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Liposomes Containing Lipid-soluble Zn(II)-bisdipicolylamine Derivatives Show Potential to be Targeted to Phosphatidylserine on the Surface of Cancer Cells Umme Ayesa, Brian D. Gray, Koon Y. Pak, and Parkson Lee-Gau Chong Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00760 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 14, 2016

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Molecular Pharmaceutics

Liposomes Containing Lipid-soluble Zn(II)-bis-dipicolylamine Derivatives Show Potential to be Targeted to Phosphatidylserine on the Surface of Cancer Cells Umme Ayesa1, Brian D. Gray2, Koon Y. Pak2, and Parkson Lee-Gau Chong 1,*

1

Department of Medical Genetics and Molecular Biochemistry, The Lewis Katz School of

Medicine at Temple University, Philadelphia, PA 19140, and 2Molecular Targeting Technologies, Inc., West Chester PA 19380

*Corresponding author: Parkson Lee-Gau Chong; Email: [email protected]; phone: 215707-4182; fax: 215-707-7536

KEYWORDS: nano carriers, MCF-7 cancer cells, targeted delivery, binding to anionic membranes, fluorescence, flow cytometry, cytotoxicity, confocal microscopy

ABBREVIATIONS: 7AAD, 7-aminoactinomycin D; Cy3, cyanine 3; DPA, zinc (II)-bis-dipicolylamine; DPACy3[22,22], zinc (II)-bis-dipicolylamine cyanine 3 [C22,C22]; FITC, fluorescein isothiocyanate; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PS, phosphatidylserine; POPS, 1palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine 1 ACS Paragon Plus Environment


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ABSTRACT Here we used a lipid-soluble Zn(II)-bis-dipicolylamine derivative as a membrane component to develop liposomal carriers that have potential to be targeted to phosphatidylserine (PS)-rich surfaces on cancer cells and to preferentially kill cancer cells without using anticancer drugs. This DPA derivative (abbreviated as DPA-Cy3[22, 22]) contains the fluorophore cyanine 3 (Cy3) and two 22-carbon chains that can be anchored into liposomal membrane bilayers. DPACy3[22, 22]/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) unilamellar vesicles (~150 nm) showed selective binding to PS-containing liposomes as demonstrated by anion exchange chromatography. This binding does not result in vesicle fusion or aggregation. Flow cytometry showed that DPA-Cy3[22, 22]/POPC liposomes have preferential binding to MCF-7 breast cancer cells over MCF-12A non-cancer cells due to 3-7 times more PS exposures on MCF-7. The extent of liposome binding with MCF-7 cells was increased by two times after cells were pre-treated with the apoptotic inducer camptothecin, which increased PS exposure to the cell surface. Moreover, our flow cytometry data also suggest that local cell membrane perturbations may occur upon liposome binding and internalization. This implies that DPACy3[22,22]/POPC liposomes alone may have a PS-dependent cytotoxic effect. This assertion was supported by the cell proliferation assay, which showed that 9.1 mol% DPACy3[22,22]/POPC liposomes exert cytotoxicity on MCF-7 cells 3.5 times higher than that on MCF-12A cells. These results indicate that DPA-Cy3[22,22]-containing liposomes hold great promise as efficient nano drug carriers.

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INTRODUCTION For years, liposomes have been utilized to passively carry small drug molecules to neoplastic tissues. To reduce toxicity of passive drug delivery and enhance specificity to diseased tissues, researchers have sought to develop active delivery systems with the ability to selectively target diseased cells over healthy cells. Targeted liposomes have a modified surface to achieve specificity for diseased cells; consequently, they have higher drug efficacy and lower toxicity.1-3 However, currently there are no FDA-approved active targeted liposomal drugs available in the market and only few that are in Phase I & II clinical trials.4,5 The challenges in developing sitespecific drug carriers include introducing greater stability and penetration and further reducing off-target side effects.6-8 Therefore, identifying the right target molecule to incorporate into liposomes and determining the most effective formulation for targeted delivery are still priorities when developing targeted liposomal carriers. Strategies have been developed to selectively target diseased cells using small molecules, peptides, and proteins incorporated on the surface of liposomal carriers.9-12 Cancer cells have different membrane characteristics than normal cells. Phosphatidylserine (PS), a negatively charged phospholipid at neutral pH, normally resides in the inner leaflet of the bilayer. However, during various diseased states such as cancers, PS translocates to the outside of the bilayer due to cellular and environmental stressors such as hypoxia, low pH, and reactive oxygen species present in the tumor micro-environment.13 The membrane organization of cancer cells, including MCF-7 human derived breast cancer cell line, changes drastically upon the activation of the membrane ABC transporter which normally maintains asymmetric lipid distribution across the cell membrane (14). Activation of the ABC transporter results in lipid flip-flop, increasing PS exposure on the outer leaflet of cell membranes.15,16 Previous studies showed significantly high



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levels of PS on the outsides of tumors, cancer cells, infected cells, injured tissues, and apoptotic cells detected using annexin V or anti-PS antibody.13,17-21 Dipicolylamine forms stable complexes with Zn2+ that strongly interact with negatively charged PS and can be attached to a fluorescent dye for the purpose of detecting PS exposure.2224

