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Brief Article
Liposomal Co-Encapsulation of Doxorubicin with Listeriolysin O Increases Potency via Sub-Cellular Targeting Zachary F. Walls, Henry Gong, and Rebecca J Wilson Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00674 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016
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Liposomal Co-Encapsulation of Doxorubicin with
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Listeriolysin O Increases Potency via Sub-Cellular
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Targeting
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Zachary F. Walls, PhD1,2*, Henry Gong3, Rebecca J. Wilson4
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1
Department of Pharmaceutical Sciences
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2
Center for Inflammation, Infectious Disease and Immunity
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3
Department of Biomedical Sciences
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Department of Biological Sciences
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East Tennessee State University
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Johnson City, TN, USA
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*Address for correspondence: Zachary F. Walls, Box 70594, Johnson City, TN 37614, USA.
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Tel: (423) 439-6236. Email:
[email protected] 13
TABLE OF CONTENT/ABSTRACT GRAPHIC
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ABSTRACT
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Liposomal doxorubicin is a clinically important drug formulation indicated for the treatment of
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several different forms of cancer. For doxorubicin to exert a therapeutic effect, it must gain
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access to the nucleus. However, a large proportion of the liposomal doxorubicin dose fails to
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work because it is sequestered within endo-lysosomal organelles following endocytosis of the
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liposomes due to the phenomenon of ion trapping. Listeriolysin O (LLO) is a pore-forming
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protein that can provide a mechanism for endosomal escape. The present study demonstrates that
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liposomal co-encapsulation of doxorubicin with LLO enables a significantly larger percentage of
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the dose to co-localize with the nucleus compared to liposomes containing doxorubicin alone.
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The change in intracellular distribution resulted in a significantly more potent formulation of
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liposomal doxorubicin as demonstrated in both the ovarian carcinoma cell line A2780 and its
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doxorubicin-resistant derivative A2780ADR.
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KEYWORDS
14 15 16
Cancer, drug-resistance, endosomal escape, pH-sensitive INTRODUCTION
Doxorubicin has been a mainstay of cancer chemotherapy since the 1970s.
1,2
Although its
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exact mechanism of action remains unknown, it has been found that doxorubicin can intercalate
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between DNA base pairs, inhibiting transcription and DNA synthesis. Conceptually, doxorubicin
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is attractive therapy for any type of cancer, since one of the defining aspects of cancer is
20
unregulated cell growth requiring accelerated genomic replication. A liposomal formulation of
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doxorubicin that alleviates many of the toxic side effects of the drug has been in routine clinical
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use for over a decade. 3 However, despite the more favorable therapeutic profile of liposomal
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doxorubicin compared to free doxorubicin regarding side effects, clinical studies have
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demonstrated no change in potency, response rate, or time to progression between liposomal and
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free doxorubicin. 4,5
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The unaltered potency of liposomal doxorubicin is thought to be due to the phenomenon of 6,7
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multi-drug resistance (MDR).
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altered intracellular pH gradients is especially germane to the liposomal delivery of weak bases
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such as doxorubicin.
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molecules are uncharged at neutral pH, enabling the drug to freely cross biological membranes.
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However, acidic environments such as endosomes and lysosomes favor conversion of the drug
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into a positively charged molecule, preventing free diffusion across membranes. Since liposomes
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typically enter cells by endocytosis, the pH gradient between endosomes and the cytosol largely
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determines how much of the drug can diffuse into the cell. Many cancer cells exhibit a large pH
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gradient between the cytosol and the endosomal compartments, effectively trapping weak base
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liposomal drugs in intracellular vesicles.
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drug delivery as highlighted by clinical evidence that the intracellular concentration of
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daunorubicin (a weak base similar to doxorubicin) is no different between patients treated with
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free daunorubicin versus liposomal daunorubicin.
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values for various cancer cells and the Henderson-Hasselbalch equation, it was calculated that
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67% – 93% of a typical liposomal doxorubicin dose is sequestered endosomally.
