaltoside


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Introduction Combretastatin A4 disodium phosphate (CA4P) is a prodrug of the vascular-disrupting agent combretastatin A4 (CA4). After administration to cultured cells or living tissues, CA4P is rapidly cleaved to CA4 by endogenous phosphatases. CA4 binds tubulin, disrupting the formation of microtubules,1 which are essential for intracellular protein trafficking, cell movement, and mitotic cell division. Disruption of microtubule formation by CA4 thus reduces cell proliferation and viability. CA4 preferentially binds tubulin in endothelial cells of tumor blood vessels.2 As a result, CA4 induces a pronounced reduction in blood flow to solid tumors, leading to extensive tumor cell necrosis.2-4 It is also thought that the primary action of CA4P comes from its effect on the three dimensional shape of newly formed endothelial cells rather than its anti-mitotic effect.5 Regardless of the underlying mechanism, CA4P is clinically more favorable than CA4 due to the significantly greater solubility of CA4P in water. CA4P induces vascular shutdown within tumors at doses less than one-tenth of the maximum tolerated dose.2 As with CA4, the antivascular effect of CA4P has been demonstrated both in animal models6,7 and human cancer patients.8,9 An important limitation of combretastatin is that it also significantly alters blood flow in many normal tissues including heart, brain, spleen, skin, and kidney.10 In the case of the brain and the heart, even minute changes in blood flow may cause serious harm to the patient.

Liposomal drugs, as opposed to free drugs, can be developed to selectively deliver drugs to the desired site in the body, thus reducing the potential side effects and increasing the therapeutic efficacy.11,12 With our collaborative group, we have previously employed liposomal CA4P in an animal model study of breast cancer.13 Our data showed that targeting anti-E-selectin conjugated immunoliposomes loaded with CA4P to MCa-4 mammary tumors in mice treated with



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therapeutic doses of ionizing radiation resulted in a significant delay in tumor growth when compared with other treatment groups such as free CA4P, tumor irradiation alone, liposomal CA4P alone and empty liposomes.13 In this study,13 radiation was used to up-regulate expression of endothelial cell adhesion molecules (e.g., E-selectin), which provided a means for targeting drugs to irradiated tissue. While this approach holds great promise, the overall efficacy of the liposomal CA4P was limited. Our data showed that the combination treatment with ionizing radiation and immunoliposomal CA4P still allowed tumors to increase in size over time, although the rate was reduced significantly.13

In order to gain necessary knowledge to develop liposomal CA4P with higher therapeutic efficacy, we have herein studied the physical and spectroscopic properties of CA4P in aqueous solution. We found that cis-CA4P can be photo-isomerized to trans-CA4P in concomitant with fluorescence intensity enhancement and that both cis- and trans-CA4P undergo fluorescence intensity self-quenching after they reach a critical concentration Cq. Moreover, we obtained spectroscopic evidence showing that both cis- and trans-CA4P in buffer can form concentrationinduced J-aggregates.

We then investigated how J-aggregate formation affected trans-CA4P behavior in liposomes. We employed liposomes made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius.14,15 PLFE is a mixture of tetraether lipids, namely, GDNT (glycerol dialkylcalditol tetraether) and GDGT (glycerol dialkyglycerol tetraether) (Figure S1, Supplemental Material).14,15 The remarkable intrinsic stability of


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archaeosomes16,17 along with their low cytotoxicity in animals,18-20 makes them appealing for drug/gene delivery.21

In the current study, we found that the initial CA4P concentration entrapped in PLFE archaeosomes, therefore the extent of J-aggregation, plays a critical role in CA4P behavior. The leakage and cytotoxicity rate constants vary with the initial entrapped CA4P concentration in a biphasic manner, reaching a local maximum slightly above the critical self-quenching concentration Cq. These findings reveal the mechanism of CA4P release from liposomes and illustrate the necessity to consider drug aggregation when designing liposomal CA4P for cancer therapy.

Materials and Methods Archaeal cells and PLFE lipids.

Cells from the thermoacidophilic archaeon Sulfolobus

acidocaldarious (ATCC #49426, Rockville, MD) were grown aerobically and heterotrophically at ~75-80°C and pH 2.5-3.0. The polar lipid fraction E (PLFE) was isolated from S. acidocaldarius dry cells as previously described.14,15 The concentrations of PLFE in stock solutions were determined based on a phosphate assay.22

Photo-isomerization of CA4P in solution. Cis-CA4P, obtained from Dr. Mohammad Kiani,13 was photo-activated23 to trans-CA4P in Tris buffer by illuminating the sample with a xenon arc lamp on an ISS K2 fluorometer (Champaign, IL) through a monochromator set at 328 nm (slit width = 8 nm).