Water soluble fluorescent zinc(II)-dipicolylamine has been used to differentiate dead and live

cells25 to detect anionic lipids on surfaces of cells with bacterial26 and viral infections18 and to image tumors in mice.23,24 Zinc(II)-dipicolylamine has also been covalently linked to pegylated lipids serving as a membrane component in liposomes that displayed high selectivity towards anionic surfaces.27 It is well known that pegylated lipids can prolong the liposome circulation time when injected intravenously.28 However, recent studies showed that the initial subcutaneous injection of pegylated lipids may elicit a PEG-specific antibody that causes accelerated blood clearance,29 and that pegylated lipid degradation may cause an increase in membrane permeability.30 It is then of interest to investigate if a lipid-soluble zinc(II)-dipicolylamine derivative without PEG can still bind to anionic membrane surfaces. In our present study, we used a different lipid-soluble zinc(II)-bis-dipicolylamine derivative (abbreviated as DPA-Cy3[22,22], Figure 1) as a membrane component to develop PS targeted liposomal carriers. DPA-Cy3[22,22] does not link to PEG, yet, it contains the fluorophore cyanine 3 (Cy3), which can serve as an optical reporter, and two 22-carbon chains that can be anchored into liposomal membrane bilayers (Figure 1). Using ion exchange chromatography, dynamic light scattering, flow cytometry, fluorescence confocal microscopy, and cell proliferation assays, we studied the interactions of DPA-Cy3[22,22]-containing liposomes with PS-rich membrane surfaces. We found that liposomes made of DPA-Cy3[22,22] and 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were stable, exhibited selective


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binding to anionic liposomal membranes, and had preferential binding to MCF-7 breast cancer cells over MCF-12A non-cancer cells due to greater PS exposure on MCF-7. The extent of liposome binding with MCF-7 cells was increased after cells were pre-treated with the apoptotic inducer camptothecin, which increased PS exposure to the cell surface. Our data also suggest that there are local cell membrane perturbations and PS-dependent cytotoxicity upon liposome binding and internalization. These results together indicate that DPA-Cy3[22,22]-containing liposomes hold great promise as efficient nano drug carriers.

MATERIALS AND METHODS Lipids and fluorescent probes.

POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-

L-serine (POPS) were purchased from Avanti Polar Lipids (Alabaster, AL). Zn(II)-bisdipicolylamine cyanine 3 [C22,22] (DPA-Cy3[22,22]) as well as Compounds 1, 2, and 3 (Comp 1, 2, and 3, respectively) were obtained from Molecular Targeting Technologies (West Chester, PA). Like DPA-Cy3[22,22], Comp 1, Comp 2, and Comp 3 retain Cy3 and two 22C hydrocarbon chains; however, unlike DPA-Cy3[22,22], they lack the DPA moiety (Figure 1). Phosphatidylserine binding protein annexin V tagged with fluorescein isothiocyanate (FITC), cell nucleus dye Hoechet 33342, and cytosolic dye calcein were all obtained from Life Technologies (Carlsbad, CA). For cell death staining, the fluorescent dye 7-aminoactinomycin D (7AAD) was purchased from BD Biosciences (San Jose, CA). MCF-7 and MCF-12A cell lines. The cell lines used for this study were grown at 37°C and 5% CO2. MCF-7 (ATCC, Manassas, VA) cells were cultured in Hyclone Dulbecco’s modified Eagle’s medium (DMEM) which contained high calcium, 4.5 g/L glucose, L-glutamine and sodium pyruvate, 10% fetal bovine serum, and 1% penicillin streptomycin (Fischer,



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Waltham, MA). MCF-12A (obtained from Fox Chase Cancer Center, FCCC) cell culture was supplemented with a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium, 20 ng/ml human epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml bovine insulin, 500 ng/ml hydrocortisone, and 5% (v/v) horse serum. All the supplements were obtained from FCCC. Cells were sub-cultured every 3 days or when ~80% confluency was reached. Cells were detached from culture dish with 0.25% trypsin-EDTA and counted using a hemocytometer. Liposome preparation.

POPS and POPC lipid stock solutions were prepared in

chloroform. Stock solutions of DPA-Cy3[22,22], Comp 1, Comp 2 and Comp 3 were made in ethanol. The POPS and POPC concentrations were determined as previously described,31 and the amount of DPA-Cy3[22,22], Comp 1, Comp 2 and Comp 3 was determined spectroscopically by measuring the absorbance at 481 nm in ethanol using the extinction coefficient 72,100 M-1cm-1. To prepare liposomes at the desired molar ratio, appropriate amounts of POPC, POPS, Comp 13, or DPA-Cy3[22,22] were pipetted into a Pyrex screw capped test tube. Lipids were dried first with nitrogen gas and subsequently under high vacuum overnight. Dried lipids were hydrated with 10 mM TES buffer (pH 7.4) containing 145 mM NaCl. The buffer was pre-warmed to ~50˚C, and the hydrated lipid dispersion was vortexed at 50˚C for ~3 min to make either POPS/POPC, POPC, Comp 1/POPC, Comp 2/POPC, Comp 3/POPC, or DPA-Cy3[22,22]/POPC multilamellar vesicles (MLVs). The MLVs were subject to 3 cycles of heating at 45˚C for 30 min and cooling at 4˚C for 30 min. Annealed MLVs were incubated for at least 2 days at 24˚C in the dark under argon. MLVs were then extruded at 50˚C through two polycarbonate membranes with 200-nm pore size at elevated nitrogen pressure (~200 psi) to create unilamellar vesicles (LUVs). LUVs were flushed with argon and stored in a desiccating chamber for > 2 days at 24˚C before use.


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Liposome size measurement. Liposome size was determined in 10 mM TES buffer (pH 7.4) containing 145 mM NaCl at 25oC by the dynamic light scattering technique on a Malvern Zetasizer Nano ZS (Worcestershire, UK). The Zave value (hydrodynamic diameter) was calculated using the Stokes-Einstein equation. Anion-exchange chromatography.

