Development of Liposomal Formulation for Delivering Anticancer Drug

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Development of Liposomal Formulation for Delivering Anticancer Drug to Breast Cancer Stem Cell-Like Cells and its Pharmacokinetics in an Animal Model Ajaz Ahmad, Sujan Kumar Mondal, Debabrata Mukhopadhyay, Rajkumar Banerjee, and Khalid M Alkharfy Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00900 • Publication Date (Web): 01 Feb 2016 Downloaded from http://pubs.acs.org on February 3, 2016

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Molecular Pharmaceutics

Development of Liposomal Formulation for Delivering Anticancer Drug to Breast Cancer Stem Cell-Like Cells and its Pharmacokinetics in an Animal Model Ajaz Ahmada,e,#, Sujan Kumar Mondalb,c,d,#, Debabrata Mukhopadhyayc, Rajkumar Banerjeeb,d,* , Khalid M. Alkharfy a,*

a

Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 11451,

Saudi Arabia b c

Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India

Department of Biochemistry and Molecular Biology, Mayo Clinic, Jacksonville, FL 32224,

USA dAcademy

#

of Scientific & Innovative Research (AcSIR), 2 Rafi Marg, New Delhi 110001, India

denotes equal contribution

*ADDRESS CORRESPONDENCE TO: Khalid M. Alkharfy, Pharm.D., Ph.D. Department of Clinical Pharmacy College of Pharmacy, King Saud University PO Box 2457, Riyadh 11451 Saudi Arabia Phone: +9661-467-7494 Fax: +9661-467-7480 Email: [email protected] Rajkumar Banerjee, Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, India Phone: +91-40-2719-1478; Email: [email protected]; [email protected] 1 ACS Paragon Plus Environment


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ABSTRACT: The objective of the present study is to develop a liposomal formulation for delivering anticancer drug to breast cancer stem cell-like cells, ANV-1, and evaluate its pharmacokinetics in an animal model. The anticancer drug ESC8 was used in Dexamethasone (Dex)-associated liposome (DX) to form ESC8-entrapped liposome named DXE. ANV-1 cells showed high-level expression of NRP-1. To enhance tumor regression, we additionally adapted to co-deliver the NRP-1 shRNA-encoded plasmid using the established DXE liposome. In vivo efficacy of DXE-NRP-1 was carried out in mice bearing ANV-1 cells as xenograft tumors and the extent of tumor growth inhibition was evaluated by tumor-size measurement. A significant difference in tumor volume started to reveal between DXE-NRP-1 group and DXE-Control group. DXE-NRP-1 group showed ~4 folds and ~2.5 folds smaller tumor volume than exhibited by untreated and DXE-Control-treated groups, respectively. DXE disposition was evaluated in Sprague−Dawley rats following an intraperitoneal dose (3.67 mg/kg of ESC8 in DXE). The plasma concentrations of ESC8 in the DXE formulation were measured by liquid chromatography mass spectrometry and pharmacokinetic parameters were determined using a non-compartmental analysis. ESC8 had a half-life of 11.01±0.29 h, clearance of 2.10 ±3.63 L/kg/h, and volume of distribution of 33.42 ±0.83 L/kg. This suggests that the DXE liposome formulation could be administered once or twice daily for therapeutic efficacy. In overall, we developed a potent liposomal formulation with favorable pharmacokinetic and tumor regressing profile that could sensitize and kill highly aggressive and drug-resistive cancer stem cell-like cells.

Keywords: ANV-1 cells, tumor, drug-sensitivity, liposomes, pharmacokinetics, cancer stem cell -like cells, Hsp90, NRP-1.

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INTRODUCTION Self-renewal mechanism of cancer cells is similar to that of stem cells and hence the concept of 1-4

cancer stem cells (CSC) has been raised.

During relapsing of cancer, the cells acquire

enhanced aggression, metastasis and drug resistance owing to a cellular phenotypic transformation called epithelial-to-mesenchymal transition (EMT), which is the hallmark of CSC and the new clones formed from it.