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reformulating liposomal doxorubicin to increase endosomal escape would dramatically increase
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the drug’s potency.
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Of the known causes of MDR exist, but the mechanism of
Because the pKa of doxorubicin is 7.34, a substantial fraction of the
9,10
This resistance mechanism influences liposomal
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Using previously reported subcellular pH
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Thus,
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Listeriolysin O (LLO) is a pore-forming protein secreted by Listeria monocytogenes following
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endocytosis that permits the bacteria to escape endosomal vesicles and replicate intracellularly.
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across cellular membranes via co-encapsulation in pH-sensitive liposomes. It does this by
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forming pores in the endosomal membrane that allow the therapeutic payload to diffuse into the
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interior of the cell.
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listeriolysin O-conjugated liposomes for the enhanced delivery of small molecules.
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on the successful reports of enhanced delivery of therapeutic molecules using LLO-containing
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liposomes, it was hypothesized that co-encapsulation of doxorubicin with LLO in pH-sensitive
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liposomes could improve the drug’s co-localization with the nucleus and enhance the
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cytotoxicity of liposomal doxorubicin. This study reports the subcellular localization of
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doxorubicin and the potency of a co-encapsulated liposomal formulation of doxorubicin with
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LLO compared to liposomal doxorubicin using a cellular model of ovarian carcinoma (A2780)
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and its drug-resistant derivative (A2780ADR).
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EXPERIMENTAL SECTION
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Protein Expression and Purification
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The listeriolysin O expression construct was provided by Professor Kyung-Dall Lee (University
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of Michigan). A hyperactive mutant of LLO containing a single amino acid substitution (C484S)
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described previously by Walls and colleagues was used in all experiments.
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plasmid consisted of the pET29b backbone carrying the gene coding for LLO in frame with a
19
polyHis tag. BL21(DE3)pLysS chemically competent E. coli (Invitrogen) were transformed with
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the plasmid and then grown in Terrific Broth (TB) containing 30 µg/mL kanamycin at 37°C with
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shaking at 225 RPM. Once the cells reached sufficient density (A600 > 0.7), protein expression
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was induced by adding IPTG at a final concentration of 1mM. The temperature was reduced to
Recombinantly produced LLO has been used with stunning success to deliver macromolecules
14–16
Further, Kullberg and colleagues have demonstrated the utility of using
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17,18
Based
The expression
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30°C and expression continued for 12 – 16 hours. Cells were then pelleted at 4000xg for 20
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minutes at 4°C. The supernatant was decanted and the cell pellet was stored at -70°C.
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LLO was purified by resuspending the frozen cell pellet in wash buffer (50mM NaH2PO4,
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300mM NaCl, 20mM imidazole) with lysozyme (final concentration 1mg/mL) and PMSF (final
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concentration (20µM) using a needle and syringe. The homogeneous slurry was then sonicated
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on ice with 6 10-second bursts. Insoluble debris was removed by centrifugation at 14,000xg for 1
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hour at 4°C. Residual particulate matter was removed from the soluble fraction using a 0.45µm
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syringe filter. The filtered soluble fraction was applied to a column containing Ni-NTA agarose
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(Qiagen) by gravity, and then washed with wash buffer containing 40mM imidazole. LLO was
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eluted from the column with wash buffer containing 250mM imidazole and then exchanged into
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HBSE (10mM HEPES, 140mM NaCl, 1mM EDTA, pH 8.4) using 10DG desalting columns
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(Bio-Rad). Protein concentration was quantified using the BCA assay kit (Pierce).
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Liposome Preparation and Characterization
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Solutions of egg phosphatidylethanolamine (ePE, Avanti) and cholesteryl hemisuccinate
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(CHEMS, Sigma) were combined at a 2:1 molar ratio and then dried under an argon stream to
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remove organic solvents. The resulting lipid films were resuspended by vortexing in a total of
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1mL containing either 0.5mg of doxorubicin or 0.5mg of doxorubicin and 1mg of LLO.
