Induction of Mitochondrial Cell Death and Reversal of Anticancer Drug

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Induction of Mitochondrial Cell Death and Reversal of Anticancer Drug Resistance via Nanocarriers Composed of a Triphenylphosphonium Derivative of Tocopheryl Polyethylene Glycol Succinate

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Yuvraj Singh,† K. K. Durga Rao Viswanadham,†,# Vivek K. Pawar,† Jayagopal Meher,† Arun Kumar Jajoriya,‡ Ankur Omer,§ Swati Jaiswal,∥ Jayant Dewangan,§ H. K. Bora,⊥ Poonam Singh,∇ Srikanta Kumar Rath,§ Jawahar Lal,∥ Durga Prasad Mishra,‡ and Manish Kumar Chourasia*,† †

Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow-226031, India Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow-226031, India § Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow-226031, India ∥ Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow-226031, India ⊥ Laboratory Animals Facility, CSIR-Central Drug Research Institute, Lucknow-226031, India ∇ CSIR-Central Electrochemical Research Institute, Karaikudi-630003, Tamil Nadu India ‡

S Supporting Information *

ABSTRACT: We have devised a nanocarrier using “tocopheryl polyethylene glycol succinate (TPGS) conjugated to triphenylphosphonium cation” (TPP-TPGS) for improving the efficacy of doxorubicin hydrochloride (DOX). Triphenylphosphonium cation (TPP) has affinity for an elevated transmembrane potential gradient (mitochondrial), which is usually high in cancer cells. Consequently, when tested in molecular docking and cytotoxicity assays, TPP-TPGS, owing to its structural similarity to mitochondrially directed anticancer compounds of the “tocopheryl succinate” family, interferes specifically in mitochondrial CII enzyme activity, increases intracellular oxidative stress, and induces apoptosis in breast cancer cells. DOX loaded nanocarrier (DTPP-TPGS) constructed using TPP-TPGS was positively charged, spherical in shape, sized below 100 nm, and had its drug content distributed evenly. DTPP-TPGS offers greater intracellular drug delivery due to its rapid endocytosis and subsequent endosomal escape. DTPP-TPGS also efficiently inhibits efflux transporter P glycoprotein (PgP), which, along with greater cell uptake and inherent cytotoxic activity of the construction material (TPPTPGS), cumulatively results in 3-fold increment in anticancer activity of DOX in resistant breast cancer cells as well as greater induction of necroapoptosis and arrest in all phases of the cell cycle. DTPP-TPGS after intravenous administration in Balb/C mice with breast cancer accumulates preferentially in tumor tissue, which produces significantly greater antitumor activity when compared to DOX solution. Toxicity evaluation was also performed to confirm the safety of this formulation. Overall TPPTPGS is a promising candidate for delivery of DOX. KEYWORDS: TPGS, mitocans, doxorubicin hydrochloride, drug resistance, nanotechnology, molecular modeling

1. INTRODUCTION Numerous causatives behind the dire state of cancer are expanding. First is the extreme level of genetic heterogeneity even among the same cadre of cancer cells inflicting a person, which implies that drug targets are often nonuniform and consequently the response produced by even the most potent cytotoxic drugs is circumstantial.1−3 Second is emergence of multidrug-resistant (MDR) cells amidst susceptible ones during the course of treatment. The exponential rate of cancer cell growth selects mutant cells which develop evasive mechanisms, such as overexpressed efflux proteins [P© XXXX American Chemical Society

glycoprotein (PgP)] or altered regulatory proteins like stathmin or topoisomerase, and as a result cancer proliferates to such an extent that sometimes proven chemotherapeutic regimens can be rendered useless. This happens even if substantial plasma drug levels are attained.4,5 Third is extreme nonspecificity among prevailing cytotoxics, and fourth is the Received: February 10, 2019 Revised: August 9, 2019 Accepted: August 12, 2019

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DOI: 10.1021/acs.molpharmaceut.9b00177 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 1. Summary of work. (A) Scheme for synthesis of TPP tagged TPGS (TPP-TPGS), so as to increase the mitochondrial affinity of TPGS. We employed carbodiimide coupling to esterify the carboxylate terminus of TPP with the hydro-alcoholic tail of TPGS. Product was confirmed via 1 H NMR. (B) Steps in rational conceptualization of a smart multifunctional carrier material based on its structural homology with excipients possessing known actions. Tocopheryl succinate (shaded blue; structural moiety also present in TPGS) is an established inhibitor of mitochondrial complex II and induces intracellular ROS generation and cell death, whereas TPGS and Solutol HS15 are inhibitors of multidrug resistance producing efflux protein “PgP” owing to their lengthy alcohol chains (shaded green). (Cand D) A functional nanocarrier developed from TPPTPGs with DOX as pay load (DTPP-TPGS) will theoretically assist the drug’s cytotoxic action by enhancing its intracellular delivery (by way of its charge and affinity for mitochondria), inhibiting the drug’s PgP efflux as well as acting on its own to induce ROS generation and cause apoptosis.