5-6

This cancer initiating stem cell-like cells (or, CSC)

provide important insight for developing effective treatments for advanced stage cancer. Conventional treatments of radiation and chemotherapy for this stage of cancer often remain ineffective as the cancer cells, possessing EMT and self-renewing clones of CSCs, acquire high aggressiveness, have heterogeneous clonal populations of differential drug responses. EMT in cancer cells, among many new phenotypic changes, also deduce drug-resistivity. Hence, targeting and killing CSC is a challenge which if overcome can lead to effective treatment for relapsing, advanced stage cancer. Heat shock protein 90 (Hsp90) is over-expressed in most of the cancers

7-10

in which it

chaperones most, if not all factors including all that are involved in proliferation and sustenance of all types of cancer cells.

5,11-12

On the other hand, Neuropilin-1 (NRP-1), the VEGF

coreceptor for VEGFR2 regulates the signaling of MAPK, PI3K/Akt, and Rho/Rac which are involved in cell migration, invasion and apoptosis besides being involved in renal cancer cell differentiation.

13-17

Hence, both Hsp90 and NRP-1 are two potential cancer targets, which are

however exploited limitedly towards developing anticancer therapeutics especially against CSCimplicated cancers. The breast cancer stem cell-like cell, ANV-1 is the product of immunoediting breast tumors in transgenic mouse. It passed through EMT to possess breast CSC character.

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We show here that

ANV-1 expresses both Hsp90 and NRP-1. Hence, we chose ANV-1 breast CSC as a model to study the effect of down-regulating Hsp90 and NRP-1 and hypothesize that effective targeting of either of these factors in CSCs may lead to the development of anticancer therapeutics against aggressive, relapsing cancer. Previously, we developed Dexamethasone-associated liposomal formulation (DX) that can selectively manipulate glucocorticoid receptor (GR) of cancer cells for delivering its genetic 3 ACS Paragon Plus Environment


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cargo.

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Dexamethasone (Dex) is a well-established synthetic ligand for the GR and due to its

structural similarities with cholesterol; Dex was directly incorporated alongside cholesterol and cationic lipid to get DX liposomal formulation. Following the establishment of the DX, we have shown successfully that DX liposomal delivery system can selectively transfect cancer cells with anti-Hsp90 miRNA encoded genes. This subsequently down-regulates Hsp90 and its client proteins, thereby leading to substantial inhibition of tumor growth both in melanoma and lung cancer model.

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Dex can however induce mesenchymal to epithelial transition (MET) by

inhibiting TGF-β induced epithelial to mesenchymal transition

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and can induce forced

restoration of gluconeogenesis in hepatoma cells thereby leading to better therapeutic efficacy.

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Although, whether induction of gluconeogenesis have any direct role to play in EMT inhibition is not clear but it is evident that selective delivery of Dex in cancer cells will be an effective anticancer strategy. Towards this, we hypothesized developing a multimodal delivery system that will carry three different drug and genetic cargoes: a) Dex, b) any other anticancer agent and c) a genetic cargo encoding anti-Hsp90 or anti-NRP-1 constructs. This will be a combination approach to treat CSC: first, both Dex and anti-Hsp90 or anti-NRP-1 construct will induce EMT reversal thereby making CSCs drug sensitive; Second, drug sensitive CSCs will be killed by the delivery system associated anticancer drug. As the anticancer drug payload, we chose to use our recently developed potent anti-breast cancer drug named ESC8.

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Stability/shelf life information

and pharmacokinetic profile of ESC8 in oral and intravenous formulations have been previously established (supplementary Information).

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For this study, we entrapped ESC8 in DX liposome

and named it as DXE. DXE being cationic liposome could electrostatically complex with anticancer gene-associated plasmid DNAs. In the present study, we delivered either anti-Hsp90 miRNA or anti-NRP-1 shRNA encoded plasmid after electrostatically complexing with DXE and we found that both the lipoplexes induced enhanced cytotoxic effect in the highly drug resistant ANV-1 cells. Further, we found that DX formulation was able to de-differentiate highly drug resistant ANV-1 cells by inverting EMT and sensitizing them against anticancer drugs. Here, we also show that the DXE lipoplex with anti-NRP1-shRNA is able to inhibit ANV-1 tumor aggression by substantial amount. The pharmacokinetic behavior of liposomal formulation DXE in plasma after an intraperitoneal (IP) administration to rats was also studied. The overall


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study reveals the plausible development of potential multimodal therapeutics against CSC-based drug resistant tumors with an acceptable in vivo disposition profile.