DEAE-Sephadex A25 anion exchange resin (GE

Healthcare Bio-Sciences AB, Uppsala, Sweden) was hydrated in the equilibrating buffer (50 mM TES, pH 7.4, containing 150 mM NaCl) and packed in a column (18 cm x 1 cm). In the first reaction, LUVs comprised of 4.8 mol% DPA-Cy3[22,22] and 95.2 mol% POPC (30 nmol) were reacted with LUVs (90 nmol of total lipid) made of POPS (50 mol%) and POPC for 3 h at room temperature. The second reaction involved DPA-Cy3[22,22]/POPC LUVs and POPC LUVs (90 nmol of POPC) with the same reaction condition as the first reaction. In each reaction, the reaction mixtures were loaded onto the column and eluted first with the equilibrating buffer and then the bound material was eluted out of the column by using the eluting buffer (50 mM TES, pH 4, containing 1M NaCl). The elution profiles were determined using Cy3 fluorescence and light scattering. Cy3 fluorescence indicates the presence of DPA-Cy3[22,22]-containing liposomes whereas the light scattering (wavelength of the incident light = 500 nm, scattering detected at the right angle through a monochromator set at 505 nm) monitors the presence of liposomes. CyQuant assays.

Effects of DPA-Cy3[22,22]/POPC liposomes on cell viability were

tested using various lipid concentrations and DPA-to-lipid molar ratios. After the MCF-7 and MCF-12A cells were plated in a 96-well plate, cells were left to adhere overnight, then, treated with DPA-Cy3[22,22]/POPC liposomes for 4, 8, 24, and 48 h. After treatment, the dead cells (floating in the growth medium) were removed. The cell proliferation assay was performed to



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determine the number of cells remaining alive (i.e. those still attached to the wells). The assay was performed at room temperature using the CyQuant Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Fluorescence intensities were measured at 530 nm with excitation at 485 nm. A standard curve was constructed for each sample set using known amounts of cells counted with a hemocytometer. Flow Cytometry.

For the purposes of quantifying the level of PS exposure in MCF-7 and

MCF-12A cells and the extent of DPA-Cy3[22,22]/POPC liposome-cell binding, we utilized flow cytometry. MCF-12A or MCF-7 cells plated in 6-well plates (~600,000 cells per well) were trypsinized for ~1 min and then counted using a hemocytometer. Cell viability was determined using trypan blue. Aliquots of 100,000 cells were washed with PBS and re-suspended in binding buffer provided in the Cell Death Assay Kit (Life Technologies, Carlsbad, CA). Staining was carried out according to the manufacturer’s instructions. Cells were treated with either different concentrations of DPA-Cy3[22,22]/POPC or 20 μM Comp 1/POPC (which is Cy3[22,22]/POPC with no DPA moiety, see Figure 1 for structures) liposomes for 15 min. Afterwards, cells were stained with annexin V-FITC for 15 min and 7AAD for 5 min before taking fluorescence measurements from cells. To increase cell PS exposure, an apoptotic inducer camptothecin (Life Technologies, Carlsbad, CA) was used. Cells were treated with 10 µM camptothecin in 6-well plates for 0, 2, and 4 h and then trypsinized and incubated with DPA-Cy3[22,22]/POPC liposomes, annexin V-FITC, and 7AAD. FITC, 7AAD, and Cy3 fluorescence intensity measurements were taken using the flow cytometer (FACS Canto cell analyzer, BD, Franklin Lakes, NJ). All measurements were completed within one hour of staining. All data were analyzed using the software FlowJo version 10 (Ashland, OR) and Flowing Software 2.0 (Turku, Finland).


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Confocal Microscopy.

Cellular binding and uptake of DPA-Cy3[22,22]/POPC liposomes

were detected using confocal microscopy. Cells (600,000 cells/well) were plated in a 6-well plate with coverslips and left to adhere overnight in cell media (DMEM high calcium with 10% FBS and 1% penstrap mixture). Cells treated with DPA-Cy3[22,22]/POPC liposomes were visualized using Cy3 fluorescence which was excited with a HeNe laser at 543 nm and detected through an emission filter LP615. Cells were also visualized with the fluorescence of the cytoplasmic dye calcein, which was excited using an argon laser at 488 nm and observed through a BP505-550 filter. The nuclear dye Hoechst 33342 was excited with a diode laser at 407 nm and its fluorescence was observed at ~440 nm using a LP420 filter. MCF-7 cells treated with camptothecin for 0-6 h were washed with 1x PBS, 1x annexin V binding buffer (provided in Live/Dead Imaging Kit, Life Technologies), and then incubated with annexin V-FITC and DPACy3[22,22]/POPC liposomes for 15 min followed by washing with 1x PBS and 1x binding buffer before cell imaging. Fluorescence signals of FITC were observed through a BP505-550 filter using 488 nm excitation from an argon laser. Cells were imaged using a Zeiss LSM 510 Meta confocal microscope (Jena, Germany) with a 40× oil objective with 1.5× digital zoom. Image data were analyzed using the Zen software (Zeiss).

Results and Discussion Vesicle stability.

Stability of DPA-Cy3[22,22]/POPC liposomes was evaluated based on

particle size and polydispersity measured by dynamic light scattering. At 3.2 and 4.8 mol% DPA-Cy3[22,22], liposome size stayed at ~150 nm (Figure 2A) and polydispersity remained at ~0.2 (Figure 2B), throughout the time period examined (7 days). In contrast, at 9.1 mol% DPACy3[22,22], the particle size increased steadily from ~160 nm to ~320 nm and polydispersity 9 ACS Paragon Plus Environment


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increased from ~0.2 to ~0.8 in 9 days, which indicates that, at this mole fraction, vesicle aggregation/fusion may occur and as a result liposomes become increasingly heterogeneous over time. Based on these data, most of the remaining experiments were performed on ≤ 4.8 mol% DPA-Cy3[22,22]/POPC.

Binding of DPA-Cy3[22,22]/POPC liposomes to PS-containing liposomes.