“tocopheryl succinate” moiety is present in tocopheryl polyethylene glycol succinate (TPGS) also, there is a strong possibility that TPGS’s conjugate with TPP+ (TPP-TPGS) might also elicit a similar mitochondrially directed apoptotic action resulting in formation of a compound capable of being classified as a mitocan (Figure 1). Also, TPGS has the unique capability to inhibit PgP (the efflux protein which constantly flushes out substrates from intracellular sites), and subjugation of the efflux pump would increase the activity of cytotoxics even against MDR cells.16,17 Doxorubicin hydrochloride (DOX) is a crucial anticancer drug in terms of efficacy and diversity of usage. It is on WHO’s List of Essential Medicines and is used in many cancers, including solid tumors, hematological malignancies, and soft tissue sarcomas. It works by intercalating DNA and also induces free radical generation by disrupting mitochondrial membrane, which triggers terminal apoptotic pathways causing cell death. Unfortunately, it is an excellent substrate for efflux

physicochemical and dispositional characteristics of several anticancer drugs, their susceptibility to first pass metabolism, aqueous instability, very high dose requirement, or challenging dosing regimen.6−9 Given this bleak outlook and ever burgeoning cancer burden worldwide, finding novel and potent anticancer drugs is very urgent.10,11 One such emergent drug class is “Mitocans” (mitochondrially targeted anticancer agents) which target malignant cell mitochondria and induce apoptosis. Tocopheryl succinate, a highly coveted representative of the vitamin E group of mitocans, alters mitochondrial complex II (CII) functioning by displacing the enzyme’s endogenous ligand ubiquinone, which generates excessive reactive oxygen species (ROS), and induces cell death. To augment tocopheryl succinate’s mitochondrial affinity, researchers have complexed it to a delocalized lipophilic cation, triphenylphosphonium (TPP).12 The said conjugate associates fervently with mitochondria of cancer cells and seems to be a focal area for next generation anticancer drugs.13−15 Since a B


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bromide (5 g, 11.68 mmol, 1 equiv), TPGS (17.48 g, 11.68 mmol, 1 equiv), and N,N-dimethylaminopyridine (DMAP) (0.57 g, 4.65 mmol, 0.4 equiv) were added and stirred magnetically for 30 min. Thereafter, an additional 20 mL of dry DCM containing predissolved DCC (2.64 g, 12.65 mmol, 1.2 equiv) was introduced into the reaction mixture and allowed to stir further for 1 h in the ice bath. The system was then shifted to room temperature and allowed to stir for 24 h more. Reaction was monitored by TLC (mobile phase 2:8 methonol:DCM) and upon completion, the reaction mixture was filtered. Two cycles of water (100 mL) and one cycle of brine (100 m) were used to wash the filtrate, which was subsequently dried using anhydrous Na2SO4, and evaporated. The crude resultant was recrystallized in diethyl ether to afford a white solid in excellent yield 90%. Final compound, 3a, (TPP-TPGS) was confirmed by 1H NMR. 2.4. Molecular Modeling. The binding tendency of TPPTPGS to mitochondrial respiratory CII protein (Protein 1ZOY) was analyzed via molecular modeling. To study the target and compound interaction: molecule was docked in target site docking using Autodock Va software which depends on empirical scoring functions and is PDBQT compatible. Additionally, Autogrid, was used to generate the grid surrounding the target protein’s active site. The two dimensional structure of the compound (TPP-TPGS) was drawn with the help of ChemDraw 11.0, and the file was saved in SDF format. The structure of the compound was then optimized by MM2 force field through Chemdraw v11. OpenBabel software was used to convert the ligand file format from SDF to PDBQT and from PDBQT to PDB.19 Three dimensional structure of Protein 1ZOY was imported from RCSB’s protein data bank. Further, AutoDockTools (ADT) (version 1.4.5) was used to create Receptor file. The docking area was fixed by a 92 × 112 × 100 three-dimensional grid centered on the Ubiquinone QP (ligand) binding site.20 The interactions between protein-TPP-TPGS complex were studied with the help of Ligplus and Pymol software. 2.5. In Vitro CII Activity. Mitochondrial complex II, or CII, or CII dependent succinate dehydrogenase (SDH) activity was measured by employing a modified MTT assay (4 h exposure to test substance dissolved in a medium supplemented with excessive succinic acid so as to ensure that only succinate ion drives the cellular respiratory process).14 Briefly MTT concentrate (4 mg/mL) was dissolved in pH 7.4 phenol red-free RPMI media containing predispersed 20 mM succinic acid to form the working MTT solution. Solutions of TPPTPGS (in DMSO), 3-nitropropionic acid (in ethanol), MitoQ (in ethanol), or 3-bromopyruvate (in PBS) in MTT solution were added to semiconfluent cells followed by coincubation for 4 h. Thereafter, supernatant was discarded and formazan crystals in the individual wells were dissolved using DMSO solution and subjected to photometric evaluation at 565 nm. 2.6. Apoptosis, Reactive Oxygen Species, and Mitochondrial Membrane Potential Disruption. For quantifying apoptosis induced by TPP-TPGS, cells were sowed in a 6 well plate and exposed to TPP-TPGS (25 μM in DMSO) for 24 h. Thereafter, cells were washed, trypsinized, and dispersed in 500 μL of binding buffer which had preadded 5 μL of PI and 5 μL of annexin V-FITC. Cells in contact with binding buffer were incubated for 30 min in the dark and subjected to flow cytometry to quantify differentially stained cells. Simultaneously, cellular ROS was detected with the probe H2DCFDA by flow cytometry in cells treated with 25