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MATERIALS AND METHODS Chemicals and General Procedures. ESC8 was synthesized following previous method.

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The cationic lipid used in DX formulation (i.e., DODEAC) was synthesized as described 24

earlier.

ESC8 and DODEAC with >99% purity were included in this study. Doxorubicin,

dexamethasone, cholesterol and RIPA buffer were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). Other formulation components of DX including dexamethasone and cholesterol were also of >99% purity. Deionized water was produced and purified in the laboratory using a Millipore water purification system (Millipore Corp., Billerica, MA, USA). Remaining all other chemicals, reagents and organic solvents were purchased either from Sigma (St. Louis, MO, USA) or from Rankem Ltd. (Mumbai, India). Antibodies. β-actin (8457S), ABCG2 (4477S) antibodies were bought from Cell Signaling Technology; Hsp90 (ab13495) was purchased from Abcam; NRP-1 (sc-7239), ID-1 (sc-488), SNAI 1(sc-271977), and secondary antibodies goat anti-rabbit IgG-HRP (sc-2030), goat antimouse IgG-HRP (sc-2005) were obtained from Santa Cruz Biotechnology; and α-SMA was purchased from Millipore (Billerica, MA, USA). Cell Culture. The ANV-1 (Mayo Clinic, USA cell line repository) cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS, Lonza, USA), 1% penicillin/streptomycin, 1% sodium pyruvate, 2.5% HEPES, and 2 mM L-glutamine at 37°C in a humidified incubator containing 5% CO2. Plasmid Constructs and Stable Transfection Using Lentiviral Particle. Artificial miRNA plasmid constructs (amiR-Hsp90 or Hsp90), which contain Hsp90 micro RNA gene and 8

its control plasmid, were made following the same method as described before. The plasmids for NRP-1 shRNA were purchased from Open Biosystems (Huntsville, AL, USA). The targeting sequence for NRP-1 was: 5’- CCA GAG AAT CAT AAT CAA CTT-3’. Preparation of liposomes. All the liposomes were prepared following previous protocol. 19

The DX liposomes contained DODEAC: Chol: Dex in 1:1:0.75 mole ratios, where DODEAC

means N, N-dioctadecyl N, N-dihydroxyethyl ammonium chloride; Chol means cholesterol and Dex means dexamethasone. For the entrapment of ESC8 drug (hydrophobic) in DX liposome, chloroform solution of ESC8 was added to the chloroform solution of lipids' mixture of DX at 0.25 molar ratio with respect to DO lipid. The organic solvent was evaporated, dried under


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vacuum and then hydrated with 5% glucose overnight. A brief bath sonication and 2 min Tiprobe sonication (Branson, CT, USA) produced the liposome DXE. Detailed physical characteristics of DXE liposome including size, charge and stability are provided as Supplementary data (Table S1 and Figure S2). All in vitro studies were carried out with 1 mM liposome (with respect to cationic lipid) while in vivo studies were done with 5 mM liposome (with respect to cationic lipid), dispersed in 5% glucose solution. Lipoplex Preparation and its Treatment. The complex of lipid-DNA (lipoplex) was prepared using previous literature.

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Briefly, for cytotoxicity study, 0.3

