Liposomal formulation of hypoxia activated prodrug for the treatment of ovarian cancer

Accepted Manuscript Liposomal formulation of hypoxia activated prodrug for the treatment of ovarian cancer

Vidhi M. Shah, Duc X. Nguyen, Adel Al Fatease, Pragnesh Patel, Brianna Cote, Yeonhee Woo, Rohi Gheewala, Yvonne Pham, Man Gia Huynh, Christen Gannett, Deepa A. Rao, Adam W.G. Alani PII: DOI: Reference:

S0168-3659(18)30592-3 doi:10.1016/j.jconrel.2018.10.021 COREL 9504

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

14 May 2018 8 October 2018 16 October 2018

Please cite this article as: Vidhi M. Shah, Duc X. Nguyen, Adel Al Fatease, Pragnesh Patel, Brianna Cote, Yeonhee Woo, Rohi Gheewala, Yvonne Pham, Man Gia Huynh, Christen Gannett, Deepa A. Rao, Adam W.G. Alani , Liposomal formulation of hypoxia activated prodrug for the treatment of ovarian cancer. Corel (2018), doi:10.1016/ j.jconrel.2018.10.021

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Liposomal Formulation of Hypoxia Activated Prodrug for the Treatment of Ovarian Cancer Vidhi M. Shah1, Duc X. Nguyen1, Adel Al Fatease1, Pragnesh Patel2, Brianna Cote1, Yeonhee Woo1, Rohi Gheewala3, Yvonne Pham3, Man Gia Huynh3, Christen Gannett1, Deepa A. Rao4, Adam WG. Alani1

T

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State

University/OHSU, Portland, Oregon, USA.

SC RI P

1

2

Izon Sciences, Cambridge, Massachusetts, USA.

3

Portland State University, Portland, Oregon, USA4School of Pharmacy, Pacific

Adam Alani, Ph.D.

MA

Corresponding Author:

NU

University, Hillsboro, Oregon, USA.

Associate Professor

ED

Department of Pharmaceutical Science Oregon State University/OHSU

AC

CE

PT

Collaborative life science building 2730 SW Moody Ave Portland, Oregon 97201 [email protected]

ACCEPTED MANUSCRIPT Liposomal Formulation of Hypoxia Activated Prodrug for the Treatment of Ovarian Cancer. In this work, a new sphingomyelin-cholesterol liposomal formulation (CPD100Li) for the delivery of a hypoxia activated prodrug of vinblastine, mon-Noxide (CPD100), is developed. The optimized liposomal formulation uses an ionophore

T

(A23187) mediated pH-gradient method. Optimized CPD100Li is characterized for

SC RI P

size, drug loading, and stability. The in vitro toxicity of CPD100Li is assessed on different aspects of cell proliferation and apoptosis of ES2 ovarian cancer under normoxic and hypoxic conditions. The pharmacokinetics of CPD100Li in mice as well as the influence of A23187 on the retention of CPD100 are assessed. The dose limiting toxicity (DLT) and maximum tolerated dose (MTD) for CPD100Li are evaluated in

NU

nude mice.

CPD100 is loaded in the liposome at 5.5 mg/mL. The sizes of CPD100Li using

MA

DLS, qNano and cryo-TEM techniques are 155.4 ± 4.2 nm, 132 nm, and 112.6 ± 19.8 nm, respectively. There is no difference between the in vitro characterization of

ED

CPD100Li with and without ionophore. Freshly prepared CPD100Li with ionophore are stable for 48 h at 4 °C, while the freeze-dried formulation is stable for 3 months under argon at 4 °C. The hypoxic cytotoxicity ratios (HCR) of CPD100 and CPD100Li

PT

are 0.16 and 0.11, respectively. CPD100Li under hypoxic conditions has a 9.2-fold lower IC50 value as compared to CPD100Li under normoxic conditions, confirming the

CE

hypoxia dependent activation of CPD100. CPD100Li treated ES2 cells show a time dependent enhanced cell death, along with caspase production and an increase in the

AC

number of cells in G0/G1 and higher cell arrest. The blood concentration profile of CPD100Li in mice without A23187 has a 12.6-fold lower area under the curve (AUC) and 1.6-fold lower circulation time compared to the CPD100Li with A23187. The DLT for both CPD100 and CPD100Li is 45 mg/kg and the MTD is 40 mg/kg in nude mice. Based on the preliminary data obtained, we clearly show that the presence of ionophore affects the in vivo stability of CPD100. CPD100Li presents a unique opportunity to develop a first-in-kind chemotherapy product based on achieving selective drug activation through the hypoxic physiologic microenvironment of solid tumors.

ACCEPTED MANUSCRIPT Keywords: hypoxia; liposomes; Mono-N-Oxide Vinblastine; pro-drug; ovarian cancer

AC

CE

PT

ED

MA

NU

SC RI P

T

Graphical abstract

ACCEPTED MANUSCRIPT Introduction Ovarian cancer is the fifth most common female cancer worldwide due to late diagnosis and development of chemoresistance[1]. The gold standard for treatment is chemotherapy with two or more drugs given intravenously (IV) every 3 to 4 weeks[2]. The standard approach is the combination of platinum compounds, such as cisplatin or

T

carboplatin, and a taxane, such as paclitaxel [3-5]. The current treatment protocol is

SC RI P

successful in reducing the tumor burden, but relapses are common[3, 6, 7] due to ineffectiveness in non-proliferative hypoxic cells[8]. The tumor core is composed of non-proliferative hypoxic cells which can lead to treatment resistance in subsequent

NU

cycles[8, 9]. The origin of these slow growing cells is a result of different levels of oxygen tension in the tumor mass[10]. To distinguish between the various levels of

MA

oxygenation, the following terms are currently used: ‘normoxia’ corresponds to atmospheric oxygen pressure and is achieved in cell culture using 20% oxygenation

ED

levels, and ‘hypoxia’ represents a lower oxygenation level as compared to normoxia and is typically simulated in cell culture at oxygenation levels below 1.5%[11, 12].

PT

However, both these terms are poorly defined, as 20% oxygen (160 mmHg) in the tissue

CE

culture is significantly higher than the 14.5% (110 mmHg) oxygen level in the lung alveoli or 5% (38 mmHg) oxygen level in peripheral tissues[13]. Thus, depending on

AC

the size of the tumor and its density, various levels of oxygenation are present, with the highest levels of hypoxia seen within the core of the tumor[14]. Researchers have classified 2% oxygen (15 mmHg) as physiological hypoxia, a level expressed by most tumor tissues, and 1% oxygen (8 mmHg) as pathological hypoxia, where the poor oxygenation disrupts normal homeostasis. Prostate and pancreatic tumors considered to be severely hypoxic express a median oxygen level of 0.4% (3 mmHg)[13, 15]. The heterogeneous levels of oxygen in the tumor cells have not been successfully targeted thus far to achieve durable tumor responses in ovarian cancer. Recently,

ACCEPTED MANUSCRIPT Cascades Pro-drugs Inc (Eugene, OR) has developed a prodrug of vinblastine, vinblastine-N-oxide (CPD100), which converts to the parent compound, vinblastine, under hypoxic conditions (Fig.1)[16]. Previous work has established CPD100 conversion to vinblastine under varying levels of hypoxic conditions in numerous cell panels[17]. However, preliminary studies in mice show that the compound has a limited

T

half-life of 26 minutes, necessitating the need for a drug delivery system to make this

ED

MA

NU

SC RI P

treatment strategy viable[16].

Figure 1. Schematic representation of bio-reduction of prodrug, CPD100, to parent drug,

PT

vinblastine, under hypoxic conditions

CE

Liposomes are a well-recognized and versatile drug delivery platform that are used to

AC

either enhance the therapeutic activity or improve the safety of numerous hydrophilic anticancer drugs [18, 19]. They are colloidal carriers (0.025 – 2.5 µm in diameter) that form spontaneously or with energy input, such as agitation (friction) or heat, upon hydration of lipids in an aqueous media [19, 20]. The colloidal structure is composed of a concentric lipid bilayer vesicle enclosing an aqueous volume capable of delivering lipophilic molecules in the bilayer or, more commonly, hydrophilic compounds in the aqueous compartment[21]. To improve the pharmacokinetics of CPD100, specifically its half-life, a sphingomyelin cholesterol (SPM/Chol) liposomal formulation,

ACCEPTED MANUSCRIPT CPD100Li, has been developed[16]. Our previous work shows that the CPD100Li with the ionophore (A23187) has improved circulation time and efficacy in a small lung cancer xenograft model compared to the control group[16]. While liposomes have tremendous potential in altering the therapeutic index and improving the pharmacokinetics of many hydrophilic compounds, the efficacy and in

T

vivo retention of the drug entrapped in the lipid vesicle is subject to a great deal of

SC RI P

variability based on the various parameters and excipients needed to form these colloidal structures[22-25]. There are two common methods for drug loading into the liposome: passive and active. Briefly, passive drug loading involves dissolving the drug

NU

and lipids in organic solvent, evaporating the solvent to form a thin film, and then hydrating the thin film with a buffer that constitutes the internal solution of the vesicle.

MA

This mixture of lipids and the drug is then passed through an extruder, which results in the formation of unilamellar vesicles. These vesicles are then passed through a column

ED

equilibrated with a solution that constitutes the external solution, typically composed of saline or sucrose, resulting in the final liposome. Active drug loading involves the same

PT

procedure mentioned above, except the drug is added to the final liposome using a pH

CE

gradient that is either established due to pH differences between the internal and external solutions, or by an ionophore-mediated pH gradient. In the ionophore-mediated

AC

method, the ionophore binds to the lipid vesicle and forms a channel for ion exchange to take place, resulting in the establishment of a pH gradient. The choice of the method for drug loading depends on the physicochemical properties of the drug and the % encapsulation needed to achieve viable dosing volumes. CPD100 is a typical hydrophilic molecule with a MW of 900 g/mole and aqueous solubility of 33 mg/mL in DI water, ideally suited for drug delivery via liposomal entrapment. Based on the physicochemical properties of CPD100, active drug loading is

ACCEPTED MANUSCRIPT the preferred method for liposomal encapsulation. In vitro assessment of CPD100Li includes formulation optimization, loading and size characterization, and stability assessment for freshly prepared and freeze-dried CPD100Li. CPD100Li is assessed in ovarian cancer cell lines by comparing the underlying mechanism and activity of CPD100 to vinblastine under normoxic and hypoxic conditions and the effect on cell

T

proliferation, cell cycle, and apoptosis. In vivo characterization of CPD100Li includes

SC RI P

pharmacokinetic, dose limiting toxicity (DLT), and maximum tolerated dose (MTD) studies. The in vivo assessment of CPD100Li also determines the necessity of incorporating the ionophore, A23187, to enhance liposomal stability. This is an

NU

important clinical assessment as the ionophore, A23187, does not currently fall in the generally regarded as safe (GRAS) category as assigned by the FDA but has been

MA

shown to be a critical ingredient for retention of chemotherapeutic agents in liposomal formulations. Establishing the pharmacokinetics, DLT, and MTD of the CPD100Li

ED

incorporating the ionophore is a critical step for further clinical development. This is the first time a liposomal formulation of a vinca alkaloid based hypoxia

PT

activated pro-drug has been proposed for the treatment of ovarian cancer, and, if

CE

successful, will be the first-in-class molecule for this disease state.

