Enhanced Cytotoxicity of Folic Acid-Targeted Liposomes Co-Loaded

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Enhanced Cytotoxicity of Folic Acid-Targeted Liposomes Co-Loaded with C6 Ceramide and Doxorubicin: In Vitro Evaluation on HeLa, A2780-ADR and H69-AR Cells. Shravan Kumar Sriraman, Jiayi Pan, Can Sarisozen, Ed Luther, and Vladimir Torchilin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00663 • Publication Date (Web): 24 Dec 2015 Downloaded from http://pubs.acs.org on December 28, 2015

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Molecular Pharmaceutics

Enhanced Cytotoxicity of Folic Acid-Targeted Liposomes Co-Loaded with C6 Ceramide and Doxorubicin: In Vitro Evaluation on HeLa, A2780ADR and H69-AR Cells Shravan Kumar Sriraman1, Jiayi Pan1, Can Sarisozen1, Ed Luther1 and Vladimir Torchilin1, 2, *

1 Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston MA 02115, USA. 2 Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.

KEYWORDS: Liposomes, combination drug therapy, nanoparticles, folic acid-targeting, doxorubicin, C6 ceramide, cancer, HeLa, A2780-ADR, H-69-AR, drug-resistance, receptor targeting, apoptosis, cell cycle arrest, nanomedicine, phase holographic imaging, imaging cytometry, cancer cell spheroids.

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ABSTRACT Current research in cancer therapy is beginning to shift towards the use of combinational drugtreatment regimens. However, the efficient delivery of drug combinations is governed by a number of complex factors in the clinical setting. Therefore, the ability to synchronize the pharmacokinetics of the individual therapeutic agents present in combination not only to allow for simultaneous tumor accumulation, but also for a synergistic relationship at the intracellular level, could prove advantageous. In the present work, we report the development of a novel folic acid-targeted liposomal formulation simultaneously co-loaded with C6 ceramide and doxorubicin (FA-(C6+Dox)-LP). In vitro cytotoxicity assays showed that the FA-(C6+Dox)-LP was able to significantly reduce the IC50 of Dox when compared to the treatment with the doxorubicinloaded liposomes (Dox-LP) as well as the untargeted drug co-loaded (C6+Dox)-LP on HeLa, A2780-ADR and H69-AR cells. The analysis of the cell cycle distribution showed that while the C6 liposomes (C6-LP) did not cause cell cycle arrest, all the Dox-containing liposomes mediated cell cycle arrest in HeLa cells in the G2-phase at Dox concentrations of 0.3 and 1µM and in the S-phase at the higher concentrations. It was also found that this arrest in the S-phase precedes the progression of the cells to apoptosis. The targeted FA-(C6+Dox)-LP were able to significantly enhance the induction of apoptotic events in HeLa cell monolayers as compared to the other treatment groups. Next, using time-lapse phase holographic imaging microscopy, it was found that upon the treatment with the FA (C6+Dox) LP, the HeLa cells underwent rapid progression to apoptosis after 21 hours as evidenced by a drastic drop in the average area of the cells after loss of cell membrane integrity. Finally, on evaluation in a HeLa spheroid cell model, treatment with the FA-(C6+Dox)-LP showed significantly higher cell death compared to the C6-LP and Dox-LP. Overall, this study clearly shows that the co-delivery of C6 ceramide and Dox using a


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liposomal platform significantly correlates with an anti-proliferative effect due to cell-cycle regulation and subsequent induction of apoptosis and thus warrants its further evaluation in preclinical animal models.



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(FOR TABLE OF CONTENTS USE ONLY)

ABSTRACT GRAPHIC


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INTRODUCTION Although significant progress has been made in the field of cancer therapeutics, the development of an effective therapy still remains elusive. It is becoming more evident that cancers are able to overcome treatments with traditional chemotherapeutic agents by the acquisition of somatic mutations leading to the development of drug resistance.1 Specific examples include the development of resistance to the treatment with EGFR antibodies, as well as anthracyclines and taxane-based drugs.2-4 Current research in cancer therapy is beginning to lay a lot of onus on the use of combinational treatment regimens involving combinations of therapies, such as radiation and radiofrequency ablation with drug treatment, as well as administering cocktails of different anti-cancer drugs.5-7 Though the use of nanoparticles to deliver single anti-cancer agents have shown little improvements in the treatment of patients with refractory tumors in the clinic, they have been associated with reduced side effects.8 Recently, our group developed a novel transferrin-targeted C6 ceramide-loaded liposome. The formulation induced an increased apoptosis of ovarian cancer cells both in vitro and in vivo.9 Our current findings have demonstrated the ability of targeted ceramide-loaded liposomes to suppress the peritoneal metastasis of ovarian cancer due to its potent action on the PI3K/AKT pathway.10 These findings clearly highlight the advantages of delivering ceramides using a liposomal platform. Similarly, the ability of folic acid-targeted (FA) nanoparticles to significantly enhance their anticancer activity has been well documented.11, 12 More recently, a number of studies by other groups have also highlighted the possible advantages of using nanoparticles in combination drug therapy.13-15 Dhule et al. has demonstrated the use of combination liposomes co-encapsulating the anti-cancer drugs C6