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Resuspended lipids were then subjected to 4 freeze/thaw cycles. Small unilamellar liposomes
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were generated using a Mini-Extruder (Avanti), with 10 passes through a 0.2µm polycarbonate
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filter followed by 10 passes through a 0.1µm polycarbonate filter. Unencapsulated LLO and
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doxorubicin were removed by size exclusion chromatography using a sepharose CL-4B column.
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Doxorubicin concentration of the liposomes was determined by fluorescence using a SpectraMax
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Gemini XS Fluorescence Plate Reader (Molecular Devices). Known doxorubicin concentrations
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were prepared in a black 96-well plate with a clear bottom alongside serial dilutions of the
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liposomes. Equal volumes of 0.2% Triton X-100 were added to all wells and the plate was
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incubated at room temperature for 20 minutes. The plate was then read using 479nm/593nm
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(Ex/Em) wavelengths.
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LLO concentration of the liposomes was determined by SDS-PAGE followed by non-specific
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protein staining. Three different volumes of liposomes were heated at 70°C for 10 minutes and
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then loaded in duplicate against known concentrations of LLO on a 4-12% Bis-Tris gel
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(Invitrogen). Samples were run at 200V in 1xMOPS for 50 minutes and then gels were fixed and
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stained with Krypton (Pierce) according to the manufacturer’s instructions. Gels were imaged
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and quantified using a G:Box (Syngene) gel documentation system.
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Confocal Fluorescence Microscopy
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A2780 and A2780ADR cells (Sigma) were plated on cover slips placed in 6-well plates at a
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concentration of 200,000 cells/mL and grown in McCoy’s 5a media supplemented with 10%
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FBS and 1% penicillin/streptomycin. 24 hours later, cells were treated with either liposomal
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doxorubicin or liposomal doxorubicin co-encapsulated with LLO at a doxorubicin concentration
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of 5 µg/mL diluted in serum-free media. 8 hours following treatment, cells were washed once
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with PBS warmed to 37°C and then fixed with 4% formaldehyde for 15 minutes at 37°C. Cells
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were washed twice with room temperature PBS and then permeabilized with 0.2% Triton X-100
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for 5 minutes at room temperature. Cells were washed twice with PBS and then blocked with
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10% heat-inactivated normal goat serum for 30 minutes at room temperature. The cover slips
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were then inverted onto 100 µL drops containing a 1:1000 dilution of α-LAMP-2 antibody
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(CD107b, BioLegend) for one hour at room temperature. Following the incubation, cells were
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washed three times in blocking solution and then incubated with a 1:1000 dilution of goat α-
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mouse antibody conjugated to Alexa Fluor 790 (Invitrogen) for 30 minutes at room temperature.
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Cells were washed three times with PBS and mounted onto slides with ProLong Gold with DAPI
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(Invitrogen). Images were acquired using an epifluorescent microscope (DMI 6000, Leica) using
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a 63 x 1.4 NA oil immersion objective.
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The co-localization rate was calculated by plotting the arbitrary fluorescent units of two different
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fluorophores for each pixel of the image. After setting thresholds for each fluorophore and
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excluding background fluorescence, the percentage of pixels containing significant fluorescence
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from each fluorophore was reported as the co-localization rate.
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Cellular Viability Assay
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A2780 and A2780ADR cells were plated in 96-well plates at a concentration of 200,000
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cells/mL and grown in McCoy’s 5a media supplemented with 10% FBS and 1 %
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penicillin/streptomycin. 24 hours later, cells were treated with either liposomal doxorubicin or
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liposomal doxorubicin co-encapsulated with LLO at a range of doxorubicin concentrations (100
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– 0.02 µg/mL) diluted in serum-free media. 48 hours after treatment, the media was changed and
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12mM MTT (Invitrogen) was added. 4 hours later, an equal volume of 10% SDS in 0.01M HCl
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was added. 4 hours later, absorbance was measured using a SpectraMax Plus (Molecular
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Devices) absorbance plate reader at 570nm.