AC

Materials and methods Materials

Vinblastine-N- Oxide mono hydrochloride salt (CPD100) is obtained from Cascade Prodrug Inc. (Eugene, OR.). Egg Sphingomyelin (SPM) is obtained from NOF America Corporation (White Plains, NY.). Cholesterol and A23187 are obtained from Alfa Aesar (Haverhill, MA.). ES2 cell line (Human Ovarian Clear Cell Carcinoma, a distinct histopathologic subtype of epithelial ovarian cancer) is purchased from American Type

ACCEPTED MANUSCRIPT Culture Collection (Manassas, VA). Cell culture supplies including Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin ethylenediamine-tetra acetic acid (EDTA), penicillin/streptomycin (Pen/Strep) and Dulbecco’s phosphate buffered saline (DPBS) are acquired from VWR (Radnor, PA). Cell culture reagents and disposables are purchased from VWR (Radnor, PA) or Thermo Scientific

T

(Fairlawn, NJ). Cell Titer-Blue® Cell Viability Assay kit is obtained from Promega Inc.

SC RI P

(Madison, WI). Vybrant Dye Cycle Green is purchased from Life Technologies (Grand Island, NY). All other chemicals are from VWR (Radnor, PA).

NU

Methods

Effect of different parameters in optimizing active CPD100 loading in

MA

SPM/Chol liposomal platform

Preparation of empty liposomes for active loading

ED

SPM/Chol (12.5 mM) at a molar ratio of 55:45, along with the A23187 as the ionophore to mediate drug loading, was used as the platform to load CPD100. Empty vesicles were

PT

prepared by dissolving the lipids in chloroform/methanol (70/30) followed by solvent

CE

removal at 50 °C for 30 min using the rotary evaporator. The lipid thin film was hydrated at 65 °C in 300 mM MgSO4 internal solution at different pHs for 30 min to

AC

form multilamellar vesicles (MLVs). The formation of unilamellar vesicles (LUVs) was achieved by extruding the MLVs 20 times through a mini-extruder (Avanti Polar Lipids, Alabaster, AL) using a polycarbonate filter with a 0.1 µm pore size at 60-65 °C. A transmembrane pH gradient was established in the LUVs by passing them through prepacked PD-10 columns (GE Healthcare UK Limited, UK) pre-equilibrated with the external solution, pH 7.4 SHE buffer. The empty liposomes were stored at 4 °C until required for drug loading.

ACCEPTED MANUSCRIPT Quantification of CPD100 by reversed-phase high-performance liquid chromatography (RP-HPLC) The concentration of CPD100 was determined using a Shimadzu HPLC system consisting of an LC-20AT pump and a SPD M20A diode array detector. The analysis used Zorbax C18 column (4.6 X 75 mm, 3.5 µm) (Agilent Technologies, Santa Clara,

T

CA) in isocratic mode with methanol/water (70/30) containing 0.1% phosphoric acid

SC RI P

and 1% methanol at a flow rate of 0.5 mL/min and injection volume of 10 µL. Column temperature was kept at 40 °C with a run time of 6 min. The CPD100 peak was monitored at 266 nm. The retention time for CPD100 was 1.8 min. Quantification of

NU

CPD100 in the liposomes was done by diluting the vesicles 20-fold in methanol prior to

MA

injection.

Optimization of parameters for active drug loading process

ED

Based on the physicochemical characteristics of CPD100, the active drug loading process was further optimized by evaluating the internal pH (A), incubation time (B),

PT

incubation temperature (C), A23718 concentration (D), EDTA concentration (E), and

CE

effect of CDP100 concentration (F). Briefly, CPD100 was added to the pre-made empty liposomes, A23187, and EDTA (Fig.2) and the mixture was pre-heated for different

AC

periods of time at varying temperatures in presence of various concentrations of A23187, and EDTA. After incubation, the drug-loaded liposome mixture was cooled on ice for 15 min. Un-encapsulated drug, EDTA, and A23187 were removed from the liposome by purification using Sephadex PD-10 columns. The drug loaded liposomes were stored at 4 °C until further characterization or use.

MA

NU

SC RI P

T

ACCEPTED MANUSCRIPT

Figure 2. Schematic of CPD100 active loading into SPM/Chol vesicle

ED

(A) Effect of internal solution pH on the drug loading of CPD100 in SPM/Chol empty

PT

liposomes

The empty liposomes were prepared as described above, and the internal pH was varied.

CE

The effect of 300 mM MgSO4 internal solution at pHs 4.0, 5.0, 6.0, 7.5, and 9.0 on the loading of CPD100 was evaluated by RP-HPLC. CPD100 was loaded at a starting

AC

concentration of 8 mg/mL with an incubation time of 60 min at 60 °C in the presence of 1 µg/mg of lipid concentration of A23187 and 33 mM EDTA. (B) Effect of incubation time on the drug loading of CPD100 in SPM/Chol empty liposomes Empty liposomes were prepared as presented above at the optimized internal pH (A). CPD100 was actively loaded at 8 mg/mL with a 60 °C incubation temperature in the presence of 1µg/mg of lipid concentration of A23187 and 33 mM EDTA. To evaluate

ACCEPTED MANUSCRIPT the effect of incubation time on the overall drug encapsulation, aliquots from the active drug loading mix were withdrawn from the drug-liposome mixture at the following time points (0, 15, 30, 60, 90 min) and placed in spin columns G- 25 (GE Healthcare, Buckinghamshire, UK) at 2000 rpm for 2 min. The eluate at each time point was quantified for amount of CPD100 using RP-HPLC.

T

(C) Effect of incubation temperature on the drug loading of CPD100 in SPM/Chol

SC RI P

empty liposomes

Empty liposomes were prepared as discussed above at the optimized internal pH (A). CPD100 was actively loaded at 8 mg/mL with an incubation time optimized in section

NU

(B) in the presence of A23187 at a concentration of 1µg/mg of lipid and 33 mM EDTA. The effect of three different temperatures (25, 45, and 65 °C) on the drug loading was

MA

evaluated. Aliquots from the drug-liposome mix were withdrawn at each temperature for three replicates and applied to G- 25 spin columns. The eluents collected at each

ED

time point were then analyzed by RP-HPLC.

PT

(D) Effect of A23187 concentration on CPD100 loading in SPM/Chol empty liposomes Empty liposomes were prepared as discussed above at the optimized internal pH (A).

CE

To assess the effect of A23187 concentration on the overall uptake of CPD100, the

AC

active drug loading process was carried out at the optimized internal pH (A), incubation time (B), and incubation temperature (C), under different concentrations of A23187 (0.0, 0.5, 1.0, 2.0 μg/mg of lipid) in the presence of 33 mM EDTA. At the end of the incubation period, the liposomes loaded under different concentrations of A23187 were analyzed using RP-HPLC. (E) Effect of EDTA concentration on CPD100 loading in SPM/Chol empty liposomes

ACCEPTED MANUSCRIPT Empty liposomes were prepared as discussed above at the optimized internal pH (A). A23187 mediated CPD100 drug loading was dependent on the concentration of EDTA for scavenging the Mg+2 for optimal uptake of CPD100 in the lipid vesicle. To evaluate the effect of the EDTA concentration, the drug loading process was carried out at the optimized internal pH (A), incubation time (B), incubation temperature (C), and

T

optimized concentration of A23187 (D) in the presence of varying concentrations of

SC RI P

EDTA (0, 10, 15, 33 mM). At the end of the incubation period, the CPD100 content in liposomes loaded under different concentrations of EDTA was analyzed using RPHPLC.

NU

(F) Effect of starting concentration of CPD100 (C0) on CPD100 loading in SPM/Chol empty liposomes

MA

Empty liposomes were prepared as discussed above at the optimized internal pH (A), incubation time (B), incubation temperature (C), optimized concentration of A23187

ED

(D), and optimized EDTA concentration (E). The effect of six different initial

PT

concentrations C0 (1, 3, 5, 7, 8 10, 15 mg/mL) on the drug loading was evaluated. CPD100 was actively loaded at the different concentrations in the presence of A23187

CE

at a concentration of 1µg/mg of lipid and 33 mM EDTA for 60 min at 60 °C. Aliquots

AC

from the drug-liposome mix were withdrawn at each temperature for three replicates and placed in G- 25 spin columns. The eluent collected at each time point was then analyzed by RP-HPLC. The data for all liposome optimization work is reported as mean CPD100 uptake ± SD for three replicates. Characterization of CPD100 loaded liposomes Size assessment

ACCEPTED MANUSCRIPT Size was assessed using both qNano (Izon Science US Ltd, Boston, MA) and Zetasizer Nano ZS (Malvern, UK; Laser 4 mW He-Ne, 633 nm, Laser attenuator Automatic, transmission 100- 0.003%, Detector Avalanche photodiode at 633nm) at 25 °C. Polydispersity Index (PDI) was also measured using Zetasizer Nano ZS. Size and lamellarity are confirmed using Cryo-TEM.

T

Particle size distribution of the samples using the qNano was determined at 25 °C by

SC RI P

tuneable resistive pulse sensing (TRPS) analysis. Samples diluted 1:20 in phosphate buffered saline had a measured final concentration of approximately 2 × 109 particles/mL. Each formulation was diluted three times, independently, to account for

NU

dilution error. For all qNano size experiments, NP150 was used for samples with a mean particle size 210 nm, and for others, NP100 was used for calibration of the

MA

pressure, pore stretch, baseline current, and blockade height. Measurements were taken at 46 mm of applied stretch, and pressure was applied to the top fluid cell using the

ED

IZON Science variable pressure module. A volume of 40 μL of the liposomal suspension was added to the top fluid cell. Each experiment was measured for 10

PT

minutes or until a minimum of 500 particles were assessed, whichever came first. The

CE

% particle concentration versus size data was collected and exported for analysis using IZON proprietary software V3.3. The data is reported as % particles/mL (concentration)

AC

versus size (nm) for each sample. Particle size and PDI measurements were performed on a Zetasizer Nano ZS at 25°C. Size and PDI measurements were made by diluting the liposomes 100x in millipore water. The Zetasizer incorporates non-invasive backscatter optics for sizing measurements at a 173° detection angle for size and PDI measurement. All data points consist of the mean of at least three determinations carried out per formulation.

ACCEPTED MANUSCRIPT To determine lamellarity, 3 µL of the CPD100Li was pipetted onto a Ted Pella 01824 Ultrathin Carbon Film on Lacey Carbon Support Film, 400 mesh, copper, and vitrified on liquid ethane using a Mark IV Vitrobot. The conditions utilized are 100% humidity, blot force 0, and blotting times between 2-4 seconds. Low-dose conditions were used to acquire images on a FEI Krios-Titan equipped with a Falcon II direct electron detector

T

(FEI, Hillsboro, OR), operating at 120 keV equipped with an FEI Eagle 4k x 4k CCD

SC RI P

camera (FEI). Cryo-TEM images were collected with a defocus range of 2-4 µm. The bolting times were 2 secs and 3 secs for empty liposomes and CPD100Li, respectively. The vesicle diameter was calculated from the images using ImageJ. The data is reported

NU

as particle size (nm) ± SD. Stability of CPD100 in liposomes

MA

The freshly prepared optimized liposomal formulation with and without ionophore was evaluated for stability at 4 oC by monitoring mean particle diameter (DLS, qNano,

ED

Cryo-TEM) and drug loading at 0, 24, and 48 h by RP-HPLC. All measurements were done in triplicate, and data is presented as Mean CPD100 concentration (conc.) ± SD for

CE

PT

loading.