We used the

DEAE-Sephadex A25 anion exchange chromatography to test if DPA-Cy3[22,22]/POPC liposomes bind to PS-containing liposomes. In this experiment, two buffer systems (the equilibrating buffer and the eluting buffer) were used (see Materials and Methods). As shown in Figure 3 (I), both 4.8 mol% DPA-Cy3[22,22]/POPC liposomes and 100 mol% POPC liposomes flowed through the column when using the equilibrating buffer, whereas POPS/POPC liposomes were retained inside the column until the eluting buffer (a low pH and high salt buffer system) was employed to release the bound molecules. This result (Figure 3 (I)) agrees with the fact that, among these three liposomes tested, only POPS/POPC liposomes carry significant negative charges on membrane surface at neutral pH. Figure 3 (II) shows that after the mixture of DPA-Cy3[22,22]/POPC and POPS/POPC liposomes was loaded onto the column, DPA-Cy3[22,22]/POPC liposomes were not observed in the flow through when using the equilibrating buffer. However, the subsequent use (pointed by the arrow) of the eluting buffer resulted in two peaks detected by light scattering and one peak by Cy3 fluorescence (Figure 3 (II)). The Cy3 fluorescence peak coincides with the first light scattering peak, suggesting that DPA-Cy3[22,22]/POPC liposomes bind to POPS/POPC liposomes resulting in a DPA-Cy3[22,22]/POPC-POPS/POPC conjugate. This conjugate was retained in the column via electrostatic attraction between the negatively charged PS on the 10 ACS Paragon Plus Environment

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conjugate surface and the positively charged diethylaminoethyl on the DEAE-Sephadex A-25 resin, until the eluting buffer was employed. Upon mixing POPC/DPA-Cy3[22,22] liposomes with POPC/POPS liposomes, the particle size did not increase as monitored by dynamic light scattering (Supporting Information, Figure S1), which indicates that vesicle fusion or aggregation did not occur. Instead, the particle size decreased by several nanometers over a time period of 40 min; thereafter, the size remained almost unchanged over 20 h (Supporting Information, Figure S1). The slight decrease in particle size may arise from membrane deformation such as domain sinking.32 As a control, we repeated the anion exchange chromatography experiment by mixing POPC liposomes with DPA-Cy3[22,22]/POPC liposomes (Figure 3 (III)). We found that, in the absence of PS-containing membrane surfaces, DPA-Cy3[22,22]/POPC liposomes were not retained by the DEAE-Sephadex A-25 resin when using only the equilibrating buffer to elute the column (Figure 3 (III)). This finding supports our conclusion that DPA-Cy3[22,22]/POPC liposomes are able to bind to PS-containing membrane surfaces.

Binding of DPA-Cy3[22,22]/POPC liposomes to PS exposed cells.

Previous studies

established that the outer membrane surface of MCF-7 breast cancer cells has a higher percentage of PS exposure than non-cancerous cells and that MCF-7 anti-cancer drug resistant cells have an even higher level of PS exposure.14,15 Before testing the binding of DPACy3[22,22]/POPC liposomes to MCF-7 cells, we checked the level of PS exposure in MCF-7 cancer cells used in our laboratory and compared it with non-cancer breast cells (MCF-12A) using the cell live/dead staining and flow cytometry. MCF-7 and MCF-12A cell lines were analyzed by staining with annexin V-FITC and 7AAD to observe PS exposure and cell death,



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respectively. We found that, compared to non-cancer MCF-12A cells (illustrated in Figure 4A, Q1), MCF-7 cancer cells (illustrated in Figure 4B, Q1) had a higher percentage (8.75 vs. 1.35 percent in this illustration) of annexin V+ 7AAD- cells, which are live cells with PS exposure. Overall, MCF-7 cancer cells bear 3-7 times higher PS exposure than the non-cancerous cell line MCF-12A (Figure 4C). Consistent with our experimental findings, PS exposure in MCF-7 cells has been previously reported to be 6-10%.15 The difference in PS exposure between cancer and non-cancer cells is potentially a way to target PS on cancer cells for drug delivery. Since the PS exposure in MCF-7 cells is 3-7 times higher than that in MCF-12A cells, we can use these two cell lines to determine if DPA-Cy3[22,22]/POPC liposomes have any preference of binding to the cells with higher PS exposure. In this experiment, MCF-7 cells were incubated with DPA-Cy3[22,22]/POPC liposomes at room temperature for one hour, followed by staining with annexin V-FITC and 7AAD. The binding of DPA-Cy3[22,22]/POPC liposomes on the cell surface was detected using flow cytometry by measuring the Cy3 fluorescence from the treated cells. The fluorescence intensities of annexin V-FITC and 7AAD were also recorded. The Cy3 fluorescence emitted by 7AAD negative cells reflects the binding or uptake of DPACy3[22,22]/POPC liposomes by live cells. Figure 5A presents the histograms of the relative numbers of cells (cell count) with Cy3 fluorescence (x-axis) after treatment with DPACy3[22,22]/POPC or Comp1/POPC liposomes. Comp 1 has the same chromophore (Cy3) and long hydrocarbon chains (C22,C22) as DPA-Cy3[22,22]; the main difference is that Comp 1 does not have the DPA moiety (Figure 1). In Figure 5A, the Cy3+ gate (blue) was defined as the area containing cells with Cy3 fluorescence (red part of the peak). The percentage of Cy3+ cells was determined based on the number of cells in the Cy3+ gate. The untreated cells showed only auto-fluorescence (peak closer to 0) and were Cy3 negative.