g/well of 96 well plates), which diluted in 50

g of pDNA (0.3

l serum free media, was electrostatically

complexed with different amount of 1 mM liposome (also diluted in 50 l serum free media) to get the different lipoplexes with lipid/DNA (+/-) charge ratios at 8:1, 4:1, 2:1, 1:1. The resultant 100 µl lipid/DNA complexes were diluted to 300 µl using 10% FBS containing media and subsequently used for the treatment. For in vivo studies, 50 µg pDNA was used for the lipoplex formation at 6:1 charge ratio (+/-). Cell Viability Study. MTS or (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfonyl)-2H-tetrazolium) assay by Promega (Madison, WI, USA) was used for the cell viability study. Briefly, 5000 Cells/well were seeded in a 96- well plate (in triplicate) with 100 µl of media 18-24 h prior to experiment. Then the media was replaced with the different treatment groups containing media and incubated for 48 h. Following this, 90 µl of MTS solution was added to each well as per manufacturers protocol and incubated at 37°C for 1 to 2 h. Then the absorbance was taken at 490 nm using Spectra Fluor PLUS (Molecular Devices, Sunnyvale, CA, USA). Results were expressed as percent viability = [A490 (treated cells)-background/ A490 (untreated cells)-background]/100. Typically, viability experiments were done in triplicates on three different days. Results of two different experiments performed on different days showed a maximum variability of ~20% depending on cell condition. Hence, IC50s of different treatment groups were calculated and compared based on a representative data obtained on a single day. Western Blot. For in vitro whole cell lysate preparation, ANV-1 cells were treated with different treatment groups maintaining same dexamethasone concentration (9 µM) in each treatment group. After 48 h of continuous treatment, cells were lysed using RIPA lysis buffer (50 mM Tris [pH 7.5], 1% NP-40, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium 7 ACS Paragon Plus Environment


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deoxycholate) with 1% proteinase inhibitor cocktails (PIC) (Cell Signaling Technology; Danvers MA, USA). Respective cell lysates were run on SDS-PAGE gel (8-15%) and subsequently protein was transferred to PVDF membrane and developed. Drug Sensitivity Assay. For this assay, in 96-well plates 5000 cells/well were seeded per well in triplicate, and kept for 18-24 h. After that cells were either kept untreated or treated with DX-Hsp90 lipoplex (amiR-Hsp90 plasmid of 0.3µg/well) and DX liposome for 24 h. Following this treatment, media was removed, and washed twice with PBS, then cells were incubated for another 48 h with fresh media carrying varying concentrations of ESC8 and doxorubicin. At the end of the treatment period, viability was determined by the MTS assay. In Vivo Tumor Model Study. 1.5 X 105 ANV-1 cells were orthotopically injected into the mammary fat pads of female mice of the FVB strain (NCI, Frederick, MD, USA). Two weeks after tumor cell inoculation, when the average tumor sizes were ~50 mm3, the mice were injected with: a) 5% glucose (as untreated group), b) DXE-NRP-1 containing 50 µg of NRP-1 shRNA plasmid, or c) DXE-Control (DXE-Cont) containing 50 µg of control plasmid. DXE-NRP-1 was also injected in a separate group of mice when the average tumor size was 330 mm3. Five injections were given to respective groups every 2 or 3 days. The tumors were measured twice a week. Tumor volumes were calculated as 0.5×a×b×b, where ‘a’ is the length and ‘b’ the breadth of the tumor. ESC8 Disposition Study. Male Sprague–Dawley rats (n=6, weighing 200–220 g) were obtained from the Laboratory Animal Center of King Saud University (Riyadh, Saudi Arabia). The rats were kept in an environmentally controlled room for one week before experimentation. All the experimental procedures were in accordance with the guidelines for the Care and Use of Laboratory Animals of King Saud University. The rats were fasted overnight before dosing but allowed free access to water during the whole experiment. Liposomal formulation of ESC8 (DXE) was dispersed in 5% glucose solution, and a dose of 3.67 mg/kg of ESC8 in liposome was injected intraperitoneally (i.p.) in rats. Heparinized blood (1 ml) was serially taken from fossa orbitalis vein at 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 72 h after drug administration. Plasma was separated from the blood samples by centrifugation at 2500 × g for 10 min and stored at -20°C pending analysis. ESC8 Plasma Assay Development. Stock solutions of ESC8 (200 µg/mL) and dextromethorphan as an internal standard (IS, 2 µg/mL) were prepared in methanol. The stock 8 ACS Paragon Plus Environment