Freeze-drying and physical stability of CPD100Li

AC

Freshly prepared CPD100Li dispersions with ionophore were freeze dried in the presence of 5% sucrose as a cryoprotectant. For this study, sucrose was added to the pH 4.0 MgSO4 internal buffer and the pH 7.4 SHE external buffer during the preparation of the empty liposomes to achieve a 5% final sucrose concentration. These liposomes were then loaded with CPD100. The purified liposomes were loaded into Wheaton® Vacule® 10 mL vials at 1 mL/vial. The vials were loaded into a shelf freeze-dryer, FreeZone® Triad™ Freeze Dry System, Labconco, (Kansas City, MO). The freeze-drying cycle consisted of the following: a Prefreeze Segment, where vials were frozen at -80 °C for 6

ACCEPTED MANUSCRIPT h; Segment 1, with primary drying, a ramp of 4 °C/min, and a hold at -55 °C for 24 h; Segment 2, with secondary drying, a ramp of 5 °C/min, and a hold at - 5 °C for 12 h; Segment 3, with holding, a ramp of 0.01 °C/min, and a hold at 0 °C for 5 h; and a last step with a temperature ramp of 0.01 °C/min and hold at 4 °C. At the end of the cycle, the vials were filled with argon or air and stoppered for storage. The samples were

T

rehydrated after freeze drying and assessed for CPD100 loading using RP-HPLC and

SC RI P

size and PDI using DLS.

For short-term stability, CPD100Li freeze dried samples with ionophore were reconstituted every week for 4 weeks in DI water and optimized for storage temperature

NU

and gas content. Three different storage and temperature conditions were evaluated: a) samples stored with argon in the head space and maintained at 4 °C, b) samples stored

MA

with argon in the head space and maintained at 25 °C, and c) samples stored with ambient air and maintained at 4 °C. Every week, samples were reconstituted with DI

ED

water and assessed for size and PDI by DLS and CPD100 loading by RP-HPLC. The water content was measured using a coulometric C20 Karl Fischer titration apparatus

PT

(Mettler Toledo, Columbus, OH).

CE

A long-term stability study for a period of 3 months and 15 months was carried out for

AC

freeze dried CPD100 with ionophore at the optimised storage gas and temperature conditions. The amount of CPD100 was determined using RP-HPLC, size and PDI were measured using the DLS, and the water content was measured using a coulometric C20 Karl Fischer titration apparatus (Mettler Toledo, Columbus, OH). For all studies mentioned above, the rehydration process was completed within 5 min by repeated vortexing. The data is presented as % CPD100 retained ± SD using RPHPLC, mean particle diameter (nm) ± SD and PDI ± SD using DLS, and % increase in

ACCEPTED MANUSCRIPT water content using a C20 Karl Fischer titration apparatus (Mettler Toledo, Columbus, OH). Drug release study Drug release of CPD100 from the optimized liposomes with and without ionophore was assessed by dialysis. 2.5 mL of each liposomal formulation was loaded into a 3 mL

T

Slide –A-Lyzer (Thermo Scientific Inc.) dialysis cassette with a MWCO of 20,000

SC RI P

g/mol. Four cassettes were used in each study. The cassettes were placed in 2.0 L of PBS buffer. To ensure sink conditions, the buffer was changed every 3 h. A sample of 50 μL is withdrawn at 0, 1, 3, 6, 24, and 48 h from each cassette and replaced with 50

NU

μL of fresh buffer. The samples were diluted 20x in methanol and injected into the RPHPLC system. Data from four replicates is presented as mean percent (%) drug release

MA

± SD. The data was further analyzed by curve fitting into a one phase exponential

ED

association function using GraphPad Prism version 6.07 for Windows.

In-vitro assessment of cell proliferation under hypoxic and normoxic conditions

PT

in ovarian cancer cells

CE

In-vitro cell viability assay

To assess the cell viability of CPD100 and CPD100Li as a function of oxygen levels,

AC

ES2 cells at a cell density of 3000 cells/well were cultured using RPMI culture medium supplemented with 10% FBS and 1% penicillin/streptomycin in a 96-well culture plate. The cells were grown at 37 °C in a humidified atmosphere of 5% CO2 (v/v) in air and allowed to attach for 3 h. Post attachment, the cells were treated with 10 mM phosphate buffer (control), empty liposomes at 12.5 mM (vehicle control), CPD100 (0.02 – 100 μM) in DMSO and CPD100Li (0.02 – 100 μM) for a total of 72 h. The treatment was studied at two levels of oxygen concentrations - normoxic 20% and hypoxic 1.5% (v/v). After 18 hours pre-incubation at either oxygen level, the plates were incubated for the

ACCEPTED MANUSCRIPT remaining 54 h under 20% O2. Cell viability was assessed using a CellTiter Blue® assay per manufacturer’s instructions. Briefly, 20 µL of the reagent was added to each well, and cells were incubated for 2 h at 37 °C. The fluorescence intensity was measured at 560Ex/590Em. Half-maximal inhibitory concentration (IC50) was determined with non-linear regression

T

analysis using GraphPad Prism version 6.05 for Windows, GraphPad Software (La Jolla

SC RI P

California, USA). All experiments were performed in quadruplicate, and data is presented as mean IC50 ± SD. The IC50 values for all groups was compared by one-way ANOVA with Dunnett’s Multiple Comparison post-test at a p-value of 0.05 using

calculated using the following equation:

NU

GraphPad Prism version 6.05 for Windows. The cytotoxicity ratios (HCR), RHypoxia was

Live cell imaging study

ED

MA

𝑅𝐻𝑦𝑝𝑜𝑥𝑖𝑎 = 𝐷𝑟𝑢𝑔 𝐼𝐶50 ( ℎ𝑦𝑝𝑜𝑥𝑖𝑐 𝑐𝑜𝑛𝑑𝑖𝑡𝑖𝑜𝑛)⁄ 𝐷𝑟𝑢𝑔𝐼𝐶50 (𝑛𝑜𝑟𝑚𝑜𝑥𝑖𝑐 𝑐𝑜𝑛𝑑𝑖𝑡𝑖𝑜𝑛) (1)[26]

Time-lapse experiments for the cytotoxic effect of the CPD100Li were performed using

PT

96-well plates to visualize the anti-proliferative effect of CPD100 and CPD100Li in

CE

ES2 cells. Each well was seeded with ES2 cells to a final density of 3000 cells/well. The assay was carried out for a total of 48 h with images collected every 20 min using

AC

Cytation 5 live cell imaging (BioTek, Winooski, VT). In all cases, cell cultures were kept at 37 °C and 5% CO2 for 3 h to permit cell attachment. Post-attachment, the cells were treated in triplicate with CPD100 or CPD100Li (20000, 2000, 200, 20, 2, 0.2 nM). Cells were stored under normoxic or hypoxic oxygen conditions at 1.5% (v/v) O2 for 18 h, then incubated for the remaining 54 h under 20% O2. Data is presented as representative images from a single well at 8, 16, 24, and 48 h for each treatment.

ACCEPTED MANUSCRIPT Real time cellular proliferation study The cellular proliferation of ES2 cells was measured using the xCELLigence real time cell analysis (RTCA) proliferation platform (ACEA Biosciences, San Diego, CA) to determine if the mechanism of the action of CPD100 and CPD100Li is different compared to vinblastine. Prior to the assay, the E-plates were coated with 50μL of

T

fibronectin (10 μg/mL) (Sigma-Aldrich), per the manufacturer’s protocol, and left in the

SC RI P

biosafety cabinet for 30 min. Briefly, the plates were aspirated and 100 μL of cell free medium was added to each well, followed by a 30 min equilibration period in the biosafety cabinet. The plates were then loaded onto the instrument, and background

NU

measurements were recorded using the RTCA Software 1.2. The kinetic study was started after cells were seeded in each plate at a cellular density of 3000 cells/well and

MA

placed into the incubator for a 3 h attachment time. Cells were then treated with 1 μL of vinblastine (3000, 300, 30, 3, 0.3 nM) along with CPD100 and CPD100Li (40000,

ED

20000, 2000, 200, 20 nM) with concentrations above, below, and at drug IC50. Posttreatment, the plates were incubated at 1.5% (v/v) oxygen level for 18 h, then in 20% O2

PT

for the remaining 54 h. At the end of 72 h the plates were analyzed using RTCA

CE

Software 1.2. Data is presented as cell index (CI) ± SD for three replicates.

AC

Clonogenic assay

The in vitro cell survival capacity after treatment was studied using the clonogenic assay. ES2 cells were plated at a density of 500 cells/well in a 6 well cell culture plate. The cells were incubated for 3 h before treatment with CPD100 (10 – 1000 nM) or CPD100Li (7– 700 nM) for a period of 18 h under 20% or 1.5% of oxygen, respectively, at 37 °C. Based on the previous study, the concentration for the treatment of CPD100 and CPD100Li was determined for achieving concentrations below the IC50 for this study. At the end of 18 h, the plates were transferred to 20% oxygen at 37 °C in

ACCEPTED MANUSCRIPT a humidified atmosphere in 5% CO2 (v/v) for a period of 10 days. After completing the incubation period, the clonogenic assay was performed following a previously published method[27]. Briefly, the media was removed, and the cells were rinsed with 10 mL of PBS. After the rinsing, the PBS was removed and 2 mL of colony fixation solution consisting of acetic acid/methanol mixture (1:7 (v/v)) was added. At the end of

T

5 min, the solution was removed and 2 mL of 0.5% crystal violet was added and

SC RI P

incubated for 2 h at room temperature. Post incubation, the plates were immersed in tap water to rinse off the crystal violet and left to air dry at room temperature for a few days. The colonies were scored and counted using an inverted microscope and ImageJ.

NU

Data is presented as survival fraction (SF) ± SD for three replicates. Survival fraction was calculated by dividing the number of colonies formed after treatment by the number

MA

of cells seeded.

In vitro experiments to determine the mechanism of action of CPD100 and

PT

Effect on cell cycle arrest

ED

CPD100Li in ovarian cancer cells

The effect of CPD100 and CPD100Li on the various phases of cell cycle was studied

CE

using Vybrant® Dye Cycle™ Green stain. ES2 cells were seeded at a density of 1 x 105

AC

cell/well in a 6-well cell culture plate and allowed to attach for 3 h. Upon attachment, the cells were treated with CPD100 or CPD100Li at the previously calculated IC50 concentrations under hypoxic conditions. Similar to the cell viability assay above, plates were incubated under hypoxic conditions (1.5% O2 level) for 18 h and returned to normoxic conditions for the remainder of the study. Effect on cell cycle was performed at 18 h (end of hypoxic incubation period), 48 h, and 72 h. At the end of each incubation period, the medium was removed, and the cells were trypsinized, collected in 2 mL Eppendorf tubes, and washed 3 times with DPBS. After the cells were washed, Vybrant

ACCEPTED MANUSCRIPT Dye Cycle Green was added to each tube according to the manufacturer’s protocol, followed by incubation for 1 h at 37 °C in 5% CO2 before analysis with the BD Accuri C6 flow cytometer (San Jose, CA). All data was analyzed using BD C6 Accuri software. The results are presented as mean % ES2 cells in each phase of cell cycle ±

T

SD for three replicates.