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Compared to untreated cells and cells treated with Comp1/POPC liposomes, MCF-7 cells treated with DPA-Cy3[22,22]/POPC liposomes displayed a higher number of cells carrying Cy3 fluorescence indicated by the histogram peak shifting to the right into the gate marked Cy3 (Figure 5A), as a result of liposome binding to cells. Cells treated with Comp1/POPC liposomes, due to the lack of the DPA moiety, exhibited only a small shift in the red peak or displayed fewer Cy3+ cells (Figure 5A). This suggests that DPA is essential for the preferential binding of DPACy3[22,22]/POPC liposomes to MCF-7 cells. The effects of liposome concentration and DPA-Cy3[22,22] mole fraction on the percentage of Cy3+ MCF-7 cells are presented in Figure 5B. Within the mole fraction range examined (0-4.8 mol%), MCF-7 cells treated with liposomes with a higher mole percent of DPACy3[22,22] experienced a larger shift in red peak (Figure 5A) and yielded a higher percent of Cy3 positive cell populations (Figure 5B). We did not determine the percent of the Cy3+ cells at DPA-Cy3[22,22] mole fractions higher than 4.8 mol%, because beyond this mole fraction liposome stability was compromised (Figure 2). Figure 5 also shows that increasing the total liposome concentration increases the percent of Cy3 positive cells. These results further support DPA-Cy3[22,22]/POPC liposomes’ selective affinity toward PS-exposed cells. According to Figure 5B, 1.0 mol% DPA-Cy3[22,22]/POPC liposomes resulted in very little binding to MCF-7 cells even at high liposome concentrations whereas incubation of MCF-7 cells with 4.8 mol% DPA-Cy3[22,22]/POPC liposomes at a concentration of 20 µM POPC for 1 h led to ~90% of MCF-7 cells to be Cy3 positive. These results, along with the stability data, suggest that among all the formulations tested in this study, 4.8 mol% DPA-Cy3[22,22]/POPC is more desirable for effective targeted delivery.



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Binding of DPA-Cy3[22,22]/POPC liposomes to MCF-7 cells pretreated with the apoptosis inducer camptothecin.

In order to further assess whether the binding of DPA-

Cy3[22,22]/POPC liposomes to cells is PS content dependent, we used camptothecin to induce pre-apoptotic PS exposure in MCF-7 cells. Camptothecin can bind DNA and inhibit the enzyme topoisomerase resulting in DNA damage and apoptosis, which leads to higher PS exposure and more cell death.33 Thus, it is expected that camptothecin-treated cells can bind more annexin VFITC and exhibit higher FITC fluorescence intensity. Indeed, our flow cytometry data showed that MCF-7 cells treated with 10 µM camptothecin for 2 h had a higher percentage of annexin V + 7AAD- cells (~13%, see Q1 in Figure 6B) in comparison to untreated cells (~7%, Q1 in Figure 6A). The membrane permeability change caused by camptothecin, if there is any, will not affect our conclusion derived from Figure 6. The data in Q1 of Figure 6B reveal not just annexin V positive cells, but annexin V positive/7AAD negative (i.e., annexin V + 7AAD-) cells. 7AAD is not membrane permeable in normal conditions. If camptothecin causes changes in membrane permeability, then those cells will become 7AAD positive and will not count toward Q1 in the dot plots (Figure 6). When DPA-Cy3[22,22]/POPC liposomes were mixed with MCF-7 cells pre-treated with camptothecin, the percent of Cy3 positive cells increased significantly (~ a factor of two) in the liposome concentration range (0-20 µM POPC) examined (Figure 6B). This result can be attributed to the increased PS exposure in the cells induced by camptothecin and demonstrates that the specific binding of DPA-Cy3[22,22]/POPC liposomes to cells is mediated by the extent of PS-exposure on the host cell membrane surfaces.


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Binding and uptake of DPA-Cy3[22,22]/POPC liposome by MCF-7 cells as visualized by confocal microscopy.

We used fluorescence confocal microscopy to visualize the binding and

uptake of DPA-Cy3[22,22]/POPC liposomes by MCF-7 cells. Figure 7 shows the microscopy images of MCF-7 cells after being treated with DPA-Cy3[22,22]/POPC, Comp 1/POPC, Comp 2/POPC, and Comp 3/POPC liposomes for 4 h. The mole fraction of DPA-Cy3[22,22], Comp 1, Comp 2, and Comp 3 in the liposomes was 4.8 mol%. Cells were stained with calcein (located in the cytoplasm) and Hoechst 33342 (in the nucleus). We found that cells treated with Comp 1/POPC, Comp 2/POPC, and Comp 3/POPC have little or no Cy3 fluorescence in the intracellular environment. In comparison, the Cy3 fluorescence is clearly observable inside the cells treated with DPA-Cy3[22,22]/POPC liposomes. After DPA-Cy3[22,22]/POPC liposomes were internalized by cells, most of the Cy3 fluorescence came from the nuclei. Our confocal data (Figure 7 and Supporting Information, Figure S2) show that liposomes comprised of the DPA moiety are taken up by MCF-7 cancer cells more readily than MCF-12A non-cancer cells. This echoes our flow cytometry data in that DPA-Cy3[22,22]/POPC liposomes have selective affinity for PS-exposed MCF-7 cells. Furthermore, the confocal images suggest that the presence of DPA plays an important role in the internalization of liposomes by MCF-7 cells. Note that although liposomes without target molecules can be internalized by cells, the presence of target molecule on liposomes can greatly enhance cellular uptake.34 To this end, our data suggest that DPA-Cy3[22,22]/POPC liposomes can be used to selectively target diseased tissues because these liposomes have specific affinity to PS-exposed cells and can be subsequently internalized into the intracellular environment. The trace amount of Cy3 fluorescence seen in MCF-7 cells incubated with POPC/Comp 13 (Figure 7) may come from non-specific binding between the positively charged POPC/Comp



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1-3 liposomes and the negatively charged PS-rich cell surface or the spontaneous transfer of the Cy3 derivatives from lipid vesicles to cell plasma membranes.35 The former is more plausible because much less Cy3 fluorescence was observed in MCF-12A (less PS exposure) cells compared to MCF-7 cells (more PS exposure) when the cells were incubated with POPC/Comp 1-3 liposomes (Figure S2 and Figure 7).