transporter PgP, which reduces its efficacy, and this is where a nanonosized delivery vehicle made out of purported “TPPTPGS conjugate” would fit in perfectly. If the anticipated mitocan activity of TPP-TPGS does exist, we will have an extremely clever delivery vehicle which will not only ward off efflux of PgP substrate, DOX, but will also be inherently cytotoxic: potentiating cytotoxic response of the very drug it protected from efflux in the first place. Furthermore, the miniscule size of nanocarrier will result in painless intravenous administration and inadvertent passive targeting toward perforated microvasculature of tumor, resulting in preferential drug accumulation. The diffuse positive charge supplied by TPP would make delivery system positively charged, thereby increasing probability of its interaction and internalization by a negatively charged cancer cell membrane. To validate the above hypothesis (diagrammatically exemplified in Figure 1), a TPP-TPGS, TPGS, and Solutol HS-15 containing nanocarrier with doxorubicin hydrochloride (DTPP-TPGS) was manufactured. The anticancer activity of DTPP-TPGS was tested in 4T1 breast cancer cells via their ability to reverse drug resistance in especially conditioned MCF-7 cells overexpressing PgP. Pharmacokinetic study was also conducted via LC-MS and fluorescence imaging. In vivo efficacy studies (in Balb/C mice carrying breast cancer) were also done to investigate the anticancer potential of DTPPTPGS.

2. EXPERIMENTAL SECTION 2.1. Materials. DOX was obtained as a kind gift from Fresenius kabi, Gurgaon, India. Tocopheryl polyethylene glycol-1000-succinate (TPGS), 3-carboxypropyl triphenyl phosphonium bromide (TPP), dialysis membrane (MWCO 10000−12000 Da), Solutol HS15, fetal bovine serum (FBS), ribonucleaseA, RPMI 1640 cell culture Medium (incomplete), antibiotic solution (penicillin/streptomycin, 0.1% v/v), and MTT dye were requisitioned from Sigma-Aldrich, MO, USA. JC-1 Dye, DAPI, Lysotracker green, Early endosomal marker (GFP), propidium iodide (PI), and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) were brought from Invitrogen, OR, USA. Triple distilled ultrapure water was produced in-house using Milli-Q plus 185 water purifier (Millipore, MA, USA). 2.2. Cell Culture. MCF-7 cells, and 4T1 cells, were indented from an institutional repository. Cells were cultured in RPMI 1640 medium with 10% FBS, 1% L-glutamine/l, and antibiotic solution in a 5% (v/v) CO2/air mixture at 37 °C. Drug resistance induction in MCF-7 cells (R-MCF-7): Growth media of parent MCF-7 cell line was supplemented with very dilute content of DOX. Cells were grown over multiple passages and division cycles, under exposition of incremental DOX concentration (0.01 to 1 μM). When cells could survive in the above quoted mediums, they were maintained in media containing 0.25 μM DOX. Induction of resistance was cross validated by gauging the comparative PgP countenance in parent and drug resistant R-MCF-7 cell lines by fluorescence microscopy and Western blotting. Prior to experimentation, drug resistant cells were cultured for 2 weeks in DOX free medium.18 2.3. Synthesis of TPP-TPGS. The scheme for reaction is given in Figure 1A. Briefly, 80 mL of dry DCM was taken in a 250 mL round-bottom flask fixed in an ice bath. To this DCM, accurately weighed (3-carboxypropyl)triphenylphosphonium C