SC RI P

Apoptosis Assay

The caspase-3/7 assay was performed to quantify the caspase activity in the ES2 cell line after treatment with CPD100 and CPD100Li using the CellEvent® Caspase 3/7 Green Detection reagent assay kit (Thermo Fisher, Brookfield, WI). The cells were

NU

seeded at 3000 cells/well in a black 96-well plate (Corning, Corning, NY). CPD100 and

MA

CPD100Li at concentrations below and around IC50 (20, 200, 2000, 20,000 nM) were used to treat ES2 cells at 1.5% and 20% oxygen levels. At the end of a 72 h incubation,

ED

the media was removed from the cells, and cells were treated with 5 µM Cell Event Caspase 3/7 Green Detection Reagent for 30 min at 37 °C. At the end of 30 min, the

PT

fluorescence was measured using a multiwall plate reader (Synergy HT, BioTek Instruments, Winooski, VT) with absorption/emission maxima at 502EX/530Em nm,

CE

respectively. A one-way ANOVA combined with Dunnett’s Multiple Comparison Test

AC

at 95 % significance level was performed, and the data is presented as Caspase 3/7 % activity ± SD for four replicates. Pharmacokinetic Study Female Swiss Webster mice were randomly divided and treated with CPD100Li (A23187) at 30 mg/kg (IV tail injection). Terminal blood samples (0.7 mL) were collected via cardiac puncture at 0, 0.25, 1, 2, 4, 8, and 24 h post dosing. The blood was immediately transferred to chilled NaF/EDTA blood tubes, inverted, mixed, and maintained on wet ice. An anticoagulated whole blood aliquot (0.25 mL) was then

ACCEPTED MANUSCRIPT added to a chilled cryovial containing 1 mL of cold 8.5% phosphoric acid and stored at 80 ± 5 °C until shipped for analysis. All bio-analytical analysis was performed by MicroConstants Inc. (San Diego, CA). The pharmacokinetics of CPD100Li (-A23187) in plasma was analyzed by non-compartmental analysis using Phoenix 64 (Certara, Princeton, NJ). The area under the concentration versus time curve from 0 to ∞ (AUC0-

T

∞), plasma volume of distribution (Vd), clearance (CL) and half-life (t1/2) were

SC RI P

calculated. Plasma sample preparation

Briefly, protein precipitation of the blood samples was done using water: methanol

NU

containing 5% zinc sulphate. The samples were vortexed then centrifuged for 10 min, followed by solid phase extraction of the supernatant[16]. The calibration standard

MA

samples were prepared by serially diluting a stock of 20 μL of CPD100 into 100 μL of blank plasma with the working range at 25-25000 ng/mL. All samples were analyzed by

ED

LC-MS/MS. LC-MS/MS analysis

PT

An Agilent HPLC 1100 Series (Santa Clara, CA) coupled to AB Sciex API 4000

CE

(Framingham, MA), tandem quadrupole mass spectrometer controlled by Xcalibur software was used for plasma analysis (Thermo Fischer Scientific, San Jose, CA). The

AC

column used was a Waters, XSELECT CSH phenyl-hexyl (100 x 2.1-mm, 5 μm) analytical column with a mobile phase of 0.1% formic acid in water and 0.1% formic acid in methanol. The mass spectrometer was operated in a source/interface set to Turbo Spray positive ionization mode using heated nitrogen.

In vivo acute toxicity studies All animal work was performed in compliance with NCI guidelines, Oregon State University, and Oregon Health & Science University IACUC Policy for End-Stage

ACCEPTED MANUSCRIPT Illness and Pre-emptive Euthanasia, based on Humane Endpoints Guidelines. Nude mice were used to test CPD100 and CPD100Li mediated acute toxicity. Previous work[16] with CPD100 and CPD100Li demonstrated a dose limiting toxicity (DLT) of 45 mg/kg for both formulations in Swiss Webster mice. Using this as the starting point, a dose seeking study was initiated by injecting one nude mouse/formulation at 45mg/kg.

T

Dose escalation or de-escalation of CPD100 and CPD100Li was performed until a

SC RI P

lethal dose was identified. Lethal dose was defined as the dose which results in a weight loss >20% or morbidity. The Maximum Tolerated Dose (MTD) was defined as the highest dose below the toxic dose that does not result in acute toxicity associated with

NU

the DLT. After identifying the lethal dose, a multi-dose acute toxicity study was initiated by exposing mice to CPD100 or CPD100Li via multiple tail vein injections

MA

(every three days for 5 injections (n =15; 5 animals/group). Groups for the multi-dose study were CPD100, CPD100Li, and empty liposome (lipid dose = 50 mg/kg). The

ED

animals were monitored for weight loss and other signs of behavioral and physical toxicity for a period of 14 days after the last dose. Data is presented as mean percent

CE

PT

(%) normalized body weight ± SD.

Results and Discussion

AC

Effect of different parameters in optimizing CPD100 loading in SPM/Chol liposomal platform

Optimization of parameters for active drug loading process Based on the hydrophilicity of CPD100, active drug loading was evaluated as a method for loading. This process involved a number of variables which affect drug loading into the lipid vesicle. The parameters kept constant were a 12.5 mM final lipid concentration, 300 mM MgSO4 internal solution, SHE pH 7.4 external solution, and

ACCEPTED MANUSCRIPT A23187 ionophore (Fig.2).

(A)Effect of internal solution pH on the drug loading of CPD100 in SPM/Chol empty liposomes To optimize CPD100 loading into SPM/Chol liposomes, the internal pH was varied while the external pH was maintained at 7.4 to mimic physiological conditions. The

SC RI P

T

extent of CPD100 encapsulation in SPM/Chol liposomes as a function of five different internal solutions is displayed in Fig.3A. At pH 4.0, the highest encapsulation of 4.0 mg/mL was achieved compared to 2.4 mg/mL at pH 6.0, 2.0 mg/mL at pH 7.5, and 1.76 mg/mL at pH 9.0. The efficiency of CPD100 entrapment within the liposome decreased

NU

with increasing internal pH. Previous work has indicated that the optimal pH of the internal solution or the optimal pH gradient across the bilayer is dependent on the drug

MA

structure and the ionizability of the molecule [23, 28, 29]. CPD100, a weakly basic water-soluble drug with a pKa of 4.9 ± 0.4 (ACD Lab/Percepta, module ACD/pKa

ED

GALAS), is retained within the lipid vesicle at lower pH values, as it exists as a charged

PT

molecule.

The results are in accordance with published work which indicates that the lower the

CE

internal pH, the higher the encapsulation of a weakly basic drug along with a lower

AC

efflux rate of the drug from the liposome [30, 31]. Other studies show that the internal and external pH gradient, which is created due to the weak acid-base buffer system, is dependent on the external buffer. However, literature suggests that the external SHE buffer at pH 7.4 is optimal for A21387–mediated transport of Mg+2[32]; thus, we did not alter the pH gradient by changing the external pH. Our findings are in agreement with the Fenske et al. theory, which states that a pH gradient established according to the pKa of the drug is essential for superior drug uptake and retention into the liposome [22, 33]. Thus, the pH gradient across the lipid bilayer plays a significant role in the

ACCEPTED MANUSCRIPT drug accumulation and needs to be optimized based on the physicochemical properties of each molecule being studied for drug loading. (B)Effect of incubation time on the CPD100 loading in SPM/Chol empty liposomes Fig.3B indicates that CPD100 loading is a time dependent process with a much faster uptake at incubation times less than 30 min. At 30 min, the CPD100 loading was 4.4

T

mg/mL. The loading ranged from 4.4 to 4.8 mg/mL for incubation times of 60 to 120

SC RI P

min. Therefore, we established an optimum incubation time of 60 min for the remainder of the study. Thermodynamic equilibrium for molecules partitioning between two phases is a time dependent phenomenon. Once equilibrium is reached, no changes in

NU

concentration are seen[34]. Thus, in the case of CPD100, the threshold time needed to achieve maximal partitioning is 30 min, and additional time does not have significant

MA

impact on the amount of drug loaded. However, to ensure full and reproducible loading, the incubation time utilized for the study is 60 min.

ED

(C)Effect of incubation temperature on the CPD100 loading in SPM/Chol empty liposomes

PT

The effect of three different temperatures (25, 45 and 65 °C) on the drug loading show

CE

that CPD100 uptake is temperature dependent (Fig.3C). The temperature effect study was capped at 65 °C, as most lipids degrade above this temperature[35]. The maximum

AC

accumulation of 5.0 mg/mL (62 %) occurred at 65 °C, with 3.6 mg/mL (55%) at 45 °C and 2.2 mg/mL (28%) at 25 °C (Fig.3C). This result is anticipated, as published work has shown that the phase transition temperature of SPM is 55 oC [36]. Below the phase transition temperature, SPM remains coupled with Chol; thus, heating the mixture above that temperature will result in fluidization of the membrane, increasing CPD100 flux, and thereby enhancing CPD100 loading[36]. Therefore, to achieve effective drug

ACCEPTED MANUSCRIPT loading, the incubation temperature for the mixture must be above the phase transition temperature of the lipid, but below its degradation temperature. (D) Effect of A23187 concentration on CPD100 loading in SPM/Chol empty liposomes The effect of A23187 concentration on CPD100 loading is presented in Fig.3D. The absence of A23187 resulted in 3.12mg/mL (39%) CPD100 loading in the liposome.

T

CPD100 concentrations of 3.7 mg/mL, 3.8 mg/mL and 3.3 mg/mL were seen in the

SC RI P

presence of 0.5 µg/mg, 1.0 µg/mg, and 2.0 µg/mg of A23187/lipid, respectively. A23187 swaps divalent Mg2+ ions inside the lipid vesicle for external protons (H+). This creates an electrochemical gradient, resulting in an acidic interior of the lipid vesicle

NU

(Fig.2). The activity of A23187 is specific to divalent metal cations with specificity in the following order: Mn2+ >Ca2+ >Mg2+[37]. Based on our results, the presence/absence

MA

of ionophore with 33 mM EDTA does not affect drug loading in this case. However, literature suggests that incorporation of drug with an ionophore mediated active loading

ED

process leads to liposomes with higher stability in vivo [29]. Thus, for our optimized formulation, we chose 1 µg of A23187/mg of lipid to achieve the necessary loading of

PT

CPD100.

CE

(E) Effect of EDTA concentration on CPD100 loading in SPM/Chol empty liposomes Effect of EDTA concentration on CPD100 loading is presented in Fig.3E. A loading of

AC

0.6 mg/mL (7.5%) at 0 mM EDTA, 1.1 mg/mL (13.5%) at 4.2 mM EDTA, 2.2 mg/mL (27.5%) at 8.3 mM EDTA, 3.76 mg/mL (46.9%) at 16.7 mM EDTA, and 4.8 mg/mL (60%) at 33.3 mM EDTA was observed. Increasing the concentration of EDTA demonstrated a proportionate increase in drug uptake. The optimum concentration of EDTA in the mixture for maximum drug loading is 33 mM EDTA. Similar to A23187, in the absence of EDTA, only 10% of the drug accumulated in the liposome after 1h of incubation. EDTA acts as a scavenger of Mg+2, which ensures that the ion exchange

ACCEPTED MANUSCRIPT continues to retain the pH gradient (Fig.2). This results in optimum drug encapsulation[38]. EDTA plays a crucial role in the encapsulation of CPD100 in the lipid vesicle. (F)Effect of starting concentration of CPD100 (C0) on CPD100 loading in SPM/Chol empty liposomes

T

The effect of starting concentration of CPD100 on the % loading into the lipid vesicles

SC RI P

is shown in Fig.3F. At a C0 of 1 mg/mL, 100% encapsulation was achieved. At a C0 of 3 mg/mL, 5 mg/mL, 7 mg/mL, 8 mg/mL, 10 mg/mL, and 15 mg/mL, the encapsulations were 1.83 mg/mL (61%), 2.55 mg/mL (51%), 4.7 mg/mL (67%), 5.1 mg/mL (64%), 4.5

NU

mg/mL (45%), and 5.4 mg/mL (36%), respectively.