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solutions were consecutively diluted with methanol to prepare working solutions just before use. Plasma ESC8 standards were prepared by spiking 20 µl of the ESC8 working standard stock solution into 180 µL of blank plasma in Eppendorf tubes resulting in plasma ESC8 concentrations of 5, 10, 15, 25, 50, 100, 250 and 500 ng/mL (for calibration) and four quality control (QC) samples with final concentrations of 5, 15, 150, 250 ng/mL. The IS solution was prepared by diluting the primary stock solution with methanol, giving a concentration of 25 ng/mL. Standard calibration and QC samples were stored at -20°C until use. Each tube was then precipitated with methanol and was centrifuged at 2500 × g for 10 min, producing a protein pellet at the bottom of the tube. The supernatant was removed and evaporated under a gentle stream of nitrogen and reconstituted with 200 µl of methanol; 10 µL aliquot was injected into a liquid chromatography and mass spectrometric (LC-MS) system. The temperature of the autosampler was kept at 4ºC and the LC-MS analysis was performed using Waters Alliance 2795HT® system (Waters Corp., Milford, MA, USA). The LC system comprised of a quaternary pump, degasser, an auto-sampler with injection loop of 50 µL, and a column heater-cooler system. Drug separation was performed on Waters Acquity UPLC BEHTM C18 column (50 × 2.1 mm, 1.7 µm) maintained at 40ºC. The mobile phase was composed of methanol: water: formic acid (70:30:0.1 v/v/v) pumped at a flow rate of 0.7 ml/min. The resulting retention times for both ESC8 and IS were approximately 5 minutes. The accuracy for ESC8 assay method ranged from 98.99 to 100.67% with a precision (CV %) of less than 5%.

Pharmacokinetic analysis. A non-compartmental pharmacokinetic analysis was used to determine the pharmacokinetic plasma behavior of ESC8 in the liposomal formulation DXE. The calculated parameters were: area under plasma concentration-time curve (AUC) using linear trapezoid method; area under the first moment curve (AUMC); mean residence time (MRT) where MRT= AUMC/AUC; volume of distribution (Vd/F) where Vd/F = (dose/AUC x Ke); total clearance (Cl/F) as dose/AUC; and the terminal elimination rate constant (Ke) which was calculated from the slope of the logarithm of the plasma concentration versus time profile. The apparent elimination half-life (T1/2) was computed as ln2×Vd/Cl. The maximum plasma concentration (Cmax) and time to maximum concentration (Tmax) were determined directly from the concentration-time curve. The pharmacokinetic parameters were calculated using Excel’s PK solver program, and data are presented as mean ± SEM.



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Statistical analysis: Data were expressed as mean with standard deviation (SD) and analyzed by a two-tailed unpaired Student’s t-test using Microsoft Excel (Seattle, WA, USA). A p value < 0.05 was considered to be statistically significant.


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RESULTS AND DISCUSSIONS Effect of DX-Liposome Mediated Co-Delivery System Carrying ESC8 Drug and

amiR-Hsp90 Anticancer Plasmid on ANV-1 Cell Viability. In comparison to other charge ratios, the lipoplex [DXE-Hsp90] with 4:1 charge ratios (+/-), carrying 0.3 µg of anti-miRHsp90 plasmid and 7 µM of ESC8, showed a significant combination cytotoxic effect. Thus, here we exhibit results with lipoplex maintaining a 4:1 charge ratio while comparing its effect with other treatments such as free drug, ESC8 (E), DX liposome, DX-Cont (i.e., carrying control plasmid), DX-Hsp90 (i.e., carrying anti-miRHsp90), and DXE-Cont lipoplex. Figure 1A, shows that the DXE-Hsp90 lipoplex could induce significant cell death (with only 20% viability) compared to free ESC8, DX liposome and DX-Hsp90 lipoplex which showed cell viability around 88%, 84%, and 77%, respectively. In addition, DXE-Control [DXE-Cont] lipoplex also exhibited cytotoxicity in ANV-1 cells. However, the cytotoxicity effect of DXE-Hsp90 was significantly higher compared to DXE-Cont. Clearly, treatment of DXE-Hsp90 lipoplex had synergistic effect over individual treatments of ESC8 and DX-Hsp90. Together, these data suggest the combined cytotoxic effect of DX-mediated co-delivery of anticancer drug and gene. Dexamethasone Associated Liposomal Delivery System Simultaneously Inhibit Epithelial to Mesenchymal Transition and Reduce Drug-Efflux Proteins in ANV-1 Cell. We hypothesized that the GR-targeted delivery system could have been able to de-differentiate cancer cells thereby sensitizing the cells to anti-cancer drug. To test the hypothesis, ANV-1 cells were treated with different treatment groups without the anticancer drug and subsequently looked for the mesenchymal phenotype-associated protein markers. Drug-associated DX lipoplex formulation [i.e., DXE lipoplex] was too sensitive for the cells to withstand the cytotoxicity and hence we chose to use only DX lipoplex [i.e., without the drug] for this mechanistic study. Western blot analysis as exhibited in Figure 1B, shows that all dexamethasone-related treatments either as free Dex or DX liposomes/lipoplexes lead to a down-regulation of EMT markers such as Id-1 and α-SMA. Expression of SNAI-1, which is a repressor of the E-cadherin (epithelial marker) was down-regulated by all treatment groups. However, free dexamethasone treatment is showing maximum inhibitory effect in cellular conditions. ABCG2 is a transporter protein responsible for drug resistance in breast cancer cells. We find that all Dex-associated treatment groups decrease the cellular ABCG2 levels by ~30-40%. Taken together all these data support 11 ACS Paragon Plus Environment