Comparing binding of DPA-Cy3[22,22]/POPC liposomes to cancer cells (MCF-7) versus noncancer cells (MCF-12A).

Figure 8A shows that, when treating cells with 2.0 mol% DPA-

Cy3[22,22]/POPC liposomes, the percentage of Cy3+ cells is higher in MCF-7 cells than in MCF-12A cells by ~30-35% at all liposome concentrations examined. However, when 4.8 mol% DPA-Cy3[22,22]/POPC liposomes were used, the difference in the percentage of Cy3+ cells between MCF-7 and MCF-12A cells was not constant with liposome concentration (Figure 8B). The difference is significantly high (~45%) at 7 and 10 µM POPC and considerably low (~20%) at higher liposome concentrations such as 15 and 20 µM POPC (Figure 8B). The observation that MCF-12A non-cancer cells treated with 15-20 µM of 4.8 mol% DPA-Cy3[22,22]/POPC liposomes exhibited nearly 60-70% Cy3+ cells is surprising. One plausible explanation of this phenomenon is that at high mole fractions (such as 4.8 mol% DPA-Cy3[22,22]) and high liposome concentrations (such as 15-20 µM), significant local membrane perturbations may follow the initial liposome binding to the outer leaflet of the cell membrane, leading to membrane leakage to external Cy3-containing liposomes. As a result, the Cy3-containing liposomes enter the cytosol and bind PS originally residing in the inner leaflet of the plasma membrane as well. If this occurs, DPA-Cy3[22,22]/POPC liposomes alone (without entrapped drugs) may have some cell killing effect associated with the initial binding to the PS exposed


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area. This would be an additional beneficial effect of using DPA-Cy3[22,22]/POPC liposomes as drug carriers to treat cancers.

Cytotoxic effect of DPA-Cy3[22,22]/POPC liposomes on cancer cells (MCF-7) versus noncancer cells (MCF-12A).

We used the Invitrogen CyQuant viability assay to test the above-

mentioned idea. The results are presented in Figure 9. Incubation of 2.0 or 4.8 mol% DPACy3[22,22]/POPC liposomes with either MCF-7 or MCF-12A cells for 48 h generated a low percentage of cell death (~ 10%) (Figure 9). Considering that the experimental errors of the CyQuant assay are about 4-8%,36 it is hard to determine whether, under this experimental condition, the toxicity is the same or different between MCF-7 and MCF-12A cells. In order to exaggerate the differential cytotoxicity effect of the DPA-Cy3[22,22]/POPC liposomes on cancer versus non-cancer cells, we used 9.1 mol% DPA-Cy3[22,22]/POPC liposomes and demonstrated that 87% of MCF-12A cells and 54% of MCF-7 cells remaining alive after 48 h of incubation with liposomes (Figure 9). This difference is significantly larger than the experimental errors; thus, the result can be taken to indicate the existence of a real differential cytotoxicity effect of DPA-Cy3[22,22]/POPC liposomes on cancer (PS-rich) vs. non-cancer (PS-poor) cells. It appears that a higher DPA density on liposomes (in the case of 9.1 mol% DPA-Cy3[22,22]) and a higher PS exposure on MCF-7 cells (Figure 4) cause a more extensive binding between the liposomes and cell membranes, which results in more cell membrane damage, increased liposome internalization, and eventually more severe cell toxicity. Although we do not plan to employ 9.1 mol% DPA-Cy3[22,22]/POPC liposomes for future liposomal drug studies due to their poor long-term stability (Figure 3), the rather high cytotoxicity against MCF-7 relative to MCF-12A cells (Figure 9) illustrates that there is a killing effect on cancer cells following the initial



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preferential binding between DPA-Cy3[22,22]/POPC liposomes and PS-rich cell membrane surfaces. Note that saposin C coupled liposomes without entrapped drugs also exhibited a significant killing effect on cancer cells.37,38

Outlook-Concluding Remarks In conclusion, we demonstrated that 2.0 mol% and 4.8 mol% DPA-Cy3[22,22]/POPC liposomes are stable and show low cytotoxicity (Figures 2 and 9). They can bind to both PScontaining liposomes and PS-exposed cells (Figures 3 and 5). The specific binding of DPACy3[22,22]/POPC liposomes to cells is established by the DPA moiety and depends upon the extent of PS exposure on the membrane surface of host cells. MCF-7 cancer cells have more PS exposure than MCF-12A non-cancer cells; therefore, DPA-Cy3[22,22]/POPC liposomes show preferential binding to MCF-7 cells rather than MCF-12A cells (Figure 7). DPACy3[22,22]/POPC liposome binding can cause toxicity to treated cells also in a PS-exposure dependent manner, with 3.5 times more cell death in MCF-7 cells compared to MCF-12A cells (Figure 9). Under pathophysiological conditions, such as cancers, tumor tissues possess significantly higher PS-exposure than normal tissues.20,21 Therefore, based on our current results, we predict that more DPA-Cy3[22,22]-containing liposomes will be accumulated in the tumors than normal tissues. However, in human bodies, there are non-cancer cells having PS-rich surfaces. The most noticeable is the activated platelets and the platelet-derived microvesicles (PMPs). This may not be an obstacle in using DPA-Cy3[22,22]-containing liposomes because platelets and PMPs promote cancer metastasis and because platelet depletion (thus PMP depletion) increases the efficacy of chemotherapy and reduces tumor growth.39,40 It has been suggested that combing


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anti-platelet treatment with therapeutic drugs may help develop a more effective way to fight cancer.41 In this regard, it is possible that our DPA-Cy3[22,22]-containing liposomes actually have some additional beneficial effect by binding to activated or apoptotic platelets and PMPs. This may outweigh their potential adverse effects due to the PS-exposure in certain normal tissues. Using nano particles capable of targeting and killing cancer cells without entrapped anticancer drugs is an attractive therapeutic approach because it avoids the side effects caused by remaining drug molecules or drug metabolites.42 DPA-Cy3[22,22]/POPC liposomes seem to fall into this category. On the other hand, DPA-Cy3[22,22]/POPC liposomes can be developed to include entrapped anticancer drugs, which altogether may exhibit a synergistic effect on cancer treatment. Of course, all these assertions will need to be tested in vivo in the future.