The % loading increased with increasing concentration until a C0 of 7 mg/mL. Further

MA

increase in C0 showed a decrease in % CPD100 uptake. Small unilamellar vesicles have lower entrapment volume, and saturation of the vesicle can lead to a decrease in %

ED

CPD100 loading[39].

Therefore, based on the above data, the optimal loading conditions for CPD100 in the

PT

SPM/Cholesterol (55/45) liposome is with ionophore (CPD100Li(+)), contains a total

CE

lipid concentration of 12.5 mM (7.5 mg/mL), has an internal solution of 300 mM MgSO4 at pH 4, and has SHE buffer at pH 7.4 as the external solution. For drug

AC

loading, optimal parameters include CPD100 at 7 mg/mL, 33 mM EDTA, and 1 µg of A23187/mg of lipid. These conditions resulted in 67% encapsulation efficiency for CPD100 in liposomes. A second optimized formulation of CPD100 without ionophore (CPD100Li(-)) was also prepared for future in vitro and in vivo characterization to determine if the presence of the ionophore influences the stability of CPD100.

ACCEPTED MANUSCRIPT Our results are consistent with published results that show the incorporation of hydrophilic weakly basic molecules into saturated neutral lipids requires lower internal

AC

CE

PT

ED

MA

NU

SC RI P

T

pH in the presence of ionophore and EDTA[40].

Figure 3. Effect of various parameters on CPD100 loading in SPM/Chol vesicle. Internal pH (A), incubation time (B), incubation temperature (C), EDTA concentration (D), A23187 concentration (E), CPD100 C0 (F). All data is presented as mean % CPD100 ± SD (n=3).

ACCEPTED MANUSCRIPT

Characterization of CPD100 loaded liposomes Size assessment Table 1: Size characterization of Fresh liposomes with A23187 (+) and without A23187 (-).

qNano DLS Mean Particle Size Zave ± SD

PDI ± SD

T

Cryo-TEM Mean Particle diameter ± SD Fresh (+) 64.3 ± 10.9 Fresh (-) 69.5 ± 22.1 The optimized CPD100Li(+) had

SC RI P

Size (nm) Instrument Sample

132 155.4 ± 4.2 0.1 ± 0.02 131 170.3 ± 20.8 0.18 ± 0.02 a mean particle size of 132 nm (TRPS) and Zave size

of 155 ± 4.15 nm (DLS) (Table 1) and a drug loading of 5.5 mg/mL. Cryo-TEM of the

NU

CPD100Li showed unilamellar vesicles with a size of 64 ± 10.87 nm along with the

MA

presence of some multilamellar vesicles (Fig.4). The CPD100Li (-) had a mean particle size of 131 nm (TRPS) and Zave size of 170 ± 20.8 nm (DLS) (Table 1) and a drug

ED

loading of 5.0 mg/mL. Cryo-TEM of the CPD100Li (-) showed unilamellar vesicles with a size of 70 ± 22.09 nm along with the presence of some multilamellar vesicles

PT

(Fig.4). In addition, the cryo images showed an absence of drug precipitation or electron dense regions in the CPD100Li which was observed for other liposomal formulations,

CE

such as in CPT-11 and doxorubicin[29, 33, 38]. Unlike DLS, TRPS analysis assesses

AC

individual particle population and concentration of the samples. The total concentration of fresh CPD100Li (+) and CPD100Li (-) was 5.88 x 1010 and 8.10 x

10

particles/mL,

respectively. The % population was about 9.0% with a mean diameter of 132 nm for fresh CPD100Li with A23187 (+) and 131 nm for CPD100Li without A23187 (-) (Fig.5A, 5B). Both formulations have less than 5 % of the vesicle population with a size > 200 nm. The size assessment of the CPD100Li is in accordance with liposomes prepared through the extrusion method[16, 41]. The optimum size for passive targeting through the EPR

ACCEPTED MANUSCRIPT effect is between 50-200 nm. The elimination rate of the liposomes from the circulation is very sensitive to the size of the lipid vesicle[18]. Particles below 10 nm will undergo rapid clearance by the kidneys, while particles larger than 200 nm will acquire a higher degree of opsonin coating, resulting in enhanced uptake via liver phagocytosis. Studies indicate that a size range between 10-200 nm is better suited to minimize uptake by the

T

reticuloendothelial system [42, 43]. Liposomes cause inadvertent activation of

SC RI P

complement system in human subjects[44-47]. Minor differences in liposome size can stimulate complement activation, which can cause cardio-pulmonary distress. Thus, a great emphasis exists on detecting size variations in the population, and complete size

NU

characterization is imperative for regulatory purposes. The capability of TRPS analysis to detect size range variation along with % population and concentration is a beneficial

AC

CE

PT

ED

MA

tool for lipid formulations, as it is independent of the dilution factor.

Figure 4: Cryo-TEM (A) CPD100Li (+), (B) CPD100Li (-)

T

ACCEPTED MANUSCRIPT

SC RI P

Figure 5 : Mean particle size vs % population and particle concentration for (A) CPD100Li (+) and (B) CPD100Li (-). Peaks in panels A and B are labeled with Population %, particles mg/mL

Stability of CPD100 in liposomes

The CPD100Li with and without ionophore was stable both in terms of particle size and

NU

drug retention in the liposomes for a period of 48 h. CPD100 loading in the presence of

MA

ionophore was 5.5 mg/mL and without ionophore was 5.0 mg/mL. [16]. The stability data shows that the presence or absence of ionophore has no effect on the

ED

retention of CPD100 in the lipid vesicles. The presence of ionophore has been shown to increase stability in vivo [29]; however, we failed to observe any effect on the in vitro

PT

stability.

CE

Freeze-drying and physical stability of CPD100Li

AC

The size and PDI of the CPD100Li freeze-dried formulation with 5% sucrose was 141.5 ± 2.9 nm and 0.09 ± 0.002, respectively, compared to a solution of CPD100Li with a size of 155.4 ± 4.2 nm and PDI of 0.1 ± 0.002 (Table 2). There was no change in the concentration of CPD100 before and after freeze drying (Table 2). Table 2: Size characterization of fresh and lyophilized liposomes with A23187 (+)

Sample

Zave ± SD (DLS) nm PDI ± SD (DLS)

Solution (+) Lyophilized (+)

155.4 ± 4.2 141.5 ± 2.9

0.1 ± 0.02 0.09 ± 0.002

CPD100 mg/mL 5.3 5.2

conc.

ACCEPTED MANUSCRIPT

Short-term stability data for storage temperature and gas content optimization is presented in Fig. 6. The data includes % CPD100 retention, size (nm), PDI, and % increase in water content. The % CPD100 retention at the end of 28 days was 93.1 ± 1.5

T

%, 88.7 ± 1.2 %, and 87.5 ± 1.31 % for CPD100Li stored at 4 °C argon, 4 °C air, and 25

SC RI P

°C argon, respectively. The size and PDI at the end of 28 days was 138.2 ± 5.3 nm (PDI: 0.01 ± 0.065), 144.6 ± 15.1 nm (PDI: 0.08 ± 0.2), and 153.6 ± 24.5 nm (PDI: 0.2 ± 0.082) for CPD100Li stored at 4 °C argon, 4 °C air, and 25 °C argon, respectively. The increase in % water content at the end of 28 days was 1 %, 21 %, and 107 % for 4

AC

CE

PT

ED

MA

NU

°C argon, 4 °C air, and 25 °C argon, respectively.

ACCEPTED MANUSCRIPT Figure 6: Stability study of freeze dried liposomes containing 5% sucrose at three different conditions- 4 °C argon, 4°C air, and 25°C argon for 28 days. (A, B, C) % CPD100 retained; (D, E, F) size (DLS); (G, H, I) PDI (DLS); (J, K, L) % increase in water content (Karl Fischer).

The % CPD100 retention, size (nm), PDI, and % increase in water content for the longterm stability data at the optimized storage temperature and gas content is shown in Fig.7. The % CPD100 retention, size, and PDI at the end of 3 months was 98.8 % ±

SC RI P

T

0.83, 142 ± 4.16 nm, and 0.11 ± 0.005, respectively, compared to 81.4 % ± 0.83, 149 ± 3.21 nm, and 0.15 ± 0.017 at 6 months. The increase in % water content at the end of 3 months was 15% and almost up to 400% at the end of 6 months.

The long-term stability data of freeze dried liposomes capped under argon and stored at

NU

4°C indicate that they were stable for a period for 3 months with no significant change in CPD100 retention, size, PDI, and water content. However, at the end of 15 months,

MA

the CPD100 retention decreased to 80% with a significant increase in water content; however, there was no significant change in size and PDI of the vesicle (Figure 7A, 7B,

AC

CE

PT

ED

7C, 7D).

MA

NU

SC RI P

T

ACCEPTED MANUSCRIPT

ED

Figure 7: Long term stability of CPD100 freeze dried liposomes stored under argon at 4 °C. (A) % CPD100 retained, (B) size (DLS), (C) PDI (DLS), (D) % increase in water content (Karl Fischer).

PT

Our results are in accordance with previously published work that shows presence of a cryoprotectant and storage conditions, such as the temperature and capping gas, play a

CE

crucial role in the chemical stability of the lipids[48]. Lipids have a phase transition

AC

temperature at which they transition from a gel phase, where the hydrocarbon chains are closely packed, to liquid crystalline phase, where the hydrocarbon chains are randomly oriented and fluid[49]. Sphingomyelin cholesterol mixture has a phase transition temperature of 55 °C[50]. The ideal shelf temperature for storage is a temperature at which the lipids exist in a non-leaky gel phase. The phase transition temperature decreases with an increase in hydration (water content)[50]. The ideal residual water content for lyophilized liposomes should be between 1-3%, to ensure increased stability[51]. A higher water content can also lead to increased oxidation in the dried

ACCEPTED MANUSCRIPT state[48]. Thus, measurement of the residual water content is critical for the stability of the lyophilized liposomes. Argon is an inert gas and therefore reduces the oxidation of lipids, a process which is catalyzed in the presence of atmospheric oxygen. The longterm stability issue, despite refrigeration and storage with argon, may be due to the increase in water content (Fig.7D), which would increase the rate of CPD100 leakage.