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ceramide and curcumin in vitro and in vivo on a human osteosarcoma xenograft model.16 Similarly, Fonseca et al. have also shown that the combined intracellular delivery of ceramide and doxorubicin allows for their synergistic effects compared to their un-encapsulated combination.17 In light of these recent findings from our group as well as others, the cytotoxic potential of novel FA-targeted liposomes co-loaded with C6 ceramide and Dox was evaluated on HeLa cells as well as on Dox-resistant ovarian carcinoma (A2780-ADR) and lung carcinoma (H69-AR) cells in vitro. Furthermore, the optimized formulations were also evaluated on a HeLa cell spheroid model. MATERIALS & METHODS Materials & Cell Culture: Folate receptor-α primary goat antibody (sc-16386), FITC-labeled secondary donkey anti-goat antibody (sc-2024) and FITC-labeled normal mouse IgG (sc-2855) (as negative control) were purchased from Santa Cruz Biotechnology (Dallas, TX). AminoPEG3400-DSPE was purchased from Laysan Bio (Arab, AL). Eggphosphatidylcholine (ePC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG), N-hexanoyl-D-erythro-sphingosine (C6 Ceramide), (lissamine rhodamine)-DPPE (Rh-PE) were purchased from Avanti (Alabaster, AL). Folic acid, dicyclohexylcarbodiimide (DCC) and Ninhydrin were purchased from Sigma-Aldrich (St. Louis, MO). The microBCA™ Protein Assay Kit, Whatman nucleopore polycarbonate membranes 19 mm at 0.2µ, 0.1µ and 0.05µ pore sizes, bovine serum albumin (BSA) and agarose were purchased from Fisher/Thermo Scientific (Waltham, MA). Spectra/Por pre-wetted 300,000 MWCO dialysis membranes and Spectra/Por 12-14 kDa MWCO dry membranes were


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purchased from Spectrum Labs Inc. (Rancho Dominguez, CA). CellTiter-Blue® and CellTiter Glo® cell viability assays were purchased from Promega Corporation (Madison, WI). Doxorubicin hydrochloride was purchased from LC Labs (Woburn, MA) and Lancrix Chemicals (Shanghai, China). Hochest33342, Yo-Pro®-1-iodide and propidium iodide were purchased from Life Technologies (Carlsbad, CA). Streptomycin (25µg/mL)/penicillin (10,000 U/mL) solution, RPMI media, Cellstripper™ solution as well as Trypsin/EDTA were purchased from Fisher/Mediatech (Manassas, VA). RPMI without folic acid was purchased from Life Technologies (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Flowery Branch, GA). Adriamycinresistant human ovarian carcinoma cells (A2780-ADR) were purchased from Sigma-Aldrich (St. Louis, MO). Human cervical cancer cells (HeLa) and adriamycin-resistant lung carcinoma cells (H69-AR) were purchased from ATCC (Manassas, VA). The HeLa, H69 and A2780-ADR cells were grown in RPMI media. Additionally, HeLa were also grown in folic acid-deficient RPMI for 2 weeks for the cell association experiments (HeLa-FA). All media were supplemented with 10% FBS and 1% penicillin-streptomycin solution. Cells were all grown at 37°C with 5% CO2. Synthesis of Folic Acid-PEG3400-DSPE: The folic acid conjugate (FA-PEG-DSPE) was synthesized by slight modification of methods described previously.18 Folic acid (4.52mg, 10.24 µmol) was added to 4mL of DMSO under constant stirring followed by 2mL pyridine. DCC (6.34mg, 30.76 µmol) was then added to the reaction solution and stirred at room temperature (RT) for 15 minutes. Amino-PEG3400-PE (20mg, 5.12 µmol) was then added to the above reaction mix. The reaction was then carried out at RT for 4 hours under constant stirring. Using TLC, disappearance of amino-PEG-PE from the reaction mix was confirmed by the Ninhydrin