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TUNEL Assay
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A2780 cells were plated on cover slips in 6-well plates at a density of 200,000 cells/mL. At 24
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hours, they were treated with either liposomal doxorubicin or liposomes co-encapsulated with
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doxorubicin and listeriolysin o at a doxorubicin concentration of 0.5 µg/mL. 72 hours after
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treatment, the cells were fixed and stained according to the Click-IT TUNEL AF647 Imaging
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Assay Kit (Invitrogen) protocol. The cover slips were mounted onto slides, cured overnight, and
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then imaged using an EVOS FL AUTO Cell Imaging System.
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In vivo pH Measurements
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A2780 and A2780ADR cells were plated at a density of 200,000 cells/mL in an 8-well
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chambered coverslip (ibidi µ-slide). 24 hours after plating, the media was aspirated and cells
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were exposed to a 2µM solution of SNARF-5F 5-(and-6)-carboxylic acid (Invitrogen). After a 25
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minute incubation at room temperature, a 2µM solution of LysoSensor Yellow/Blue DND-160
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(Invitrogen) was added to the cells. After a 5 minute incubation at room temperature, both
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solutions were aspirated and Live Cell Imaging Solution (Invitrogen) was added. Cells were
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imaged using an epifluorescent microscope (DMI 6000, Leica) at the following wavelengths: Ex:
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405nm, Em: 440nm ± 25nm and Em: 540nm ± 25nm; Ex: 552nm, Em: 580nm ± 25nm and Em:
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640nm ± 25nm. Fluorescence intensity was measured across 3 cells for each sample. The
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following ratios were then calculated for each pixel: Em540nm/Em440nm and
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Em640nm/Em580nm. The ratios from all 3 cells were averaged and compared using the two-
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tailed paired t-test.
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Statistical Analysis
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All statistical tests were performed in Prism 6 for Mac OS X (GraphPad). Wilcoxon rank-sum
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(Mann-Whitney) was used to determine the significance of differences between encapsulation
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efficiencies. MTT assay data was fit to sigmoidal dose-response (variable slope) curves and then
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the best-fit logEC50 values were compared for significance using the sum-of-squares F test.
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RESULTS
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Liposome Preparation and Characterization
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Recombinant LLO expression and purification, as well as pH-sensitive liposome construction
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were accomplished by the methods established by Provoda and colleagues. 20 These methods
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have been used for the co-encapsulation of LLO with many different macromolecules and
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involve the passive encapsulation of water-soluble molecules in the core of PE:CHEMS::2:1
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liposomes. LLO encapsulation efficiency, as determined by SDS-PAGE, was found to be 9.8 ±
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3.6 % (average ± SEM). This value was consistent with previous reports of LLO encapsulation
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efficiency. 21 Doxorubicin encapsulation efficiency was determined by measuring the intrinsic
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fluorescence of the drug in purified formulations. It was found that the encapsulation efficiency
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of liposomal doxorubicin was 43.4 ± 3.2 % and the encapsulation efficiency of liposomal
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doxorubicin co-encapsulated with LLO was 40.8 ± 5.6 % (average ± SEM). Although the
15
encapsulation efficiency was generally lower for the co-encapsulated formulations, the
16
difference was not statistically significant (Mann Whitney test, p > 0.99).
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Fluorescence Microscopy and Co-localization
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To determine whether co-encapsulation of doxorubicin with LLO could improve the co-
19
localization of the drug with the nucleus, confocal fluorescence microscopy was performed on
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cells treated with the two different formulations. A2780 ovarian carcinoma cells showed a stark
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contrast in doxorubicin localization between the two formulations. Liposomal doxorubicin was
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primarily sequestered in the lysosomes (identified by LAMP-2 staining, Figure 1A). However,
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when doxorubicin was delivered by liposomes co-encapsulated with LLO, there was a high
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proportion of co-localization with the nucleus (identified by DAPI staining, Figure 1B).