Acknowledgment This work was supported in part by NSF grant DMR1105277. The authors would like to thank Dr. Jose Russo for providing MCF-12A cells and cell culture medium, Drs. Madesh Muniswamy and Stefania Gallucci for the use of their instruments, and Mary Mintzer, Nicholas Hoffman, Jun Xu, and Marita Chakhtoura for technical support.



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17. Riedl, S.; Rinner, B.; Asslaber, M.; Schaider, H.; Walzer, S.; Novak, A.; Lohner, K.; Zweytick, D. In search of a novel target - phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy. Biochim. Biophys. Acta 2011, 1808, 2638-2645. 18. Ran, S. and Thorpe, P.E. Phosphatidylserine is a marker of tumor vasculature and a potential target for cancer imaging and therapy. Int. J. Radiat. Oncol. Biol. Phys. 2002, 54, 1479-1484. 19. Zwaal, R.F.A.; Comfurius, P.; Bevers, E.M. Surface exposure of phosphatidylserine in pathological cells. Cell. Mol. Life Sci 2005, 62, 971-988. 20. Stafford, J.H. and Thorpe, P.E. Increased exposure of phosphatidylethanolamine on the surface of tumor vascular endothelium. Neoplasia 2011, 13, 299-308. 21. Utsugi, T.; Schroit, A.J.; Connor, J.; Bucana, C.D.; Fidler, I.J. Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res. 1991, 51, 3062-3066. 22. Koulov, A.V.; Stucker, K.A.; Lakshmi, C.; Robinson, J.P.; Smith, B.D. Detection of apoptotic cells using a synthetic fluorescent sensor for membrane surfaces that contain phosphatidylserine. Cell Death Differ. 2003, 10, 1357-1359. 23. Smith, B.A.; Akers, W.J.; Leevy, W.M.; Lampkins, A.J.; Xiao, S.; Wolter, W.; Suckow, M.A.; Achilefu, S.; Smith, B.D. Optical imaging of mammary and prostate tumors in living animals using a synthetic near infrared zinc(II)-dipicolylamine probe for anionic cell surfaces. J. Am. Chem. Soc. 2010, 132, 67-69.


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24. O'Neil, J.E. and Smith, B.D., Anion recognition using dimetallic coordination complexes. Coordination Chemistry 2006, 250, 3068. 25. Lakshmi, C.; Hanshaw, R.G.; Smith, B.D. Fluorophore Linked Zinc (II) Dipicolylamine Coordination Complexes as Sensors for Phosphatidylserine Containing Membranes. Tetrahedron 2004, 60, 11307-11315. 26. Leevy, W.M.; Gammon, S.T.; Jiang, H.; Johnson, J.R.; Maxwell, D.J.; Jackson, E.N.; Marquez, M.; Piwnica-Worms, D.; Smith, B.D. Optical imaging of bacterial infection in living mice using a fluorescent near-infrared molecular probe. J. Am. Chem. Soc. 2006, 128, 1647616477. 27. Turkyilmaz, S.; Rice, D.R.; Palumboa, R.; Smith, B.D. Selective recognition of anionic cell membranes using targeted liposomes coated with zinc(II)-bis(dipicolylamine) affinity units. Org. Biomol. Chem. 2014, 12, 5645-5655. 28. Allen, T.M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo. Biochim. Biophys. Acta, 1991, 1066, 29-36. 29. Zhao, Y.; Wang, C.; Wang, L.; Yang, Q.; Tang, W.; She, Z.; Deng, Y. A frustrating problem: accelerated blood clearance of PEGylated solid lipid nanoparticles following subcutaneous injection in rats. European J. Pharm. Biopharm. 2012, 81, 506-513. 30. Nakamura, K.; Yamashita, K.; Itoh, Y.; Yoshino, K.; Nozawa, S.; Kasukawa, H. Comparative studies of polyethylene glycol-modified liposomes prepared using different PEGmodification methods. Biochim. Biophys. Acta. 2012, 1818, 2801-2807.



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31. Tejera-Garcia, R.; Connell, L.; Shaw, W.A.; Kinnunen, P.K. Gravimetric determination of phospholipid concentration. Chem. Phys. Lipids 2012, 165, 689-695. 32. Hamada, T.; Miura, Y.; Ishii, K.; Araki, S.; Yoshikawa, K.; Vestergaad, M.; Takagi, M. Dynamic processes in endocytic transformation of raft-exhibiting giant liposomes. J. Phys. Chem. B. 2007, 111, 10853-10857. 33. Liu, L.F.; Desai, S.D.; Li, T.K.; Mao, Y.; Sun, M.; Sim, S.P. Mechanism of action of camptothecin. Ann. N. Y. Acad. Sci. 2000, 922, 1-10. 34. Mossalam, M.; Dixon, A.S.; Lim, C.S. Controlling subcellular delivery to optimize therapeutic effect. Ther. Deliv. 2010, 1, 169-193. 35. Schutz, G.J.; Kada, G.; Pastushenko, V.P.; Schindler, H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. The EMBO journal 2000, 19, 892-901. 36. Venegas, B.; Zhu, W.; Haloupek, N.B.; Lee, J.; Zellhart, E.; Sugar, I.P.; Kiani, M.; Chong, P.L.-G. Cholesterol supelattice modulates combretastatin A4 disodium phosphate (CA4P) release from liposomes and CA4P cytotoxicity on mammary cancer cells Biophys. J. 2012, 102, 20862094. 37. Kaimal, V.; Chu, Z.; Mahller, Y.Y.; Papahadjopoulos-Sternberg, B.; Cripe, T.P.; Holland, S.K.; Qi, X. Saposin C coupled lipid nanovesicles enable cancer-selective optical and magnetic resonance imaging. Mol. Imaging Biol. 2011, 13, 886-897.