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the fact that at least at cellular level, all Dex-associated treatments including liposome-associated ones have the potential to reverse EMT and drug resistance in highly drug resistant and aggressive ANV-1 cells. DX-Mediated Sensitization of ANV-1 Cells to Anticancer Drugs. To support the above-mentioned hypothesis, we examined if in the presence of DX or DX-Hsp90, either hydrophobic (ESC8) or hydrophilic drug [doxorubicin (Dox)] exhibit any additional toxic effect on ANV-1 cells. Earlier it has been shown that free Dex has the ability to induce better therapeutic efficacy in cancer cells [17]. It is hence interesting to know if the liposome or liposome-associated Dex behaves similarly. Our Western blot studies predicted that not only free Dex but also liposome/lipoplex associated Dex exhibited the ability to reduce drug resistivity. For this, first ANV-1 cells were treated either with DX or DX-Hsp90 for 24 h followed by the treatment with respective anti-cancer drugs ESC8 or Dox for additional 48 h. MTS assay-based cellular viability results showed that DX or DX-Hsp90 pretreatment followed by ESC8 treatment indeed decreased the IC50 value by ~3.3 fold when compared to the free ESC8 drug (Figure 1C). Typical IC50s (obtained from a representative graph) for 'DX+ESC8' was 8.0 µM and for 'DXHsp90 + ESC8' was 4.7 µM with respect to ESC8 drug concentration (15.5 µM IC50). Similar type of result was also found with Dox treatment where DX-Hsp90 pretreated group showed a sharp fall in cell viability to ~30 % at 0.2 µM Dox concentration (Figure 1D) while free Dox did not show any significant amount of cytotoxicity at sub-micromolar concentration (IC50 >2µM). Typical IC50s for 'DX + Dox' was ~2.0 µM and for 'DX-Hsp90 + Dox' was 0.12 µM with respect to Dox concentration. These results further support the fact that cationic liposomeassociated Dex (i.e., DX) either alone or in association with anti-Hsp90 gene has the ability to induce drug-sensitivity in ANV-1 cells. DXE-NRP-1 Lipoplex Induces Systematic Cytotoxicity in ANV-1 cells. NRP-1 is a membrane bound co-receptor for vascular endothelial growth factor (VEGF) and semaphorin. It is well known for its involvement in vascularization and progression of cancer. Recently we showed the plausible link between NRP-1 and EMT in renal cell carcinoma.

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ANV-1 cells

show high-level expression of NRP-1. We also find that ANV-1 undergoes MET type similar phenotypic change when its NRP-1 receptor is knocked down. Transfection of NRP-1 shRNA in ANV-1 cells led to α-SMA and Id-1 down-regulation (unpublished data; Supplementary Figure S1 for review purposes only). Among antimiR-Hsp90 and NRP-1 sh-RNA encoded plasmids, it 12 ACS Paragon Plus Environment