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In order to quantify the increase in nuclear doxorubicin localization due to LLO co-
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encapsulation, line scans of fluorescence intensity through individual cells were made and
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separated by channel. These quantitative measurements showed a high correlation between
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intensity for the LAMP-2 and doxorubicin channels in cells treated with liposomal doxorubicin
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(Figure 2A), and a high correlation between intensity for the DAPI and doxorubicin channels in
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cells treated with the co-encapsulated formulation (Figure 2B).
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For a more comprehensive quantitative measurement of doxorubicin subcellular localization,
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scatter plots were generated for the entire field of view. For cells treated with liposomal
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doxorubicin, the co-localization rate between doxorubicin and DAPI fluorescence was 3.2%
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(Figure 3A, left), and the co-localization rate between LAMP-2 and doxorubicin fluorescence
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was 100.0% (Figure 3A, right). For cells treated with the co-encapsulated formulation, the co-
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localization rate between doxorubicin and DAPI fluorescence was 99.6% (Figure 3B, left), and
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the co-localization rate between LAMP-2 and doxorubicin was 2.1% (Figure 3B, right).
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Fluorescence microscopy performed on A2780ADR cells treated with the two different
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doxorubicin formulations showed trends similar to those observed for the A2780 cells
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(Supplementary Figure 1). The co-localization rate between LAMP-2 and doxorubicin decreased
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from 72.7% for cells treated with liposomal doxorubicin to 1.23% for cells treated with
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liposomal doxorubicin co-encapsulated with LLO. The co-localization rate between doxorubicin
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and DAPI increased from 65.6% for cells treated with liposomal doxorubicin to 78.6% for cells
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treated with liposomal doxorubicin co-encapsulated with LLO. In addition to the observed
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nuclear localization of doxorubicin in A2780ADR cells treated with the co-encapsulated
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formulation, punctate staining was also seen throughout the cytoplasm. These areas did not,
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however, overlap with LAMP-2 staining.
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Effect of Co-encapsulated Formulation on Cellular Viability
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The effect of liposomal doxorubicin co-encapsulated with LLO on cellular viability was
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determined by treating cells with serial dilutions of either liposomal doxorubicin or liposomal
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doxorubicin co-encapsulated with LLO. After a 48-hour treatment period, cells were assayed for
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viability with MTT. Data was fit to sigmoidal dose-response curves with variable slopes by
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constraining the top and bottom best fit values (Prism 6, GraphPad Software, Figure 4). It was
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found that co-encapsulation of doxorubicin with LLO significantly lowered the EC50 for both
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cell lines compared to liposomes containing doxorubicin alone (extra sum-of-squares F test, p
0.05, Supplementary
4
Figure 3).
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The pore-forming activity of LLO is pH-dependent and thus the protein is thought to only exert
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an effect in the acidic environment of endolysosomal organelles. 22 Other studies have shown that
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liposomes containing LLO alone result in limited cellular toxicity. 20 In agreement with these
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reports, it was found that the dose-response curves for liposomal LLO and liposomal doxorubicin
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co-encapsulated with LLO were statistically different (Supplementary Figure 2, p < 0.0001),
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with only the highest concentrations of liposomal LLO causing a reduction in cellular viability.
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In vivo pH Measurement
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To determine whether the magnitude of reduction in EC50 corresponded to the pH-gradient in
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each cell line, live cells were exposed to pH-sensitive ratiometric fluorophores and then imaged
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using a confocal microscope. LysoSensor Yellow/Blue DND-160 was used to measure the
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lysosomal pH while SNARF 5F 5-(and-6)-carboxylic acid was used to measure the cytosolic pH
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of A2780 and A2780ADR cells. No significant difference in lysosomal pH was observed
17
between the two cell lines, but a statistically significant difference in cytosolic pH was observed
18
(Figure 5, p