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38. Qi, X.; Chu, Z.; Mahller, Y.Y.; Stringer, K.F.; Witte, D.P.; Cripe, T.P. Cancer-selective targeting and cytotoxicity by liposomal-coupled lysosomal saposin C protein. Clin. Cancer Res. 2009, 15, 5840-5851. 39. Demers, M.; Ho-Tin-Noe, B.; Schatzberg, D.; Yang, J.J.; Wagner, D.D. Increased efficacy of breast cancer chemotherapy in thrombocytopenic mice. Cancer Research 2011, 71, 1540-1549. 40. Dymicka-Piekarska, V.; Gryko, M.; Lipska, A.; Korniluk, A.; Siergiejko, E.; Kemona, H. Platelet-derived microparticles in patients with colorectal cancer. Journal of Cancer Therapy 2012, 3, 898-901. 41. Demers, M. and Wagner, D.D. Targeting platelet function to improve drug delivery. OncoImmunology 2012, 1, 100-102. 42. Chung, M.-F.; Chen, K.-J.; Liang, H.-F.; Liao, Z.-X.; Chia, W.-T.; Xia, Y.; Sung, H.-W. A liposomal system capable of generating CO2 bubbles to induce transient cavitation, lysosomal rupturing, and cell necrosis. Angew. Chem. Int. Ed. 2012, 51, 10089-10093.



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Figure 1. Structure of DPA-Cy3[22,22] and its derivatives Compounds 1, 2 and 3.


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Figure 2. Time dependence of Zave (A) and polydispersity (B) of DPA-Cy3[22,22]/POPC liposomes at three different mole fractions (3.2, 4.8, and 9.1 mol% DPA-Cy3[22,22]). Standard deviations calculated from three measurements are smaller than the data symbols (see Supporting Information, Table S1).



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Figure 3. Anion exchange chromatography showing affinity of DPA-Cy3[22,22]/POPC liposomes to PS containing liposomes. (I) DPA-Cy3/POPC, POPC, and POPS/POPC liposomes eluted separately. (II and III) DPA-Cy3/POPC liposomes incubated with POPS/POPC liposomes or POPC liposomes, respectively, for 3 h at room temperature before loading onto the column. Samples were first eluted with 2 mL of the equilibrating buffer (see Materials and Methods). Arrow indicates the time when the elution buffer was used.


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Figure 4. Flow cytometry analysis of (A) MCF-12A and (B) MCF-7 cells stained with annexin V-FITC and 7AAD. Annexin V-FITC binds pre-apoptotic and late apoptotic cells with PS exposure, and 7AAD stains necrotic and dead cells. The number in each quadrant indicates the percentage of cells in that quadrant: Q1: annexin V + 7AAD – (pre-apoptotic); Q2: annexin V + 7AAD+ (dead); Q3: annexin V- 7AAD+ (necrotic); and Q4: annexin V- 7AAD- (alive). PS exposed live cells are located in Q1. (C) Percent of total annexin V + live cells (or PS exposed live cells or Q1) in MCF-12 and MCF-7 cell lines. Standard deviations are from 4 independent measurements, and the comparison of these data gives a **p value of < 0.05.



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Figure 5. (A) Histogram of Cy3 positive MCF-7 cells after being treated with Comp 1/POPC (4.8 mol%) or DPA-Cy3[22,22]/POPC (2.0 and 4.8 mol%) liposomes for 1 h. [POPC] = 20 µM. Untreated cells with auto-fluorescence were used as a control to set Cy3+ gates (blue). More Cy3 positive cells (red, in Cy3+ gate) were detected when cells were treated with DPACy3[22,22]/POPC liposomes. (B) Effect of liposome concentration (in terms of liposomal POPC concentration) on the percentage of Cy3 positive cells (determined from the number of cells in the Cy3+ gate) after cells were treated with liposomes. 30 ACS Paragon Plus Environment

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Figure 6. Dot plots of MCF-7 cells, in the absence (A) and presence (B) of 10 µM apoptotic inducer camptothecin, stained with annexin V-FITC (y-axis) and 7AAD (x-axis). (C) Effect of liposome concentration (in terms of POPC concentration) on the percentage of MCF-7 cells carrying Cy3 fluorescence after the cells were treated with camptothecin for 2 h and subsequently incubated with 2 mol% DPA-Cy3[22,22]/POPC liposomes for 1 h.



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Figure 7. Confocal microscopy images of MCF-7 cells after being treated with DPACy3[22,22]/POPC, Comp 1/POPC, Comp 2/POPC, and Comp 3/POPC liposomes for 4 h. Cells were stained with calcein (cytoplasm) and Hoechst 33342 (nucleus). “Untreated cells” were the cells not treated with liposomes. Scale bar = 20 µm.


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Figure 8. The effect of liposome concentrations on the percentage of Cy3 positive cells (from total live cells) detected from flow cytometry after MCF-7 (circles) and MCF-12A (triangles) cells were treated with (A) 2.0 mol% and (B) 4.8 mol% DPA-Cy3[22,22]/POPC liposomes for 15 min, followed by staining with annexin V–FITC and 7AAD.



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Figure 9. Cytotoxicity of DPA-Cy3[22,22]/POPC liposomes to MCF-7 (A) and MCF-12A (B) cells. Liposomes containing 2.0, 4.8, and 9.1 mol% DPA-Cy3[22,22] were used. Lipid concentration in each CyQuant assay well was 10 µM. Cells were treated with liposomes for 048 h.


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