T

However, there is no change in size and PDI till 3 months. Beyond that time, at 15

SC RI P

months, the PDI significantly increased but still measured below 0.15, indicating a monodisperse population (Fig.7B&C). The PDI value indicates the level of dispersity in the sample. The ideal value, < 0.1, indicates a monodisperse solution, and a value > 0.7

NU

indicates a polydisperse sample which will not be suitable for DLS measurements. A lower PDI around 0.1 is ideal for nano-formulations, as increases in the value are

MA

related to the toxicity associated with complement activation[52]. The size and PDI suggest that the use of 5% sucrose as cryoprotectant can stabilize the lipid vesicle, but

ED

the leakage of CPD100 accelerated due to higher concentrations of CPD100 encapsulated in the vesicle. Numerous researchers have studied other sugars such as

PT

trehalose, mannitol, lactose etc. for their cryoprotectant effect; however, in in this study,

CPD100Li.

CE

we investigated only sucrose, as it constitutes the final purification solution for

AC

Drug release study

The release of CPD100 from the liposomes with (+) and without (-) A23187 is reported in Fig.8. The release of CPD100 from both liposomes was primarily in the first 6 h. The half-life for CPD100 released in the presence and absence of A23187 is identical, with a goodness of fit value (r2) of 0.98 and 0.96 respectively. The r2 values indicate that the release of these prodrugs from the liposomes follows a one-phase association with a calculated

half-time.

Previous

work has

attributed formulation

stability to

ACCEPTED MANUSCRIPT sphingomyelin, a saturated acyl chain resulting in a rigid lipid, which along with cholesterol, results in stable vesicles[53, 54]. Other studies have indicated that the addition of A23187 and EDTA may also contribute to the overall retention of drug

ED

MA

NU

SC RI P

T

molecules in the liposomes in vivo[55].

PT

Figure 8: Release kinetics of CPD100 from CPD100Li with (+) or without (-) A23187. Data is presented as mean ± SD of 4 replicates.

CE

In vitro assessment of cell proliferation under hypoxic and normoxic conditions in ovarian cancer cells

AC

In vitro cell viability assay To confirm that the liposomal formulation does not modify the anti-proliferative effect of CPD100, both the free and the encapsulated drug were evaluated in ovarian ES2 cells under 20% and 1.5% oxygen. The IC50 values of CPD100 and CPD100Li at 20% and 1.5% are depicted in Fig.9A. The IC50 values of CPD100 at 20% and 1.5% were 25077 ± 9.17 nM and 4063 ± 1.5 nM, respectively. The HCR ratios for CPD100 and CPD100Li were 0.16 and 0.11, respectively.

ACCEPTED MANUSCRIPT Cancer cells, especially ovarian, demonstrate a significantly higher growth rate with higher infiltration and colony and sphere formation potential when subjected to a short period of hypoxia followed by normoxia compared to cells under normal conditions [56]. Subjecting the cells to hypoxia (hours to days) leads to cellular adaptation due to decrease DNA repair, which increases genomic instability manifesting as defective

T

repair and chromosomal aberrations, resulting in decreased sensitivity to chemotherapy

SC RI P

and radiotherapy. Moreover, it also leads to activation of hypoxia-driven downstream pathways which increase resistance, and clonal selection resulting in expression of aggressive tumor phenotypes and, subsequently, increased metastasis[57, 58].

NU

Therefore, the assay was designed to subject the cells to 18 h of hypoxia followed by a return to normal conditions. There was a 10-fold lower IC50 for both CPD100 and

MA

CPD100Li in ES2 cells subjected to 1.5% oxygen for 72 h (data not shown) compared to the IC50 obtained for 18 h hypoxia/54 h re-oxygenation assay, which aligns with

ED

published work.

The incorporation of CPD100 in the liposomal formulation did not change the potency

PT

of the prodrug, indicating that incorporation of CPD100 in the liposome does not

CE

decrease its efficacy. The lower IC50 of CPD100 under hypoxic conditions compared to values under normoxic conditions clearly demonstrate higher toxicity in hypoxia,

AC

confirming the hypoxic activation of CPD100, in agreement with the previously reported study[16, 17]. However, empty liposomes showed no decrease in cell viability of ES2 cells. As a reference, according to the NCI-60 human tumor cell line screening database, the concentration of vinblastine that kills 50% of the test cells/animals with a single exposure (LC50) in ovarian cancer cell lines is 0.025 nM. The HCR ratio indicates that the liposomal entrapment of CPD100 does not affect the conversion of the prodrug to the parent compound. Interestingly, commercially available liposomal

ACCEPTED MANUSCRIPT formulations of vinca alkaloids, such as vincristine and vinorelbine, showed higher IC50 values compared to the free drug. The discrepancy in the potency may lie in the encapsulation

of

the

prodrug,

CPD100,

which

has

significantly

different

physicochemical properties relative to vinblastine, the parent compound. However, this

CE

PT

ED

MA

NU

SC RI P

T

warrants further evaluation.

AC

Figure 9: (A) IC50 values of CPD100 and CPD100Li in 20% and 1.5% O2 in ES2 cells (n=4). (B) Live cell images of the ES2 cells treated with CPD100 and CPD100Li. Real time monitoring of ES2 cell proliferation exposed to different concentrations of (C) CPD100, (D) CPD100Li, and (E) Vinblastine.

Live cell imaging study To visualize the anti-cell proliferative effect of the lipid formulation, live cell images were collected (Fig.9B). Clearly, at the end of the hypoxic period (around 18 h) in the CPD100Li-treated images, cells were rounder and lower in cell density compared to the control cells over a period up to 48 h.

ACCEPTED MANUSCRIPT This study confirmed the results from the cell viability study that showed decreased cell proliferation with increases in concentration.

Real-time cellular proliferation study A real-time kinetic proliferation study was conducted at concentrations above, below,

T

and around IC50 for vinblastine, CPD100, and CPD100Li at two oxygen levels (20%

SC RI P

and 1.5%). This study was conducted to verify if the mechanism for the cytotoxic activity of CPD100 and CPD100Li (Fig.9D, 9E) differs from the parent, vinblastine (Fig.9C). The graphs clearly show that at the concentration above the IC50 value, for both CPD100 (40000 nM) and CPD100Li (20000 nM), there is significantly lower CI as

NU

a function of time compared to control ES2 cells. Also, as the concentration was

MA

increased in both groups, the cell proliferation decreased, resulting in lower impedance values, and thus a lower CI. These results are aligned with the stationary cell

ED

proliferation study mentioned in a prior section. The kinetic profile of CPD100 and CPD100Li was identical to that of vinblastine.

PT

Each compound or drug has its own characteristic kinetic profile with respect to its interaction with the cells. Thus, the kinetic trace of an unknown compound can be

CE

compared to the profile of a known compound with an identified mechanism of action

AC

to deduce the similarity or dissimilarity between the compounds[59]. Since CPD100 converts to vinblastine under hypoxic conditions, we wanted to evaluate if the mechanism of action of the pro-drug modified the activity of the parent. This study highlighted that the N-Oxide chemical modification did not change the mode of action of the drug. However, the IC50 values of CPD100 at hypoxic levels are not similar to that of vinblastine (NCI- LC50), possibly due to incomplete conversion of CPD100 to the parent compound, thereby producing higher IC50 values for CPD100 and CPD100Li.

ACCEPTED MANUSCRIPT Clonogenic assay The ability of ES2 cells to retain their proliferative ability and propagate post treatment is tested by a clonogenic assay [27, 60]. Compared to the control cells, all the treated cells showed a greater reduction in the survival fraction (Fig.10). The control colonies showed a lower survival fraction (SF) of 8.6 (1.5% O2) in hypoxia compared to

T

14.4(20% O2) at normoxia. For both oxygenation groups, CPD100 and CPD100Li

SC RI P

showed a decrease in colonies with an increase in concentration. Colonies at the same concentration for both oxygenation groups showed a decrease in SF at 1.5% O2 compared to 20% O2. The SF values for CPD100 and CPD100Li are comparable at 20%

NU

O2, but at 1.5% O2, the CPD100Li compared to CPD100 SF values are as follows: control (0 μM) SF (12.6 vs 8.6); 1 μM CPD100 is 3.2 vs 0.7 μM CPD100Li is 1.8; 0.1

MA

μM CPD100 is 5.8 vs 0.07 μM CPD100Li is 2.8; and, 0.01 μM CPD100 is 10.6 vs 0.007 μM CPD100Li is 3.8 (Fig.10). The trend clearly shows that the liposomes under

ED

hypoxic conditions are more potent than CPD100. Published studies have shown that clonogenic assays are accurate for predicting drugs

PT

likely to be active in clinical settings[61]. It has also been used for rapid screening of

CE

new anti-neoplastic agents, as well as for predicting resistance[62]. A clonogenic assay is a predictive test for chemosensitivity of anti-tumor agents and is analogous to

AC

bacterial culture for antibiotic sensitivity[62]. The lower toxicity of the drug and liposomal formulation under normoxia confirms that the presence of an oxygen gradient is crucial for CPD100 to convert to the parent compound.

NU

SC RI P

T

ACCEPTED MANUSCRIPT

MA

Figure 10: Clonogenic assay for CPD100 and CPD100Li at 20% and 1.5% oxygen levels. Survival fraction expressed as a function of concentration, (A) CPD100 and (B) CPD100Li. (n=3). Representative images of colonies treated with different concentrations of (C) CPD100 and (D) CPD100Li at 20% and 1.5% oxygen levels.

ED

Effect on cell cycle arrest

The effect of an IC50 dose of CPD100 and CPD100Li (Fig.11) on different phases of the

PT

cell cycle was studied as a function of time in ES2 cells. After 18 h of incubation time,

CE

the % of cells in S phase treated with CPD100 at 20% O2 was 1.6 compared to 1.0 at 72 h. The % of cells in G0/G1 phase treated with CPD100Li was 2.9 at 18 h compared to

AC

0.96 at 72 h. The % of cells in S phase treated with CPD100 and CPD100Li at 1.5% O2 was 0.72 and 0.38 at 72 h, respectively. The % of cells in G2/M phase for CPD100 at 20% O2 was 8.36 at 18 h compared to 25 at 72 h, and the % of cells for CPD100Li in the same phase was 13.2 at 18 h compared to 24.36 at 72 h. However, at 1.5% O2, the % of cells in G2/M phase treated with CPD100 was 3.65 at 18 h compared to 3.86 at 72 h, and the % of cells treated with CPD100Li was 3.64 at 18 h compared to 3.48 at 72 h. At the end of 72 h only the CPD100Li 1.5% oxygen was significantly different than the control group for the G2/M phase. No significant difference was observed between the

ACCEPTED MANUSCRIPT control and treatment group as a function of time and oxygen levels in the G0/G1 and S phases of the cell cycle. The parent compound, vinblastine, binds to the ends of microtubules, suppressing their dynamic instability and causing their depolymerization[63]. Vinblastine induces cells to accumulate in mitosis prior to the induction of apoptosis[64]. The biological

T

consequence of interfering with microtubule dynamics causes a G1 and G2/M arrest,

SC RI P

inhibition of cell proliferation, and induction of apoptosis[64]. The analysis revealed that the liposomal formulation along with hypoxia allowed the cell to move through the cell cycle, but many cells are not able to divide and instead accumulated in the G 0/G1

NU

and G2/M phase of the cell cycle. This data complements the real-time proliferation

PT

ED

action to the parent, vinblastine.

MA

study completed with xCELLigence that shows CPD100 has a similar mechanism of

CE

Figure 11: Cell cycle analysis of ES2 cells treated with CPD100 and CPD100Li at IC 50 concentration as a function of time. * indicates statistical significance as determined by a one-way analysis of variance (one-way ANOVA) with a p-value of 0.05.