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Fluorescence, absorbance, and NMR measurements.

Fluorescence excitation and emission

spectra as well as self-quenching were taken on an ISS K2 (Champaign, IL) or SLM DMX-1000 (Urbana, IL) fluorometer under constant stirring. Self-quenching was assessed by examining the fluorescence intensity at different concentrations of both cis- and trans-CA4P using an excitation of 328 nm and an emission of 400 nm. Absorption spectra were measured on the Perkin-Elmer Lambda 25 spectrometer (Shelton, CT). 1H-NMR spectra were recorded on a Bruker Avance-III 500 spectrometer (Billerica, MA) at 500 MHz using 10 mg of CA4P dissolved into 0.93 mL of D2O (24.4 mM CA4P).

Liposome preparation.

PLFE stock solution was made in chloroform/methanol/water

(66/31/7, v/v/v). Unilamellar vesicles of PLFE dispersed in Tris buffer (50 mM Tris, 10 mM EDTA, with or without (for the cytotoxicity study) 0.02% NaN3, pH 7.2) were prepared as described.24 In the leakage and cytotoxicity studies, the buffer that was used to make liposomes contained the desired amount of CA4P. Thus, the initial CA4P concentration entrapped in the interior compartment of the liposomes can be varied as needed. The hydrodynamic diameters of the liposomes were measured at 25oC by photon correlation spectroscopy using a Malvern Zetasizer 1000HAS spectrometer (Wores, UK). The light source was a 10 mW He-Ne laser (633 nm) and the scattered light was measured at 90° to the incident beam.

Separation of free CA4P from liposomal CA4P.

Immediately before the cytotoxicity assay or

the CA4P leakage experiment, free CA4P was removed by a Sephadex G-50 gel filtration column, the fractions containing liposomes with entrapped CA4P were pooled, and the amount of CA4P entrapped in liposomes was determined, as previously described.25


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Drug leakage assay.

Leakage of entrapped CA4P from liposomes was monitored by

measuring the increase of CA4P fluorescence intensity due to the relief of self-quenching. Specifically, 200 µL of liposomal CA4P dispersions collected from Sephadex G-50 (described above) was diluted with 2 mL of 50 mM Tris buffer (pH 7.2) containing 10 mM EDTA and 0.02% NaN3. The samples were excited at 328 nm, and the fluorescence intensity at 400 nm (Ft) was recorded as a function of time on the ISS K2 fluorometer. At the end of each leakage experiment, the sample was mixed with the detergent n-tetradecyl-β-D-maltoside (TDM) (Anatrace, Maumee, OH) (final TDM concentration in the cuvette = 100 µM) for 20 min to release all the entrapped CA4P, providing the maximal fluorescence intensity (Fmax). The equation F = + B(1 − ), where k is the rate constant of leakage, t is time, and A and B are constants, was used to fit the normalized fluorescence intensity F (= Ft/Fmax). For each drug concentration examined, the leakage assay was repeated three times.

MCF-7 cell line.

Human MCF-7 breast cancer cells (from ATCC) were cultured in T-75

flasks at 37°C with 5% CO2 in Dulbecco's modification of Eagle's medium (DMEM 1x, with 4.5g/L glucose, L-glutamine and sodium pyruvate) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cell medium was replaced every two days. When ~80% confluency was reached, cells were detached using 0.25% trypsin-EDTA, rinsed with growth medium, and collected after centrifugation at 1000 rpm for 5 min at 25°C. Cell numbers were counted by a hemocytometer.



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Cytotoxicity assays.