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spray. The reaction mix was dried under nitrogen and then placed under a rotary evaporator for 1 hour to remove the pyridine. After addition of 8mL DI water, the solution was centrifuged at 5000 RPM for 15 minutes to remove trace insolubles. The supernatant was then dialyzed against DI water (5 x 2L) over 48 hours using an MWCO of 300,000 Da to ensure complete removal of the free folic acid. The dialyzate was then freeze dried and dissolved in a 90:10 mixture of chloroform: methanol (1 mg/mL) and stored at -80°C until further use. Preparation of Liposomes: The liposomes were prepared by the thin-film hydration technique (See schematic in Fig 1). A lipid film consisting of ePC, cholesterol and C6 ( where required) at the mole ratios shown in Table 1 for the various liposomal formulations was obtained by removal of chloroform using rotary evaporation followed by freeze drying on a Freezone 4.5 L Freeze Dry system (Labconco, Kansas City, MO) for a minimum of 4 hours. For preparation of the C6 ceramide liposomes (C6-LP), the film was rehydrated with phosphate buffered saline (PBS) pH 7.4 to obtain a lipid concentration of 10 mg/mL. The solution was sequentially extruded through 200, 100 and 50 nm polycarbonate membranes. Similarly, for the preparation of the Dox (Dox-LP) as well as C6 and Dox liposomes [(C6+Dox)-LP], the film was rehydrated with 250 mM ammonium sulfate (pH 5.6) so as to maintain a lipid concentration of 15 mg/mL. The solution was vortexed and then sequentially extruded through 200, 100 and 50 nm polycarbonate membranes. The liposomes were then dialyzed against PBS pH 7.4 using a MWCO of 12-14kDa for 1 hour at RT to exchange the outer liposomal buffer. The liposomes were then incubated with Dox (5 mg/mL stock solution in 0.9% NaCl) for 1 hour at 65°C, at a 7.5 mol % of Dox to total lipid. Following this, the unencapsulated Dox (if any), was removed by dialysis against PBS pH 7.4 using a MWCO membrane of 12-14 kDa for 4 hours at RT. The volumes were then finally adjusted with PBS pH 7.4 to have a final liposomal lipid


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concentration of 10 mg/mL in solution. The PEG2000-DSPE (PEG) and the FA-PEG-DSPE were then added to all the liposomes [C6LP, Dox-LP and (C6+Dox)-LP] by the post-insertion method at 3 and 0.5 mol%, respectively. Briefly, a thin film of the PEG and FA-PEG-DSPE was obtained by the removal of organic solvent by drying under a stream of nitrogen gas followed by freeze-drying for a minimum of 4 hours. The PEG/FA-PEG-DSPE film was then hydrated with the respective drug-loaded liposomal solution and incubated at 37°C overnight to allow for complete incorporation of the PEG chains onto the liposome.

Figure 1: Schematic of liposomes preparation



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Table 1. Composition of Liposomes Formulation

ePC

Cholesterol

C6 Ceramide

PEG2K-DSPE

FA-PEG3.4K-DSPE

C6-LP

70 mol%

15 mol%

15 mol%

3 mol%

-

Dox-LP

70 mol%

30 mol%

-

3 mol%

-

(C6+Dox)-LP

70 mol%

15 mol%

15 mol%

3 mol%

-

FA-(C6+Dox)LP

70 mol%

15 mol%

15 mol%

3 mol%

0.5 mol%

Characterization of Liposomes: Particle size and zeta potential analysis was carried out using an N4 Coulter particle size analyzer and Zetaplus (Brookhaven Instruments Corporation, Holtsville, NY) respectively. For particle size analysis, 5µL of the liposomal solution was mixed with 995µL of 1mM potassium chloride (KCl). While for zeta potential, 50µL was mixed with 1.5mL of 1mM KCl. For the determination of liposomal Dox content, the liposomes were dissolved in methanol at a dilution factor of 50 and the absorbance was measured at 480 nm. The drug concentration was determined by comparison with a standard curve of free Dox in methanol (0-60 µg/mL). Folate Receptor Characterization: For the characterization of the folate receptor (FR) α, cells were detached using Cellstripper solution (10 mins incubation at 37ºC). Detached cells were then neutralized with complete media and centrifuged at 2000 RPM, 5 mins. Cell pellets were then resuspended in 3% BSA/PBS and counted. 200,000 cells were taken in each centrifuge tube. To the control, 100 µL 3% BSA/PBS was added; to the control antibody group, 100 µL of normal mouse IgG1-FITC was added (final dilution of 1:20 in cell suspension). To the FR antibody group 100 µL of the anti-FR primary antibody was added (final dilution of 1:16 in cell suspension), washed and incubated with the secondary FITC-labeled antibody (final dilution of