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Molecular Pharmaceutics μM TPP-TPGS for 6 h along with an extra group of cells which in addition to TPP-TPGS were simultaneously subjected to polyethylene glycol-SOD (500 U/ml; Sigma). Disruption of mitochondrial membrane potential was evaluated via flow cytometry. Briefly, cells were treated with TPP-TPGS (25 μM in DMSO) for 6 h. JC-1 dye was used as the fluorescent stain, and carbonyl cyanide m-chlorophenyl hydrazine (CCCP) acted as positive control. 2.7. Development of Nanocarrier (DTPP-TPGS). DOX loaded nanocarrier (DTPP-TPGS) was prepared by modifying a previously described method.21 Briefly, 60 mg of polymer (TPP-TPGS), 20 mg of TPGS, and 10 mg of Solutol HS-15 were solubilized in 3 mL of ethyl acetate (O phase). Separately, 10 mg of drug was dissolved in 1 mL of water to form the first aqueous phase (W1). Thereafter, under probe sonication (Sonics, USA, 40% amplitude for 3 min, narrow tip), W1 was added dropwise to the O phase (in an ice bath) to form a primary W/O emulsion. After a standing period of 2 min, a second aqueous phase, W2 (3 mL of 5 mg/mL NH4Cl solution) was similarly introduced dropwise to the primary emulsion under probe sonication (10 min at 30% amplitude) to form a W1/O/W2 multiple emulsion. Formulation was thereafter evaporated under vacuum to eliminate organic phase (ethyl acetate) and fully ripen the nanocarriers. Unentrapped drug and dissolved NH4Cl were removed by dialyzing against water to arrive at the final working formulation (DTPP-TPGS). This formulation was subjected to a variety of experiments as discussed next. For comparative purposes blank nanocarriers of TPP-TPGS (NTPP-TPGS) were prepared as above without addition of drug. 2.8. Cytotoxicity Study and Scratch Migration Assay of DTPP-TPGS. In vitro cytotoxicity of the developed formulation was assessed using the MTT assay. Cells were seeded (0.5 × 104 cells/well) in a 96 well plate. After priming, they were treated with DOX solution, DTPP-TPGS, at differing concentrations, and NTPP-TPGS in amounts which mimicked DTPP-TPGS. Once 24 h had elapsed, wells were drained and adhered cells were subjected to 0.5 mg/mL MTT solution (in culture media) for 4 h further. Precipitated formazan crystals were solubilized using DMSO, and the colored solution so formed was evaluated spectrophotometrically at 572 nm. Inhibitory concentrations (IC50) of tested groups were determined using nonlinear regression. The protocol followed for cell migration assay is briefly described next. After 80% cell confluence had been attained in 6-well plates, a clear and uniform scratch was engraved through the cell layer using a sterile pipet tip. Subsequently, cells were treated with culture medium with or without treatments (MCF-7, 4T1 cells with DOX concentration equivalent to 100 nM, and R-MCF-7 cells with DOX concentration equivalent to 1 μM), and the scratched area was photographed at regular intervals at 0, 12, and 24 h under a light microscope at 10× objective. 2.9. Cell Uptake Study. Cellular uptake of DTPP-TPGS in R-MCF-7 and 4T1 cells was determined using flow cytometry. Cells were sowed in a 6 well plate (0.5 × 105 cells/well) and treated with DOX solution and DTPP-TPGS for 6 h, respectively. Following treatment, cells were washed with PBS, trypsinized, centrifuged for 2 min, rewashed, collected in sampling tubes, and subjected to flow cytometry analysis to measure median cell fluorescence produced by DOX uptake (FACS Calibur, software Cell Quest Pro). For

investigating the mechanism of uptake, adherent R-MCF-7 cells (106 cells/well) were pretreated with different uptake inhibitors respectively (250 μM monodansyl cadaverine, 15 min, blocks clathrin-mediated endocytosis; 5 μM rottlerin, 45 min, blocks macropinocytosis; 200 μg/mL colchicine, 3 h, blocks pinocytosis; 20 μg/mL cytochalasin B, 3 h, blocks phagocytosis; 50 μg/mL nystatin, 30 min, blocks caveolaemediated endocytosis; 15 μg/mL polyinosinic acid, 45 min, blocks scavenger receptor; and 100 μM dynasore, 45 min, blocks dynamin-dependent endocytosis). After pretreatment, cells were further incubated with DTPP-TPGS for 6 h and fluorescence emanating from 10000 cells was calculated using FACS as above.22 2.10. Western Blot Analysis for PgP, Apoptotic, Antiapoptotic Proteins: Intracellular ATP Assay and Rhodamine 123 Efflux Assay. To determine the variation in levels of PgP induced by developed formulation, R-MCF-7 cells were sowed in 6 well plates (105 cells/well) and treated with DOX solution, DTPP-TPGS (equivalent to 1 μM DOX), and NTPP-TPGS. After 24 h of treatment, cells were washed thrice with chilled PBS, lysed (using SLS) and treated to a cocktail of protease inhibitors. Cellular lysate was pelleted by spinning it at 10000 rpm for 5 min. Protein extract (40 μg) so obtained was resolved using 10% SDS−PAGE electrophoresis and transferred onto nitrocellulose membranes which were subsequently blocked and treated with appropriately diluted primary antibodies for Pgp, BAX, Bcl-2, survivin (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Calbiochem, San Diego, CA). After washing, membranes were treated with horseradish peroxidase (HRP) conjugated goat secondary antibody. Protein bands were detected by a chemiluminescence system (Merck Millipore). For ATP assay, R-MCF-7 cells were cultured in 6-well plates (105 cells/well) for 24 h at 37 °C and then treated with 25 μg/ mL TPP-TPGS, TPGS, and equivalent NTPP-TPGS dispersion for a duration of 12 h. Subsequently generated intracellular ATP was quantified using bioluminescence based Sigma-Aldrichʼs ATP Determination Kit. In rhodamine efflux assay, a different set of R-MCF-7 cells were pretreated with 25 μg/mL TPP-TPGS, TPGS, equivalent NTPP-TPGS for 24 h and verapamil (5 μM) for 12 h. Cells were then incubated with rhodamine 123 (10 μg/mL) for 45 min, washed thrice with PBS, detached, spun, and subjected to flow cytometric analysis to measure rhodamine 123 associated fluorescence as it is a substrate for PgP. 2.11. Relative Cell Uptake, Endosomal Trafficking, Generation of ROS via Confocal Microscopy. For cell uptake, R-MCF-7 cells were grown on poly L-lysine coated coverslips in 6-well plates and treated with 500 nM DTPPTPGS and DOX solution respectively for 6 h. Thereafter, cells were fixed in formaldehyde (3.7% v/v in PBS), permeabilized [Triton-X (0.5% v/v)], and mounted on slides with Fluoroshield DAPI. Endosomal trafficking was also observed. Briefly cells were grown to semi confluency and transfected with CellLight Early Endosomes-GFP, BacMam 2.0 (Thermo Fisher Scientific, OR, USA) for 24 h. Thereafter cells were treated with 500 nM DTPP-TPGS for 30 min and processed as before. Since terminal lysosomal vacuoles appear only after 2−3 h of material engulfment, cells were grown to 70% confluence and treated with 500 nM DTPP-TPGS for 3 h. The media was then aspirated off, and treated cells were further treated with Lysotracker green-DND (ThermoFisher Scientific, OR, USA), D