AC

Apoptosis Assay Caspase 3/7 expression was studied in ES2 cells to determine whether the cell death is driven by apoptosis. The data for caspase 3/7 activity in ES2 cells is shown in Fig.12. The percent of caspase expression for cells treated with 20 nM of CPD100 and CPD100Li at 20% was 108% and 123%, respectively, whereas it was 177% and 172%, respectively, for cells treated at 1.5% O2. The percent of caspase in cells treated at 2000 nM CPD100 and CPD100Li at 20% O2 is 141% and 165%, respectively, whereas it is 204% and 234%, respectively, for the treatment at 1.5% O2. The increase in caspase3/7

ACCEPTED MANUSCRIPT is dose and oxygen dependent. However, there was no significant difference in the caspase 3/7 production between the drug and the liposomal formulation under normoxic conditions, which follows the trend seen in previous experiments. Meanwhile, there is a significant difference in levels of caspase 3/7 expression for both CPD100 and CPD100Li as a function of concentration at 1.5% O2.

T

Vinblastine causes alterations of p53 and DNA fragmentation, thus exhibiting an anti-

SC RI P

proliferative effect via the induction of apoptosis through a caspase-dependent pathway[65, 66].

In summary, the oxygen gradient causes the conversion of CPD100 to the parent

NU

compound, vinblastine. The anti-proliferative assessment of CPD100 via the cell viability and clonogenic assay showed that CPD100Li at 1.5% O2 is more potent than

MA

CPD100Li at 20% O2. The real time proliferation curves, cell cycle, and apoptosis assays signify that the mechanism of action of CPD100, both for the drug and its

AC

CE

PT

ED

liposome formulation, is similar to that of the parent compound, vinblastine.

Figure 12: Caspase 3/7 % expression as a function of different concentrations of CPD100 and CPD100Li.

Pharmacokinetic Study The plasma concentration - time curve of CPD100Li (-A23187) compared to the blood profile of CPD100Li (+A23187) following a single IV dose (30 mg/kg) in mouse

ACCEPTED MANUSCRIPT plasma [16] is shown in Fig.13 and Table 3. The pharmacokinetic profile for CPD100Li (-A23187) showed a Cmax of 35675 ng/mL, AUC of 6644 h*ng/mL, and half-life of 3.3 h, whereas CPD100Li (+A23187) showed a Cmax of 54300 ng/mL, AUC of 84221 h*ng/mL, and half-life of 5.5 h [16]. This data clearly demonstrates that in the presence of ionophore, there is sustained release of CPD100 from the liposome, whereas in the

T

absence of ionophore, there is minimal retention of CPD100 in the vesicle, resulting in a

SC RI P

quick release of CPD100, highlighted by the low AUC and shorter circulation time compared to CPD100(+ A23187).

The results are in accordance with several works which indicated that the presence of

NU

A23187 in the formulation may be fundamental in drug retention and longer circulation in vivo[55]. When irinotecan, a water soluble weakly basic drug, is encapsulated in

MA

liposomes in the presence of A23187, a similar trend in pharmacokinetics and retention of drug in the liposomes is seen[67, 68]. A23187 clearly has a crucial role, not only in

ED

drug loading, but also in the in vivo stability and retention of the drug in the liposome. On the contrary, the in vitro release data shows no difference in the release of CPD100

PT

from the CPD100Li with and without A23187 (Fig.8). This clearly indicates that flux of

CE

the CPD100 out of the liposome is not the main factor that governs the PK profile of the CPD100Li. Based on this data, we speculate that other in vivo parameters influence the

AC

CPD100Li PK profile and that A23718 plays a critical role. However, the exact mechanism by which A23718 exerts its effect is poorly understood.

T

ACCEPTED MANUSCRIPT

SC RI P

Figure 13: Pharmacokinetic profile of CPD100[16], CPD100Li (+ A23187)[16] and CPD100Li (- A23187) in mouse plasma at a dose of 30 mg/kg. Table 3: Pharmacokinetic parameters of the CPD100Li with (+) A23187, CPD100Li (-) A23187 and CPD100.

CPD100Li(+)[16]

CPD100Li(-)

5.5

3.3

h

0.08

AUC

h*ng/mL

15162

Cmax

ng/mL

68567

In vivo acute toxicity studies

84221

6644

54300

35675

MA

T0.5

NU

CPD100[16]

ED

The maximum tolerated dose for the free CPD100 and CPD100Li in nude athymic mice

PT

was 40 mg/kg. Based on the pharmacokinetic study, mice were dosed at 40 mg/kg every 3 days for 5 times and observed for physical and behavioral changes. No treated mice

CE

exhibited any signs of acute toxicity, such as change in behavior, eating habits, weight

AC

loss (> 20%), or death during the study or 14 days after the last dose. CPD100Li showed an identical MTD to CPD100. Empty liposomes administered at 50 mg/kg lipid dose were also studied for toxicity, as lipids have been implicated in complement activation[69]. Another study showed that empty liposomes treated identically for drug loading except for the inclusion of drug were found to be toxic when dosed to animals. Based on the process of elimination, it was concluded that the EDTA resulted in the toxicity. As a result, the final concentration of EDTA in the liposome was decreased from 33 to 15 mM.

ACCEPTED MANUSCRIPT A previous study performed in Swiss Webster mice showed similar values for DLT and

SC RI P

T

MTD, thus proving that both values are independent of the mouse strain [16].

NU

Figure 14: Normalized body weight of mice over time after i.v. injection of CPD100 and CPD100Li on days 0, 3, 6, 9, and 12, as indicated by arrows. The horizontal dashed line represents weight loss ≥ 20% body weight as an indication of toxicity.

MA

Conclusion

Optimization of the process and parameters for producing drug loaded liposomes is

ED

critical. A23187, a non-GRAS excipient, plays a crucial role in the in vivo circulation

PT

and retention of CPD100. The ES2 ovarian cancer cell data clearly highlights that the in vitro mechanism and efficacy of CPD100 is similar to vinblastine at hypoxic levels. The

CE

results of this study provide vital information for continuing the development of CPD100Li, a first-in-class liposomal formulation of a hypoxia activated prodrug for the

AC

treatment of ovarian cancer. Acknowledgments We would like to thank Oregon Nanoscience and Microtechnologies Institute (ONAMI) for funding the study and Cascades Prodrugs Inc. for the generous gift of the drug, CPD100. Electron microscopy is performed at the Multiscale Microscopy Core (MMC) with technical support from the Oregon Health & Science University (OHSU)-FEI Living Lab and the OHSU Center for Spatial Systems Biomedicine (OCSSB).

ACCEPTED MANUSCRIPT References

AC

CE

PT

ED

MA

NU

SC RI P

T

[1] S. Li, B. Sheng, M. Zhao, Q. Shen, H. Zhu, X. Zhu, The prognostic values of signal transducers activators of transcription family in ovarian cancer. Biosci. Rep. 37(4). [2] D.L. Clarke-Pearson, Clinical practice. Screening for ovarian cancer. N. Engl. J. Med. 361(2) (2009) 170-177. [3] M. Fung-Kee-Fung, T. Oliver, L. Elit, A. Oza, H.W. Hirte, P. Bryson, Optimal chemotherapy treatment for women with recurrent ovarian cancer. Curr Oncol 14(5) (2007) 195-208. [4] C. Schumann, S. Chan, O. Khalimonchuk, S. Khal, V. Moskal, V. Shah, A.W. Alani, O. Taratula, Mechanistic Nanotherapeutic Approach Based on siRNA-Mediated DJ-1 Protein Suppression for Platinum-Resistant Ovarian Cancer. Mol Pharm 13(6) 2070-2083. [5] G. Corrado, V. Salutari, E. Palluzzi, M.G. Distefano, G. Scambia, G. Ferrandina, Optimizing treatment in recurrent epithelial ovarian cancer. Expert Rev Anticancer Ther 17(12) (2017) 1147-1158. [6] O.W. Foley, J.A. Rauh-Hain, M.G. del Carmen, Recurrent epithelial ovarian cancer: an update on treatment. Oncology (Williston Park) 27(4) 288-294, 298. [7] G. Corrado, V. Salutari, E. Palluzzi, M.G. Distefano, G. Scambia, G. Ferrandina, Optimizing treatment in recurrent epithelial ovarian cancer. Expert Rev Anticancer Ther. [8] J.M. Brown, Hypoxic cytotoxic agents: a new approach to cancer chemotherapy. Drug Resist Updat 3(1) (2000) 7-13. [9] W.R. Wilson, M.P. Hay, Targeting hypoxia in cancer therapy. Nat Rev Cancer 11(6) (2011) 393-410. [10] P. Vaupel, F. Kallinowski, P. Okunieff, Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 49(23) (1989) 6449-6465. [11] S.R. McKeown, Defining normoxia, physoxia and hypoxia in tumoursimplications for treatment response. Br. J. Radiol. 87(1035) 20130676. [12] P. Ebbesen, K.U. Eckardt, F. Ciampor, E.O. Pettersen, Linking measured intercellular oxygen concentration to human cell functions. Acta Oncol. 43(6) (2004) 598-600. [13] S.R. McKeown, Defining normoxia, physoxia and hypoxia in tumoursimplications for treatment response. Br. J. Radiol. 87(1035) (2014) 20130676. [14] J.M. Brown, Tumor microenvironment and the response to anticancer therapy. Cancer Biol Ther 1(5) (2002) 453-458. [15] Y. Takakusagi, S. Kishimoto, S. Naz, S. Matsumoto, K. Saito, C.P. Hart, J.B. Mitchell, M.C. Krishna, Radiotherapy Synergizes with the Hypoxia-Activated Prodrug Evofosfamide: In Vitro and In Vivo Studies. Antioxid Redox Signal 28(2) (2018) 131140. [16] V.M. Shah, D.X. Nguyen, A. Alfatease, S. Bracha, A.W. Alani, Characterization of pegylated and non-pegylated liposomal formulation for the delivery of hypoxia activated vinblastine-N-oxide for the treatment of solid tumors. J Control Release 253 (2017) 37-45. [17] J.F.W.K. John G. Curd, Alshad S. Lalani, Paul B. Westberg, Bradford S. Goodwin, William David Henner,, Treatment of hyperproliferative diseases with vinca alkaloid n-oxide and analogs, USA Patent 2007.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI P