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1:20). Antibody incubation steps were done at 4ºC for 30 minutes following which the cell suspensions were centrifuged at 2000 RPM, 5 mins and washed with 3% BSA/PBS. Cell pellets were then resuspended in 3% BSA/PBS, kept on ice and immediately analyzed by flow cytometry (BD FACS Calibur®, Bedford, MA). Cell Association Studies: The liposomes were labeled with 1 mol % Rh-PE, which was added along with the other lipids prior to the film hydration, and the PEG and FA-PEG-DSPE were added by post insertion as described earlier. 75,000 cells were seeded per plate of a 12-well plate 24 hours prior to the experiment. Following this, the cells were treated with the formulations at a liposomal concentration of 0.1 mg/mL for 4 hours. The formulations were then washed off with PBS and the cells were trypsinized, washed and maintained as a cell suspension in PBS on ice. Cells were then immediately analyzed for their mean fluorescent intensity by flow cytometry. All samples were normalized with a control cell population based on their mean fluorescent intensity and analyzed. In Vitro Cytotoxicity Experiments on Monolayers: 3,000 cells were seeded in each well of a 96-well plate 24 hours prior to the experiment. Formulations were sterile filtered and incubated with the cells for 4 hours, washed off and replaced with media. The cell viability was then measured 48 hours later using the CellTiter-Blue® cell viability assay. The plates were read at an excitation wavelength of 530 nm and emission of 590 nm using a BioTek Synergy HT plate reader (BioTek Instruments Inc., VT). Analysis of Apoptosis and Cell Cycle Distribution: 3,000 cells per well were seeded in black-walled polystyrene 96-well plates 24 hours prior to the experiment. Formulations were sterile filtered and incubated with the cells for 48 hours. Hoechst33342 (5µg/mL) followed by



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the markers, Yo-Pro-1 (0.12µg/mL) for early apoptosis and propidium iodide (1µg/mL) for late apoptosis/necrosis were diluted in media and all added together directly onto the cells. After incubation at 37°C for 30 minutes, the stained cells were analyzed without washing using the iCyte imaging cytometer (Compucyte Corp., Westwood MA). A 40x objective lens was used with 0.25 µm spatial resolution in two-pass scanning. In the first pass, the 405 nm laser was used to excite Hoechst and fluorescence was collected through a 440 /30 bandpass filter. In the second pass, the 488 nm argon laser was used, with a 515/30 bandwidth filter for green YoPro-1 fluorescence, and a 650 nm long pass filter for the red propidium iodide fluorescence. Cells were segmented using Hoechst fluorescence, and total cellular DNA fluorescence was quantified. For the cell cycle distribution, live single cells were gated into the G1, S and G2 phases based on their combined DNA content and nuclear area. For the analysis of apoptotic events, cells were gated based on their green Yo-Pro-1 and red propidium iodide signal and quantified by random segmentation. For the experiments involving the phase holographic imaging (PHI) system, 50,000 HeLa cells were seeded in MatTek (Ashland, MA) glass-bottom dishes (35 mm petri dish, 14mm Microwell). After 24 hours, cells were untreated or treated with the FA-(C6+Dox)-LP at a C6/Dox concentration of 1.6/0.8 µM. The formulation was diluted in media and sterile filtered before the addition to the cells. Following this, the cells in both treatment groups were analyzed in parallel using the Holomonitor® M4 (PHI, Sweden) with images being captured at 5 minute intervals (20X objective) for 48 hours. After analysis, using the Hstudio® data analysis software (PHI, Sweden) 20 cells were chosen at random at the earliest time point. The average area occupied by these cells was then tracked at 5 minute intervals over the 48 hour time point and evaluated.


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Spheroid Penetration and Analysis of Spheroid Cytotoxicity: HeLa cell spheroids were prepared by the liquid overlay method as described previously.19 Briefly, 1.5% w/v agar in serum free media was prepared and sterilized. 50 µL of this was added to each well of a 96-well flat-bottom plate. Plates were then allowed to cool down for 45 minutes under UV. 7,500 HeLa cells were then added to each well. The plates were then centrifuged for 15 minutes at 1500 RCF at RT. Once they were formed and compact in appearance (typically 3-5 days after seeding) the resulting spheroids were then treated with the formulations (at a C6/Dox concentration of 40/20 µM) for 96 hours. Following this, the spheroids were harvested, centrifuged at 2000 RPM for 5 minutes and washed with PBS. The cell viability of the resulting cell suspensions was analyzed using the CellTiter Glo® assay according to the manufacturer’s protocol. Data was generated in triplicates with each triplicate consisting of 5 spheroids (total of 15 spheroids for each data point) for increased sensitivity of measurements. Statistical Analysis Data was generated in triplicates and expressed as mean +/- S.D. Statistical analyses were performed using one-way ANOVA followed by post-hoc analyses. Significance was determined by a P-value < 0.05 (denoted by *), P

Enhanced Cytotoxicity of Folic Acid-Targeted Liposomes Co-Loaded

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