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is established that NRP-1 has a link with EMT transition; thus as a tumor-regressing strategy, we chose to deliver the NRP-1 sh-RNA-encoded plasmid using the established DXE liposome. This is expected to maximize de-differentiation (mesenchymal to epithelial phenotypic change) leading to reduction in aggression, followed by sensitization of tumor cells against encapsulated ESC8 drug. To test this hypothesis, we first tested if DX-mediated delivery of NRP-1 shRNA could lead to a down-regulation of NRP-1 in ANV-1 cells. We found a prominent downregulation of NRP-1 in DX-NRP-1-shRNA treated ANV-1 cells (Figure 2A). Then we treated the ANV-1 cells with either ESC8 free drug or with lipoplexes such as DXE-NRP-1 shRNA plasmid (DXE-NRP1) and DXE-Control shRNA plasmid (DXE-Cont). The cytotoxicity data (Figure 2B) showed that DXE-NRP-1 induced a huge amount of cell killing (≤ 5% viability) when compared to other treatment groups. Incidentally, the free ESC8 showed significantly low cytoxicity in ANV-1 cells. DXE-NRP-1 Lipoplex’s Effect in ANV-1 Tumor Bearing Mice. Next, ANV-1 tumors were generated by orthotopic inoculation of ANV-1 cells in the mammary fat pads of mice. Two weeks after cell inoculation, when the average size of the tumor was about 50-60 mm3, intraperitoneal injections of respective lipoplex formulations (i.e., DXE-NRP1 or DXE-Cont, containing corresponding plasmids) were started. As shown in (Figure 2C), at the initial stage when injections were continued both the treatment groups showed a similar pattern of inhibition of tumor progression. However, at later stages when injections were no longer given, the tumor size began to increase but at a much more controlled rate in the DXE-NRP-1 treatment group than in the DXE-Cont group. A significant difference in tumor volume started to reveal between DXE-NRP-1 group and DXE-Cont group. On the 38th day DXE-NRP-1 group showed ~4 fold and ~2.5 fold smaller tumor volume than that exhibited by untreated (UT) and DXE-Cont-treated groups, respectively. The typical tumor sizes on day 38 were as follows: 2320±490 mm3 (UT), 1488±266 mm3 (DXE-Cont) and 662±395 mm3 (DXE-NRP-1).

It is a challenge to contain the tumor in the later stage as at this stage tumors undergo phenotypic changes (EMT), which not only helps in generating heterogeneous populations of varied aggression but also evolve drug resistance in tumor mass-associated cells. To check if our liposome, which succeeded in reversing the EMT in vitro, will be able to contain the tumor in the later stage when the maximum aggression is observed, we formed another group of tumorbearing mice. In this, when the average tumor size was 330 mm3, ESC8-NRP-1 injection began. 13 ACS Paragon Plus Environment


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The inhibition of the aggressiveness was immediately visible after two injections and this effect was continued for 38 days, after which mice were sacrificed as the average tumor volume of UT group exceeded 2000 mm3. It is to be noted that when the tumor size in UT group was near ~330 mm3 and allowed the tumor to increase for 11 days uninhibited, its size increased to ~ 2000 mm3. However, for mice bearing 330 mm3 tumor when DXE-NRP-1 treatment began, the tumor size after 11 days remained largely unchanged. Hence, the tumor aggression could be reduced by 6 folds. It seems DXE-NRP-1 has a substantial high anti-tumor effect even when tumor aggression is at its highest level. ESC8 Disposition in Vivo. Plasma concentration−time curve of ESC8 in rat plasma following a single i.p. dose (3.67 mg/kg) in DXE formulation was estimated from peak area of LC-MS chromatograms of injected samples as shown in Figure 3. The pharmacokinetic profile of ESC8 in the liposomal DXE formulation showed a Cmax of 118.44±3.63 µg/L at 4 h postdosing and AUC0-36 of 1559.34 ±28.28 µg/L*h. The AUC determines the bioavailability of the drug for a given dose of the formulation. The average value of Cl/F is 2.10 ±3.63 L/kg/h, and Vd/F equals 33.42 ±0.83 L/kg. This has resulted in a T1/2 of about 11.01 ±0.29 h and a MRT of ~16 h. The plasma profile showed that ESC8 in formulation is released in a sustained manner, which indicates that once or twice dosing would be sufficient to maintain its therapeutic efficacy. The pharmacokinetic parameters are listed in Table 1. We previously validated an orally administered solid-lipid nanoparticle, containing ESC8 and other components of mostly non-cationic in nature, for the anticancer activity in mice bearing triple negative breast cancer (TNBC) as xenograft tumors.

23

The liposomal formulation,

DXE in the current study was evaluated for pharmacokinetic profile of ESC8 in healthy rats with an i.p. injection. It has been established that administration of liposome formulation would enhance drug absorption and systemic bioavailability.