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Cytotoxicity was determined using both the CyQuant assay and the

live/dead cell imaging kit. In the CyQuant assay, MCF-7 cells were resuspended into complete growth medium, plated into a 96-well microplate (~10,000 cells per well) and maintained in a serum-containing medium for > 12h, until the cells adhered to the wall of the microplate wells. After the MCF-7 cells were treated with liposomal CA4P for the desired time period at 37oC, the dead cells (floating in the growth medium) were removed. The total volume (growth medium plus vesicle dispersions in buffer) in each well was 200 µL. The cell proliferation assay was performed to determine the number of cells remaining alive (defined as those still attached to the well). The assay was performed at ~24oC using the CyQuant kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Fluorescence intensities at 530 nm were measured on a Spectra Max M5 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation at 485 nm. A standard curve was constructed for each sample set using known amounts of MCF-7 cells counted with a hemacytometer so that the fluorescence intensities could be converted to the cell numbers. Typically, 0.5 nmole of CA4P in liposomes was added to each of the 96-wells in the microplate, which had ~10,000 cells per well at time zero. Cytotoxicity of archaeosomal CA4P against MCF-7 cells was also determined via a Nikon Eclipse TE-2000U fluorescence microscope using a live/dead cell imaging kit (Molecular Probes, Eugene, OR). The cytotoxicity rate constant k’ was calculated by fitting the cytotoxicity data into an exponential decay equation: % surviving cells = A1 (1-e(-k’t)) + yo, where A1 and k’ were floating parameters and yo was fixed at 100.

Results and Discussion


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Photo-activation of CA4P.

Figure 1A shows that when CA4P in buffer was excited with light

at 328 nm, the fluorescence intensity detected at 400 nm underwent a steady increase over time in a concentration dependent manner. When the excitation iris of the fluorometer became more widely open (Figure 1B, illumination increased by ~25-fold), the fluorescence intensity of CA4P was enhanced with a faster rate, reaching a plateau earlier. This photoactivation effect was observable in different buffer systems (Figures 1 and 2). The blockage of the light exposure for 24 hours (Figure S2) after the sample was excited continuously by light at 328 nm for 72 hours did not reverse the fluorescence enhancement.

A possible reaction mechanism responsible for the fluorescence enhancement is shown in Scheme 1 (Figure 2),26 in which an intermediate is generated as a resonance structure. This intermediate is followed by an oxidant (including oxygen)-induced ring closure.26 To test if this mechanism is plausible, we have repeated the fluorescence enhancement experiment using the degassed buffer. As shown in Figure 2, virtually the same fluorescence enhancement profile was obtained for CA4P in degassed MOPS buffer and in un-degassed buffer. This result argues against Scheme 1 to be the underlying reaction mechanism of the photon-induced fluorescence enhancement.

Another possible reaction mechanism is a photon-induced cis-to-trans isomerization (Scheme 2 in Figure 3), which has been previously reported to occur in many stilbenes and stilbenoids.27 The NMR chemical shift (δ) of the two protons on the ethylene bridge of cis-stilbenes is known to be ~6.5 ppm whereas the chemical shift of the two ethylene protons on the trans-stilbenes is ~7.0 ppm.27,28 It is clear from Figure 3A that, before UV light exposure, the 1H NMR spectrum



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of CA4P (a stilbenoid) in D2O (δ = 4.75 ppm) displays two doublets at δ = 6.5 ppm, which correspond to the two protons on the ethylene bridge of the cis-CA4P. Upon UV light exposure, two additional doublets at δ = 7.0 ppm appear (Figure 3B), which correspond to the two protons on the ethylene bridge of trans-CA4P. Note that, for 1H NMR measurements, a high CA4P concentration was needed (24.4 mM in our case). At such high CA4P concentrations, exposure to the xenon arc lamp UV light in the ISS K2 fluorometer cannot convert all the cis-CA4P molecules to trans-CA4P in days. This explains why both cis-ethylene proton and trans-ethylene proton chemical shifts are observable in the NMR spectra (Figure 3B). In short, the appearance of two doublets at δ = 7.0 ppm (Figure 3B) is strong evidence for the occurrence of a cis-to-trans isomerization on CA4P when it is exposed to UV light.

It is noted from a recent study that CA4 (the lipid soluble form of CA4P) can also undertake photon-induced cis-to-trans isomerization, with the cis-isomer being more cytotoxic and having a lower fluorescence quantum yield.29

Comparison of fluorescence properties of trans-CA4P and cis-CA4P in solution.

As

shown in Figure 4, there is a very small difference in the excitation and emission spectra of CA4P in buffer before and after photon-induced activation. For un-activated CA4P (cis-CA4P) in buffer, λex,max = 339 nm and λem,max = 397 nm. For photon-activated CA4P (trans-CA4P), λex,max = 342 nm and λem,max = 395 nm. The small blue shift in emission maximum suggests that trans-CA4P is slightly more hydrophobic than cis-CA4P, which is consistent with the conclusion derived from the HPLC elution profile (Figure S3).