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2.15. Toxicity Study. Balb/C mice weighing approximately ∼20 g were used to determine the acute toxicity potential of the formulation. Testing was performed as per institutionally approved protocol (10 mg/kg DOX) with three equivalent doses spaced 72 h apart. Twenty animals were divided into groups of five animals each. The first group was intravenously administered PBS and acted as control. Simultaneously, DOX solution, DTPP-TPGS, and NTPPTPGS were administered to the second, third, and fourth groups, respectively. On the 15th day, animals were euthanized for organ collection. Excised organs were examined histologically. Briefly, organs were fixed in formalin (10% in PBS). Fixed organs were thereafter embedded in paraffin wax blocks and sliced into thin sections using automatic micro tome (Leica, model- 2155). The sections were counter-stained with hematoxylin and eosin and photographed microscopically (Eclipse 50i, Nikon, Japan).

and processed as before. ROS generation in 4T1 cells after treatment with NTPP-TPGS, DOX solution, and DTPP-TPGS (equivalent to 200 nM DOX) for 3 h was observed by staining cells for 5 min in dark conditions with H2DCFDA (10 μM). H2O2 was used as a positive control. Prepared slides were observed under an Olympus FV1200MPE confocal microscope. Images were acquired at 60× zoom. 2.12. Cell Cycle Distribution and Apoptosis after Treatment with DTPP-TPGS. To gauge changes in cell cycle distribution brought about by treatment of DTPP-TPGS, DOX solution, and NTPP-TPGS in R-MCF-7 (500 nM equivalent of DOX), MCF-7 (100 nM equivalent of DOX), and 4T1 (100 nM equivalent of DOX) cells, a PI based flowcytometry assay was used. Briefly, cells were planted in six well plates (106cells/well). After priming, cells were exposed to above the quoted formulations for 24 h. Thereafter, cells were washed, detached, pelleted, rewashed, and fixed overnight using chilled ethanol (70% v/v). After fixation, cells were spun again and resuspended in 200 μL of PBS and sequentially treated with ribonuclease A (100 μg/mL) and PI (50 μg/mL) for 10 and 15 min, respectively. Fluorescence emanating as a consequence of PI stained cells was analyzed by flow cytometry. The type and extent of apoptosis induction by treatments similar to that in the cell cycle assay was quantified as per protocol described in preceding sections. 2.13. In Vivo Antitumor Efficacy. About 5−7 week old female Balb/C mice (weighing 15−20 g) were subcutaneously injected with 4T1 cells (106 cells/mice) in the mammary fat pad region of the lower right abdominal quadrant to induce breast cancer generation. Post inoculation, a 10 day waiting period was observed for tumors to become palpable. Subsequently animals were screened on the basis of tumor volume (∼100 mm3) and divided into four groups. Beginning from the 12th day after cell inoculation, different animal groups were administered DOX solution, NTPP-TPGS, DTPP-TPGS, and PBS via tail vein at a dose equivalent to 4 mg/kg DOX. Three doses were administered at intervals of 72 h. The study was culminated by sacrificing animals on the 27th day. Antitumor efficacy was measured by gauging tumor dimensions during as well as on the day of study termination. 2.14. Pharmacokinetics. Breast cancer bearing Balb/C mice (as developed in antitumor efficacy studies) were used for pharmacokinetic and organ distribution study of intravenously administered DOX solution and DTPP-TPGS (dose equivalent to 4 mg/kg for both groups). Approximately, 100 μL of blood was drawn from the retero-orbital plexus of dosed animals into heparinized microcentrifuge tubes (Eppendorf, Germany) at predetermined time points (0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 24, 48, and 72 h). Samples were centrifuged in a cooling centrifuge to separate plasma. A parallel dosed animal set was sacrificed for organ collection at 2, 6, 10, 24, 30, and 72 h post dosing. A protein precipitation method was used to extract drug from plasma and tissue samples. Briefly, plasma or tissue homogenate (20 μL; blank, spiked or test) was treated with 180 μL of solvent blend [70% acetonitrile:30% ammonium acetate buffer (pH 3.5, 0.01 M)]. The resulting mixture was vortexed and centrifuged to precipitate the protein matrix. Subsequently supernatant (100 μL) was transferred into HPLC vials and injected into an analytical system (Refer to Supporting Information for LC-MS configuration). Win Nonlin 6.0 (Pharsight Co., Mountain View, CA, USA) was used to derive the pharmacokinetic parameters from the obtained sample concentrations.