T

[18] T.M. Allen, P.R. Cullis, Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev 65(1) (2013) 36-48. [19] H.I. Chang, M.K. Yeh, Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy. Int J Nanomedicine 7 (2012) 4960. [20] A. Akbarzadeh, R. Rezaei-Sadabady, S. Davaran, S.W. Joo, N. Zarghami, Y. Hanifehpour, M. Samiei, M. Kouhi, K. Nejati-Koshki, Liposome: classification, preparation, and applications. Nanoscale Res Lett 8(1) (2013) 102. [21] N. Maurer, D.B. Fenske, P.R. Cullis, Developments in liposomal drug delivery systems. Expert Opin Biol Ther 1(6) (2001) 923-947. [22] S.C. Semple, R. Leone, J. Wang, E.C. Leng, S.K. Klimuk, M.L. Eisenhardt, Z.N. Yuan, K. Edwards, N. Maurer, M.J. Hope, P.R. Cullis, Q.F. Ahkong, Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity. J. Pharm. Sci. 94(5) (2005) 1024-1038. [23] I.V. Zhigaltsev, N. Maurer, Q.F. Akhong, R. Leone, E. Leng, J. Wang, S.C. Semple, P.R. Cullis, Liposome-encapsulated vincristine, vinblastine and vinorelbine: a comparative study of drug loading and retention. J Control Release 104(1) (2005) 103111. [24] M.S. Webb, T.O. Harasym, D. Masin, M.B. Bally, L.D. Mayer, Sphingomyelincholesterol liposomes significantly enhance the pharmacokinetic and therapeutic properties of vincristine in murine and human tumour models. Br. J. Cancer 72(4) (1995) 896-904. [25] M. Chountoulesi, N. Naziris, N. Pippa, C. Demetzos, The significance of drugto-lipid ratio to the development of optimized liposomal formulation. J Liposome Res 110. [26] S. Strese, M. Fryknas, R. Larsson, J. Gullbo, Effects of hypoxia on human cancer cell line chemosensitivity. BMC Cancer 13 331. [27] N.A. Franken, H.M. Rodermond, J. Stap, J. Haveman, C. van Bree, Clonogenic assay of cells in vitro. Nat Protoc 1(5) (2006) 2315-2319. [28] L.D. Mayer, M.B. Bally, H. Loughrey, D. Masin, P.R. Cullis, Liposomal vincristine preparations which exhibit decreased drug toxicity and increased activity against murine L1210 and P388 tumors. Cancer Res. 50(3) (1990) 575-579. [29] A.S. Taggar, J. Alnajim, M. Anantha, A. Thomas, M. Webb, E. Ramsay, M.B. Bally, Copper-topotecan complexation mediates drug accumulation into liposomes. J Control Release 114(1) (2006) 78-88. [30] N.L. Boman, L.D. Mayer, P.R. Cullis, Optimization of the retention properties of vincristine in liposomal systems. Biochim. Biophys. Acta 1152(2) (1993) 253-258. [31] N.L. Boman, D. Masin, L.D. Mayer, P.R. Cullis, M.B. Bally, Liposomal vincristine which exhibits increased drug retention and increased circulation longevity cures mice bearing P388 tumors. Cancer Res. 54(11) (1994) 2830-2833. [32] W.L. Erdahl, C.J. Chapman, R.W. Taylor, D.R. Pfeiffer, Effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems. Biophys. J. 69(6) (1995) 2350-2363. [33] P.R. Harrigan, K.F. Wong, T.E. Redelmeier, J.J. Wheeler, P.R. Cullis, Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients. Biochim. Biophys. Acta 1149(2) (1993) 329-338.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI P

T

[34] G.L. Mosley, C.D. Yamanishi, D.T. Kamei, Mathematical modeling of vesicle drug delivery systems 1: vesicle formation and stability along with drug loading and release. J Lab Autom 18(1) 34-45. [35] A. Bjorkbom, T. Rog, K. Kaszuba, M. Kurita, S. Yamaguchi, M. Lonnfors, T.K. Nyholm, I. Vattulainen, S. Katsumura, J.P. Slotte, Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol. Biophys. J. 99(10) 33003308. [36] P.J. Quinn, Structure of sphingomyelin bilayers and complexes with cholesterol forming membrane rafts. Langmuir 29(30) 9447-9456. [37] S.C. Semple, R. Leone, J. Wang, E.C. Leng, S.K. Klimuk, M.L. Eisenhardt, Z.N. Yuan, K. Edwards, N. Maurer, M.J. Hope, P.R. Cullis, Q.F. Ahkong, Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity. J. Pharm. Sci. 94(5) (2005) 1024-1038. [38] S.A. Abraham, K. Edwards, G. Karlsson, S. MacIntosh, L.D. Mayer, C. McKenzie, M.B. Bally, Formation of transition metal-doxorubicin complexes inside liposomes. Biochim. Biophys. Acta 1565(1) (2002) 41-54. [39] S. Kalepu, S. Kumar Kt, S. Betha, M. Varma M, Liposomal drug delivery system - A Comprehensive Review, 2013. [40] D.B. Fenske, K.F. Wong, E. Maurer, N. Maurer, J.M. Leenhouts, N. Boman, L. Amankwa, P.R. Cullis, Ionophore-mediated uptake of ciprofloxacin and vincristine into large unilamellar vesicles exhibiting transmembrane ion gradients. Biochim. Biophys. Acta 1414(1-2) (1998) 188-204. [41] S.G. Ong, M. Chitneni, K.S. Lee, L.C. Ming, K.H. Yuen, Evaluation of Extrusion Technique for Nanosizing Liposomes. Pharmaceutics 8(4). [42] G. Storm, S. O. Belliot, T. Daemen, D. D. Lasic, Surface modification of nanoparticles to oppose uptake by the mononuclear phagocyte system, 1995. [43] D.C. Litzinger, A.M. Buiting, N. van Rooijen, L. Huang, Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)containing liposomes. Biochim. Biophys. Acta 1190(1) (1994) 99-107. [44] S.M. Moghimi, A.J. Andersen, D. Ahmadvand, P.P. Wibroe, T.L. Andresen, A.C. Hunter, Material properties in complement activation. Adv Drug Deliv Rev 63(12) 1000-1007. [45] J. Szebeni, F. Muggia, A. Gabizon, Y. Barenholz, Activation of complement by therapeutic liposomes and other lipid excipient-based therapeutic products: prediction and prevention. Adv Drug Deliv Rev 63(12) 1020-1030. [46] J. Szebeni, The interaction of liposomes with the complement system. Crit. Rev. Ther. Drug Carrier Syst. 15(1) (1998) 57-88. [47] P.P. Wibroe, D. Ahmadvand, M.A. Oghabian, A. Yaghmur, S.M. Moghimi, An integrated assessment of morphology, size, and complement activation of the PEGylated liposomal doxorubicin products Doxil(R), Caelyx(R), DOXOrubicin, and SinaDoxosome. J Control Release 221 1-8. [48] J. Yu, T.J. Anchordoquy, Effects of moisture content on the storage stability of dried lipoplex formulations. J. Pharm. Sci. 98(9) (2009) 3278-3289. [49] H.L. Wu, Y.J. Sheng, H.K. Tsao, Phase behaviors and membrane properties of model liposomes: temperature effect. J. Chem. Phys. 141(12) 124906. [50] P.R. Maulik, G.G. Shipley, N-palmitoyl sphingomyelin bilayers: structure and interactions with cholesterol and dipalmitoylphosphatidylcholine. Biochemistry (Mosc). 35(24) (1996) 8025-8034. [51] T. Kodama, M. Takeuchi, N. Wakiyama, K. Terada, Optimization of secondary drying condition for desired residual water content in a lyophilized product using a

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI P

T

novel simulation program for pharmaceutical lyophilization. Int. J. Pharm. 469(1) 5966. [52] H. Harashima, K. Sakata, K. Funato, H. Kiwada, Enhanced hepatic uptake of liposomes through complement activation depending on the size of liposomes. Pharm. Res. 11(3) (1994) 402-406. [53] D. Scherfeld, N. Kahya, P. Schwille, Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcholines and cholesterol. Biophys. J. 85(6) (2003) 3758-3768. [54] N. Kahya, P. Schwille, How phospholipid-cholesterol interactions modulate lipid lateral diffusion, as revealed by fluorescence correlation spectroscopy. J Fluoresc 16(5) (2006) 671-678. [55] C. Li, J. Cui, Y. Li, C. Wang, L. Zhang, W. Guo, J. Wang, H. Zhang, Y. Hao, Y. Wang, Copper ion-mediated liposomal encapsulation of mitoxantrone: the role of anions in drug loading, retention and release. Eur. J. Pharm. Sci. 34(4-5) (2008) 333344. [56] D. Liang, Y. Ma, J. Liu, C.G. Trope, R. Holm, J.M. Nesland, Z. Suo, The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells. BMC Cancer 12 (2012) 201. [57] R.G. Bristow, R.P. Hill, Hypoxia and metabolism. Hypoxia, DNA repair and genetic instability. Nat Rev Cancer 8(3) (2008) 180-192. [58] E.M. Hammond, M.J. Dorie, A.J. Giaccia, Inhibition of ATR leads to increased sensitivity to hypoxia/reoxygenation. Cancer Res. 64(18) (2004) 6556-6562. [59] Z. Xi, S. Khare, A. Cheung, B. Huang, T. Pan, W. Zhang, F. Ibrahim, C. Jin, S. Gabos, Mode of action classification of chemicals using multi-concentration timedependent cellular response profiles. Comput Biol Chem 49 23-35. [60] V. Rajendran, M.V. Jain, In Vitro Tumorigenic Assay: Colony Forming Assay for Cancer Stem Cells. Methods Mol. Biol. 1692 89-95. [61] D.H. Kern, C.A. Bertelsen, B.D. Mann, M.A. Campbell, D.L. Morton, A.J. Cochran, Clinical application of the clonogenic assay. Ann. Clin. Lab. Sci. 13(1) (1983) 10-15. [62] A.P. Simmonds, E.C. McDonald, Ovarian carcinoma cells in culture: assessment of drug sensitivity by clonogenic assay. Br. J. Cancer 50(3) (1984) 317-326. [63] Y.N. Cao, L.L. Zheng, D. Wang, X.X. Liang, F. Gao, X.L. Zhou, Recent advances in microtubule-stabilizing agents. European journal of medicinal chemistry 143 (2018) 806-828. [64] B.L. Salerni, D.J. Bates, T.C. Albershardt, C.H. Lowrey, A. Eastman, Vinblastine induces acute, cell cycle phase-independent apoptosis in some leukemias and lymphomas and can induce acute apoptosis in others when Mcl-1 is suppressed. Molecular cancer therapeutics 9(4) (2010) 791-802. [65] E. Calvino, M.C. Tejedor, P. Sancho, A. Herraez, J.C. Diez, JNK and NFkappaB dependence of apoptosis induced by vinblastine in human acute promyelocytic leukaemia cells. Cell Biochem. Funct. 33(4) 211-219. [66] D.A. Rao, G. Mishra, B.S. Doddapaneni, S. Kyryachenko, I.H. Wierzbicki, D.X. Ngyuen, V. Shah, A.M. Al Fatease, R.G. Alany, A.W.G. Alani, Combinatorial Polymeric Conjugated Micelles with Dual Cytotoxic and Antiangiogenic Effects for the Treatment of Ovarian Cancer. Chemistry of Materials 28(17) (2016) 6068-6079. [67] E. Ramsay, J. Alnajim, M. Anantha, J. Zastre, H. Yan, M. Webb, D. Waterhouse, M. Bally, A novel liposomal irinotecan formulation with significant antitumour activity: use of the divalent cation ionophore A23187 and copper-containing liposomes to improve drug retention. Eur. J. Pharm. Biopharm. 68(3) (2008) 607-617.

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

MA

NU

SC RI P

T

[68] V. Shah, R.A. Bellantone, D.R. Taft, Evaluating the Potential for Delivery of Irinotecan via the Buccal Route: Physicochemical Characterization and In Vitro Permeation Assessment Across Porcine Buccal Mucosa. AAPS PharmSciTech (2016). [69] J. Szebeni, C.R. Alving, L. Rosivall, R. Bunger, L. Baranyi, P. Bedocs, M. Toth, Y. Barenholz, Animal models of complement-mediated hypersensitivity reactions to liposomes and other lipid-based nanoparticles. J Liposome Res 17(2) (2007) 107-117.