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In making liposomal drugs, it is a common practice to maximize drug loading inside the vesicles. Thus, it is of interest to study how the fluorescence properties of CA4P are affected by its concentration so we may utilize CA4P’s fluorescence signals properly to understand the molecular actions of CA4P and to optimize the design of liposomal CA4P for achieving a higher therapeutic efficacy.

Figure 5 shows that the fluorescence intensity of both trans- and cis-CA4P do not change monotonically with CA4P concentration. At low concentrations, the fluorescence intensity increases with increasing [CA4P]. Beyond a critical concentration (Cq), the fluorescence intensity decreases with concentrations. The Cq values for cis-CA4P and trans-CA4P at different pHs and temperatures are listed in Table 1. Note that, for the data presented in Figures 4, 5 and S5, the cis-CA4P samples were exposed to UV light for less than 1 min. Under this condition, the photon-induced cis-to-trans isomerization was negligible.

Linking self-quenching of CA4P fluorescence to J-aggregate formation.

Figures 6A and 6B

show that there is a pronounced red shift in excitation spectra and an increase in spectral band sharpness as [trans-CA4P] increases. These spectral changes are known to occur when dye molecules form J-aggregates.30 This assertion is supported by absorption spectra. The absorption spectrum of trans-CA4P in buffer is broad and can be deconvoluted into three Gaussian peaks (Figure 7). Peak 1, Peak 2 and Peak 3 appear at ~250, 290, and 325 nm, respectively (Figure 7). Peak 3 is virtually not observable at low [trans-CA4P] (e.g., 50 µM); however, as [trans-CA4P] increases, Peak 3 increases steadily and significantly (Figure 7 and S4A). The ratio of the absorbance at 325 nm (Peak 3) to the absorbance at 290 nm (Peak 2) also shows a steady



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increase with increasing [trans-CA4P] (Figure S4B). Clearly, the absorption spectra also reveal a red shift with increasing [trans-CA4P] in support of J-aggregate formation. Like trans-CA4P, cis-CA4P exhibits a red shift in excitation spectrum and an increase in excitation band sharpness as the concentration increases (Figure S5), suggesting that J-aggregation occurs for cis-CA4P too. In J-aggregates, molecules are paralleled aligned, with the transition dipole moments of the chromophore coherently coupled, resulting in delocalization of electronically excited states over many (typically 3-50) monomeric chromophores.31,32

To estimate the size of the CA4P J-aggregates, we have employed 90° static (time average) light scattering to check for the possibility of CA4P J-aggregates to grow into microcrystal size. Park et al.33 previously demonstrated that a fluctuation parameter, defined as the standard deviation of the intensity fluctuation divided by the average intensity (σ/mean), underwent an abrupt increase when cholesterol microcrystals (> 20 nm)34 were formed. The abrupt increase in this parameter at 66.7 mol% cholesterol in phosphatidylcholine bilayers matched with the direct detection of microcrystals by X-ray diffraction at the same mole fraction.33 Using this method, we found that

σ/mean for CA4P in buffer does not undergo any dramatic increase with increasing CA4P concentration over a large concentration range examined (0-8 mM) (Figure S6). This result suggests that J-aggregates of CA4P do not grow into cholesterol microcrystal-like particles (> 20 nm).34 In addition, we were unable to obtain any meaningful dynamic (real time) light scattering data from the Malvern Zetasizer 1000HAS spectrometer (Wores, UK), even when using high CA4P concentrations (e.g., > 1 mM). This suggests that CA4P J-aggregates are probably smaller than 2-4 nm, which is the lower detection limit of this instrument. These results also imply that,


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unlike many other anticancer drugs such as bexarotene, crizotinib, and sorafenib, CA4P does not form colloid aggregates, which have the size ranging from 50-500 nm.35

The red shift in the excitation wavelength at maximum intensity (λex,max) varies with [transCA4P] most dramatically at ~200-300 µM with an onset concentration < 200 µM (Figure 6C), which is close to the critical self-quenching concentration Cq (188 and 150 µM at 25oC and 60oC, respectively, and at pH 7.2). This result suggests that J-aggregation begins to occur below Cq and the self-quenching of CA4P fluorescence is related to the extent of CA4P J-aggregation. It is possible that the fluorescence self-quenching arises from extensive J-aggregate formation and that, when [trans-CA4P] exceeds Cq, J-aggregates increase sharply.