3. RESULTS AND DISCUSSION Literary sources suggest mitochondrial complex II (CII) to be a highly invariant target especially considering the heterogeneity of cancer cells.23 Murphy et al. had previously tagged a lipophilic cationic group on antioxidants, to generate mitochondrially active compounds.24 Also Dong et al. had shown that lipophilic cationic compounds were selectively internalized by mitochondria of cancer cells, due to inherently greater electrochemical gradient: a difference of at least 30 mV against normal cells.25 The premise that the mitochondrial membrane potential is much more in cancer cells has already been exploited using a TPP (diffuse lipophilic cation) tagged compound which was found to be ten times more selective for cancer cells than normal cells.26 We therefore conjugated TPP to TPGS (which not only has PgP inhibitory activity but is structurally similar to a proven mitocan vitamin E succinate) in anticipation that it will lead to a multifunctional compound: which possesses mitochondrial complex II directed cytotoxic activity as well as PgP inhibitory activity, and ultimately can be used as a carrier for DOX. Our aim, therefore, was to raise the cytotoxic potential of a standalone drug DOX, by using a carrier which itself has a preferential mitochondrial complex directed activity. The first objective in this direction was to synthesize TPP-TPGS (3a) by providing a diffuse positive charge to TPGS. We proceeded as per the scheme in Figure 1A by exploiting the carboxylate residue of TPP and the hydroxyl group of TPGS and conducted an esterification reaction catalyzed by DCC in dry conditions. The final compound was obtained in high yield (90%) and isolated as a white solid. Product formation was confirmed by 1H NMR (refer to the Supporting Information). 3.1. Molecular Modeling and Binding to CII. Affinity between synthesized copolymer TPP-TPGS and mitochondrial complex II (CII) was estimated using published crystal structure of mammalian CII (1ZOY) due to its high sequence identity with human CII (95−97% for individual subunits). The investigated binding site, i.e. proximal Ubiquinone Qbinding site (QP) has been reported as important for electron funnelling.27 A total of two dockings were performed. For each docking run, nine conformations of TPP-TPGS were generated. Only those conformations which had greater than −7.0 kcal/mol energy score and compatible interactions were taken. The interactions clearly showed potential of TPP-TPGS to bind strongly with the active site of protein and thereby possibly possess CII inhibitory properties. As seen from 2D E


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Figure 2. Molecular modeling. 2D and 3D confirmations of TPP-TPGS and mitochondrial complex II interaction at the Qp binding site. The TPP headgroup acts as a charged anchor which guides TPP-TPGS in intracellular spaces toward the mitochondria, while the PEG chain conveniently slithers along contours of CII allowing greater penetration of the tocopheryl succinate moiety into the binding pocket and formation of multiple hydrogen bonds. The interaction energy of TPP-TPGs and CII complex was comparable to endogenous ligand ubiquinone.