25

However, in comparison with our

previous oral delivery system in how the blood residence of ESC8 would vary when incorporated in a typical gene delivery system containing cationic lipids was not apprehensible. Moreover, it was vitally important to understand the plasma concentration of ESC8 when it was delivered, not as a naked drug, but using a drug-sensitizing liposomal delivery system for tumor. The premise was that as the DX-liposome inherently drug-sensitizes tumor, the drug-cargo ESC8 would have the maximum anti-tumor effect. Pharmacokinetic parameters such as Ke, T1/2 and Cl/F of ESC8


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at 3.67 mg/kg i.p. represent slight differences when compared with oral administration of ESC8 (20 mg/kg) in our previous study.

23

The variability in these parameters could be due to a number

of factors such as different formulation components, variations in dose and route of administration, longer sampling time and using more specific and sensitive assay method (i.e., LC-MS) in our study. The apparent Vd is an important indicator of the extent of drug distribution into body fluids and tissues. A large Vd usually indicates that the drug distributes extensively into the body tissues and fluids. ESC8 liposomal formulation has a Vd/F of 33.42 ±0.83 L/kg, indicating a wide distribution, and therefore, better tissue penetration of the drug to reach the target site of action. It is established that formulation administered through i.p. is absorbed through the portal circulation.

26-27

The mechanisms encompassed in such uptake include the

diffusion of particles through mucus and accessibility to epithelial interaction, an enterocyte surface and cellular trafficking and exocytosis and systemic dissemination. We have earlier shown that DX formulation is able to efficiently and selectively deliver genetic cargo and regulate glucocorticoid responsive element (GRE)-promoted genes in only cancer cells.

15

There are many drug-metabolizing genes that are regulated through GRE.

Clearly, the cancer cell-selectivity that DX and its related formulations [such as DXE] possess could be hypothetically useful to manipulate drug sensitivity selectively in cancer cells. Cancer stem cells (CSC) should not be an exception as these cells [or any newly formed clonal population from CSC] show the usual pattern of EMT up-regulation distinctly shown by aggressive cancers. The DXE formulation carrying Dex in the first place should have drugsensitized the CSCs, which were possibly havocked by formulation-associated ESC8. The antitumor effect was eventually compounded by the genetic cargo that sought to down-regulate EMT-inducing factor NRP-1. At the end, the side effects of the formulation in non-specific, nontumorigenic organs are expected to be minimal as the Dex-associated liposome (DX or DXE) is tumor-selective in its anticancer action. Taken together, these data describe the new strategy of treating drug resistant cancer stem cells by simultaneous inhibition in the aggressiveness of tumor mass with phenotypic change followed by anticancer therapeutic treatment. The pharmacokinetics profile suggests that the ESC8 disposition is linear, which would be helpful for the development of a suitable delivery system and determine rational clinical administration.



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ASSOCIATED CONTENT

Supporting Information. Stability information of ESC8 and for lipoplexes, hydrodynamic diameter and zeta potential of lipoplexes.

AUTHOR INFORMATION Corresponding Authors

Department of Clinical Pharmacy, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia, *Email: [email protected], Phone: +9661-467-7494, Fax: +9661-467-7480 and Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, India, *Email: [email protected]; [email protected], Phone: +91-40-2719-1478

ACKNOWLEDGEMENT

This project was funded by National Plan for science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (12-MED-2897-02) to KA, and CSIR-Mayo Clinic partnership project through Council of Scientific and Industrial Research (CSIR), Government of India funds (CMPP003) to RB and Mayo Clinic Foundation to DM. SKM thanks CSIR, Govt. of India for the doctoral fellowship. We are thankful to Prof. Keith Knutson from Mayo Clinic for ANV-1 cells as a kind gift. Associated Content


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Figure 1. (A) Cytotoxicity studies of ANV-1 cells after 48h of continuous treatment either with ESC8 (E) drug or with DX liposome or with DX-Cont, DX-Hsp90, DXE-Cont, DXE-Hsp90 lipoplexes while maintaining ESC8 concentration at 7 µM in every ESC8 carrying treatment groups; ** denotes p

Development of Liposomal Formulation for Delivering Anticancer Drug

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