Effect of initial [trans-CA4P] on drug leakage rate constant.

The entrapped J-aggregates

must affect the rate of drug diffusion across the liposomal membrane. It is then of interest to interrogate how the initial CA4P concentration entrapped inside the vesicles affects the drug leakage and consequently the drug’s cytotoxicity.

To address this question, we varied the initial entrapped concentrations of trans-CA4P ([transCA4P]i) in liposomes comprised of 100 mol% PLFE and then examined the changes in drug leakage using the method described in Materials and Method. We found that the leakage rate constant k initially increased with increasing [trans-CA4P]i up to ~250-500 µM (Figure 8). Thereafter, k decreased with increasing [trans-CA4P]i (Figure 8). Note that we did not determine the leakage rate constant of cis-CA4P because the fluorescence enhancement due to cis-to-trans



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isomerization would be concomitant with the relief of fluorescence self-quenching due to drug leakage, making it difficult to determine accurately the leakage rate constant.

The biphasic effect of initial trans-CA4P concentration on the leakage rate constant (Figure 8) can be explained as follows. At low [trans-CA4P]i (e.g., < 0.15 mM, Stage 1 in Figure 9), CA4P molecules inside the vesicles are probably almost exclusively in the monomeric form. They can diffuse across the liposomal membranes, but due to the low osmotic pressure and the tight/rigid packing of PLFE liposomal membranes,16,17 the leakage rate constant is low at this stage. As [trans-CA4P]i increases to an intermediate concentration (e.g., 0.15 mM, Stage 2 in Figure 9), the leakage rate constant increases significantly probably due to increased osmotic pressure. It is likely that, at this stage, some CA4P J-aggregates are formed, however, the majority of CA4P is still monomeric. At higher [trans-CA4P]i (> 0.5 mM; Stage 3 in Figure 9), J-aggregate becomes increasingly more abundant. It is likely that J-aggregates cannot diffuse out of the PLFE liposomes as the volume fluctuation of PLFE liposomes is extremely low.36 J-aggregates have a coherent length in the order to 3-50 monomers32 and are more tightly packed than Haggregates.37,38 Most likely, J-aggregates need to be dissociated into monomers before spontaneous diffusion across the membrane can occur. As a result, the leakage rate constant decreases steadily with increasing [trans-CA4P]i at high [trans-CA4P]i.

Effect of initial entrapped trans-CA4P concentration in PLFE liposomes on cytotoxicity on MCF-7 cancer cells (i) as revealed by the CyQuant assay.

The effect of [trans-CA4P]i on cytotoxicity of

liposomal CA4P against MCF-7 cells as monitored by the CyQuant assay kit is presented in


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Figure 10. Figure 10A shows the time dependence of PLFE archaeosomal CA4P on cytotoxicity with different initial entrapped-trans-CA4P concentrations. As illustrated in Figure 10B, the time trace can be fitted to an exponential decay function (see Materials and Methods). In the case of [CA4P] = 250 µM (Figure 10B), the fitted cytotoxicity rate constant k’ is 0.281 ± 0.017 10-4s-1. Figure 10C shows that the k’ value varies with the initial entrapped-trans-CA4P concentration in a biphasic manner, showing a maximum at ~0.25-0.5 mM. This result is consistent with the drug leakage rate constant data (Figure 8). However, understandably, after 48 hours the greatest cytotoxicity was seen from the sample with the highest amount of CA4P present (5 mM).

(ii) as revealed by Invitrogen DEAD/LIVE viability assay.