3.2. In Vitro CII Activity. To validate results of molecular modeling, we tested and found that breast cancer cells accumulated ROS (Figure 3A) and underwent significantly greater apoptosis (Figure 3B) when exposed to TPP-TPGS. Pretreatment of cells with SOD (which is a free radical quencher) suppressed ROS generation and also decreased apoptotic population. TPP-TPGS (25 μM) was further assessed for its ability to alter mitochondrial membrane potential of cancer cells (Figure 3C), and it caused mitochondrial depolarization in more than 30% cells. Previous studies on mitochondrially targeted vitamin E succinate revealed that it could bind to the ubiquinone binding site in CII and act as competitive inhibitor of succinate dehydrogenase.14,15,25 We consequently followed suit and investigated whether TPP-TPGS with its similar structure interferes with ubiquinone binding to CII. We utilized succinate in culture medium at a concentration (20 mM) high enough to enter cells and induce mitochondrial respiration via CII. Cells (MCF-7, R-MCF-7, and 4T1) were

and 3D confirmations of ligand protein interaction in Figure 2, TPP-TPGS was found to dock into the proposed QP site. The TPP headgroup despite being excluded from the lipid bilayer formed multiple hydrogen bonds with Tyr 91 and Try173 (dashed lines) and therefore acted as a charged anchor. Also the lengthy polyethylene glycol chain looped conveniently along the contours of the binding site, increasing penetration of the tocopheryl succinate headgroup into the bilayer. The residues Trp-B173, His-B216, Ser-C42, Arg-C46, Tyr-D91, and Asp-D90 are the conserved residues and have been found responsible for their specific substrate binding and catalytic function of the protein CII.28 Figure 2 clearly shows that TPPTPGS is actively involved in forming H bonding with the Trp B173, His-B216, Tyr-D91, and Asp-D90 which correspond to ubiquinone binding amino acids in the active site of the protein. The interaction energy of TPP-TPGS (−16.4 kcal/ mol) was comparable to that of ubiquinone (−15.7 kcal/mol), thereby further substantiating the probability of TPP-TPGS binding and affecting the function of CII. F


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Figure 3. In vitro CII activity of TPP-TPGS. (A) ROS generation, (B) apoptosis, and (C) mitochondrial membrane depolarization produced by TPP-TPGS in MCF-7, R-MCF-7, and 4T1 cells. Cells were treated with TPP-TPGS at 25 μM for different time periods and assessed for DCFDA, annexin V, and JC1 green associated fluorescence. It was found that TPP-TPGS produced significantly greater ROS generation, apoptotic cell death, and mitochondrial membrane potential disruption in all three cell types when compared to untreated control. A free radical quencher super oxide dismutase was able to reduce ROS load and apoptotic population in TPP-TPGS treated cells. Data is reported for n = 3 samples. (D and E) Ability of TPP-TPGS to interfere in CII activity via inhibition of succinate dehydrogenase enzyme of breast cancer cells was studied by estimating MTT reduction in RPMI fortified with 20 mM succinic acid, after a 4 h incubation period. 3-Bromopyruvate (3BP) and 3-nitropropionic acid (3NPA) treated cells were used as positive control. In (D) cells were preincubated for 60 min with MitoQ at indicated concentrations before addition of TPP-TPGS for a similar 4 h incubation and MTT reduction assessment. Reported as mean ± s.d. (%) reduction of MTT absorbance relative to untreated control.

reduction also occurred after treatment with TPP-TPGS (Figure 3D and E). Also, MitoQ, a mitochondrially directed derivative of ubiquinone, which preferentially binds to CII, overcame inhibition in MTT reduction by TPP-TPGS. These results suggested that TPP-TPGS inhibits CII associated SDH activity. Literary sources report that when α-tocopheryl succinate displaces ubiquinone from its binding site on CII,

tested for SDH activity after subjecting them to varying concentrations of TPP-TPGS with 3-nitropropionic acid (3NPA) and 3-bromopyruvate (3BP) acting as positive controls. Because of overt dependency of cells on succinate to drive CII-mediated MTT reduction, treatment with known SDH inhibitors 3NPA and 3BP inhibited MTT reduction in a dose-dependent manner. Interestingly, inhibition of MTT G


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Figure 4. Physicochemical characteristics of DTPP-TPGS. (A) Particle size and (B) zeta potential of DTPP-TPGS by DLS and electrophoretic mobility. (C and D) TEM and AFM images of DTPP-TPGS. Particles were fine ( DOX solution. To summarize in vitro experiments, it would be rational to infer that DTPPTPGS is a potent inhibitor of PgP and opens up auxiliary pathways (CII mediated ROS generation) for increasing DOX’s efficacy in drug resistant breast cancer cells. 3.5. Cell Uptake and Endosomal Trafficking. Confocal microscopy was used to investigate relative uptake and intracellular movement of DTPP-TPGS. Given time, DOX has a natural tendency to accumulate in the nuclear region due to its propensity to intercalate DNA. However, in microscopy

studies it was observed that after 6 h, the DOX content of DTPP-TPGS had near perfect colocalization with the DAPI stained nucleus, whereas DOX solution had fainter visibility in the nuclear region of R-MCF-7 cells (Figure 6A and B). This is a direct consequence of endocytosis and inhibition of PgP-efflux offered by a carrier, which as discussed before leads to greater cytotoxic action in R-MCF-7 cells. To further investigate endosomal trafficking of DTPP-TPGS, we labeled early endosomes by a GFP labeled antibody (Figure 6B). After an incubation period of 30 min, cytosolic fluorescence emanating from DTPP-TPGS was found to overlap with GFP labeled endosomes, which implied rapid internalization of drug carrier. However, for successful nuclear delivery of DOX, L