To verify the results obtained from

the CyQuant viability assay, we employed the Live/Dead microscopy kit from Invitrogen (Molecular Probes, Eugene, OR) to study MCF-7 cell death induced by CA4P entrapped in PLFE liposomes. Figure 11A shows the fluorescence microscopy images obtained from cells treated with PLFE liposomal CA4P for 8 hrs. It can be seen from these images that the number of dead cells (red) relative to live cells (green) was most abundant when the MCF-7 cells were treated with PLFE liposomal CA4P at [trans-CA4P]i = 0.25 and 0.5 mM. At [trans-CA4P]i = 0.1, 1 and 5 mM, the relative number of dead cells is decreased. Similar images were recorded at other time points, so that we have the data showing the time dependence of the percent of surviving cells (defined in Materials and Methods) arising from different [trans-CA4P]i (illustrated in Figure 11B). Like the CyQuant assay data, the time dependence of the microscopy viability data can be best fit to the equation: % surviving cells = A1(1-e(-k’t)) + yo, (red line in Figure 11B), which yielded the cytotoxicity rate constant k’. The k’ values at different [transCA4P]i are summarized in Figure 11C, which shows that k’ varies with [trans-CA4P]i in a



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biphasic manner, displaying a maximum near 0.25 and 0.5 mM. This biphasic behavior observed from the Live/Dead microscopy kit is consistent with the cytotoxicity rate constant data obtained from the CyQuant assay (Figure 10) and agrees with the trend observed from the drug leakage experiment, which also displays a biphasic change with the initial CA4P concentration entrapped inside the vesicles, with the largest leakage rate constant k at 0.25 mM and 0.5 mM trans-CA4P (Figure 8).

Conclusion In summary, our present data suggests that cis-CA4P can be photo-isomerized to trans-CA4P, in conjunction with fluorescence intensity enhancement. This result indicates that, in order to avoid complications in data interpretation, trans-CA4P as opposed to cis-CA4P should be employed when using CA4P fluorescence to monitor the drug behavior. Further, our data reveals the mechanism of CA4P release from liposomes. When trans-CA4P is at high concentrations inside the liposomes, the drug tends to form J-aggregates. The biphasic change in leakage rate constant with trans-CA4P concentration (Figure 8) supports the idea that J-aggregate does not move across the liposomal membrane but the monomeric form does. J-aggregate formation reduces not only the overall rate of drug release from liposomes, but also the effectiveness of the liposomal CA4P in killing mammalian cells (Figures 10 and 11). These findings validate the use of maximal CA4P loading (~30 mM) in our previous liposomal CA4P studies on rodent models,13 which would favor a slower spontaneous release and force more drug molecules to reach the target. The current data paves the way for future development of archaeosomal CA4P. In short, this study demonstrates the necessity to consider drug concentration and J-aggregate formation when designing liposomal CA4P for cancer therapy.


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Acknowledgment This work was supported by NSF grants (DMR1105277 and CBET1350841) and DOD Breast Cancer Research Program (BC063769). The authors thank Dr. Mohammad Kiani for providing CA4P and Drs. Yuri Persidsky, David Dalton, Charles Debrosse, and Ana Gamero for their instrument and technical support.



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Figure Legends: Figure 1. The photon-induced fluorescence enhancement for CA4P in 50 mM Tris buffer (pH 7.2) containing 10 mM EDTA and 0.02% NaN3 was examined at three different CA4P concentrations: 10, 25 and 75 µM. Temperature = ~24oC. The fluorescence intensity was measured on an ISS K2 fluorometer with the iris fixed at narrow (A) and wide (B) opening.

Figure 2: A test to determine if oxygen plays a role in the photon-induced CA4P fluorescence enhancement. 50 µM CA4P in un-degassed (blue) and de-gassed (red) MOPS buffer (1 M MOPS, 1 M citric acid, 0.02% NaN3, pH 7.2) was employed. λex = 328 nm; λem = 400 nm; temperature ~24oC. Degassing was achieved by heating and stirring the solution under vacuum overnight, followed by flushing with argon gas.

Figure 3. (A) 1H NMR spectrum of 24.4 mM CA4P in D2O before UV light exposure. (B) 1H NMR spectrum of 24.4 mM CA4P in D2O after being exposed to light at 328 nm for three days. The changes in the NMR spectrum can be explained by the light-induced cis-to-trans isomerization (Scheme 2).

Figure 4. Uncorrected excitation (dashed lines, λem = 400 nm) and corrected emission (solid lines, λex = 328 nm) spectra of photon-activated CA4P (blue) and un-activated CA4P (red) in 50 mM Tris, 10 mM EDTA, 0.02% NaN3, pH 7.2 at ~24oC. [CA4P] = 50 µM. The spectra are normalized to unity at the excitation and emission maximum.


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Figure 5. Self quenching behavior of both trans- and cis-CA4P. Fluorescence intensity at 400 nm was measured at 25, 37 and 60°C and pH 3.0 and 7.2, with the excitation wavelength set at 328 nm. CA4P samples were suspended in MOPS:citric acid buffer. Each sample was prepared from a stock of 30 mM CA4P and kept away from light until the time of fluorescence measurements

Figure 6. (A) and (B) illustrate the red shift in uncorrected excitation spectrum with increasing [trans-CA4P] at 25 and 60oC, respectively. (C) shows how the wavelength of the excitation maximum (λex,max) varies with [trans-CA4P]. Emission was measured at 430 nm. Buffer: 50 mM Tris, 10 mM EDTA, 0.02% NaN3 at pH 7.2.

Figure 7. Absorption spectrum of trans-CA4P in buffer (50 mM Tris, 10 mM EDTA, pH 7.2) at different drug concentrations. Temperature = ~25oC. Slit width = 1 nm. Scan rate = 60 nm/min. Each absorption spectrum (black) is deconvoluted into three Gaussian peaks (Peak 1, red; Peak 2, green; Peak 3, blue) using Origin v. 8.5.

Figure 8. Effect of the initial concentration of entrapped trans-CA4P on the drug leakage rate constant k of PLFE liposomes. Temperature = 25oC; Buffer: 50 mM Tris, 10 mM EDTA, pH 7.2. Error bars are the standard deviations from three independently prepared samples.

Figure 9. Schematic explanation of the effect of [trans-CA4P]i on drug leakage rate constant. (1) At the low entrapped concentrations, CA4P is virtually completely in monomeric form and is slowly released via spontaneous diffusion. At this stage, osmotic pressure is low. (2) At the



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intermediate concentrations, osmotic pressure is increasingly significant, resulting in increased leakage rate constant. Few aggregates are present, however, CA4P is still primarily monomeric. (3) At high concentrations, CA4P is primarily in aggregate form. CA4P fluorescence is highly quenched, when the aggregation (coherent length) is extensive. Aggregates cannot diffuse out of the liposomal membrane so leakage rate is slow. Aggregates must dissociate before spontaneous transmembrane diffusion can occur.

Figure 10. (A) Effect of [trans-CA4P]i in PLFE liposomes on the percentage of surviving MCF7 cells over time, monitored by the CyQuant viability assay kit. (B) The CyQuant assay data obtained from PLFE liposomes with [trans-CA4P]i = 250 µM were fitted by the equation (red line): % surviving cells = A1(1-e(-k’t)) + yo, where k’ is the cytotoxicity rate constant and yo was fixed at 100 (see Materials and Methods). The fitted parameters are: A1= - 41.2 ± 0.76 and k’ = 0.281 ± 0.017 10-4s-1, with R2 = 0.995 and χ2 = 1.30. (C) Effect of [trans-CA4P]i in PLFE liposomes on the cytotoxicity rate constant k’. Buffer: 50 mM Tris, 10 mM EDTA, pH = 7.2; vesicle size ~200 nm; temperature = 37°C; n = 4

Figure 11. (A) Fluorescence images showing the effect of initial CA4P concentration [CA4P]i on cytotoxicity against MCF-7 cells after 8 hrs PLFE liposomal CA4P treatment. Red and green indicate dead and live cells, respectively. Images were taken by a Nikon Eclipse TE-2000U fluorescence microscope. (B) Live/Dead microscopy data obtained from PLFE liposomal CA4P with [trans-CA4P]i = 250 µM were fitted to the equation: % surviving cells = A1(1-e(-k’t)) + yo (red line). The fitted parameters are: A1= - 41.0 ± 0.95 and k’ = 0.292 ± 0.023 10-4s-1, with R2 = 0.992 and χ2 = 2.07. (C) Effect of [trans-CA4P]i on the cytotoxicity rate constant k’ of PLFE


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liposomal CA4P against MCF-7 cells. Buffer: 50 mM Tris containing 10 mM EDTA (pH = 7.2); vesicle size ~200 nm; temperature = 37°C; Error bars are the standard deviations (n=4).



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Table 1. Critical self-quenching concentration (Cq) of CA4P in buffer Cq (µM) cis-CA4P trans-CA4P Temperature pH 3.0 pH 7.2 pH 3.0 pH 7.2 (oC) 25 250 215 200 188 37

200

215

200

203

60

200

225

260

150


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Concentration-Induced J-Aggregate Formation Causes a Biphasic

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