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Figure 9. Results of pharmacokinetic and in vivo imaging studies. (A−D) Comparative pharmacokinetic profiles of DOX solution, and DTPPTPGS (Dose 4 mg/kg for both groups) administered intravenously in tumor bearing mice. Values reported as mean ± s.d. (E) List of important pharmacokinetic parameters obtained by noncompartmental modeling of plasma and tissue distribution data. (F) Live in vivo images of animals treated with DTPP-TPGS and DOX solution. We see selective localization of DOX loaded in DTPP-TPGS in tumor and tumor associated regions (encircled) in comparison to DOX solution. After 24 h DOX solution does not produce signals, whereas DTPP-TPGS still has epifluorescence spots in the tumor region.

escape from endosomal vesicles is mandatory, as these are terminally pooled into degradative lysosomes. It has been found that delivery vectors constructed out of positively charged polymer such as PEI have the capability to buffer endosomal pH via a “proton sponge effect” and cause fluidization of the late endosomal membrane causing expulsion of endosomal contents.34,35 Since DTPP-TPGS also possessed an overtly positively charge, we observed its relative compartmentalization with acidic lysosomal vacuoles after incubation of cells with formulation for 3 h. It was found that DOX present in DTPP-TPGS was sitting independently in the nuclear region away from green acidic compartments present

cytosolically (Figure 6C). Therefore, it was concurred that apart from intrinsic cytotoxic action and PgP inhibitory activity, TPP-TPGS owing to its positive charge also supported timely endosomal escape of its payload which results in rapid intracellular DOX availability and consequently much greater cytotoxic activity. 3.6. Cell Cycle, Apoptosis, ROS Generation. Results (Figure 7A) suggest that DOX solution causes cell arrest (81.8% and 87.8% in MCF-7 and 4T1 cells) in the G2M phase which was significantly greater than control cells. However, DOX solution did not produce the same extent of arrest in G2M phase (41.5%) in case of R-MCF-7 cells. On the M


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body’s tendency to lose weight during chemotherapy is a welcome relief. To summarize the toxicity data, DTPP-TPGS presents a very mild toxic potential when compared to standard DOX solution due to its ability to control the release and pharmacokinetic distribution of DOX. 3.8. Pharmacokinetics. In pharmacokinetic study (Figure 9A−E), plasma levels of DOX obtained after i.v. administration of DOX solution corresponded to its dissolution pattern. A Cmax of 1054 ± 57.3 ng/mL was observed at 5 min after dosing. Thereafter plasma levels fell off rapidly as dispositional forces took over and drug was eliminated with a half-life of 5.39 ± 0.76 h. Contrastingly, animals treated with DTPPTPGS had a dividedly 9-fold lower Cmax of 157.5 ± 13.69 ng/ mL. We suspect this difference is a consequence of control exerted by the carrier system. For DTPP-TPGS, elimination changed drastically, resulting in 5-fold prolongation of half-life (25.8 ± 1.27 h) compared to DOX solution. Long half-life of drug raises the probability of drug load DTPP-TPGS reaching the tumor. Extended MRT (31.35 ± 3.6 h), lower clearance (2034 ± 69.8 mL/h/kg), and more than 2-fold greater AUC0‑∞ (1965.5 ± 108.4 h*ng/mL) of drug delivered by DTPP-TPGS in comparison to DOX solution point to sustained release of drug The tumor pharmacokinetic profile of DTPP-TPGS (Cmax 65.3 ± 10.7 ng/g and AUC0‑∞ 1513 ± 91.4 h*ng/g) was much more favorable than DOX solution (Cmax 10.45 ± 2.7 ng/g, AUC 0‑∞ 367 ± 58.4 h*ng/g). Further DTPP-TPGS accumulated to lower levels in heart (Cmax 62.9 ± 18.3 ng/g, AUC0‑last 2792 ± 135.6 ng/g*h) in comparison to DOX solution (AUC0‑last 3960 ± 144.8 ng/g*h, Cmax 139.4 ± 18.4 ng/g). We propose the advantages in pharmacokinetic behavior of DTPP-TPGS are a consequence of its (1) size (below 100 nm) and (2) material of construct; positively charged architecture will ensure greater tumor uptake. Live animal in vivo images were acquired for tumor carrying animals to portray fate of drug carried by DTPP-TPGS after intravenous administration (Figure 9F). Signal intensity was correlated to the relative location of DOX in the body of the animal. After 6 h, the extent of drug localization in and around the tumor tissue was lower for animal treated with DOX solution than for animal treated with DTPP-TPGS. Similarly, in a manner analogous to pharmacokinetic data, cardiac sequestration in the case of DTPP-TPGS was much lesser than in DOX solution. Additionally there was incremental drug accumulation in the tumor region which was greater than that produced at 6 or 12 h by DOX solution. At 24 h, DOX solution was not detectable and seemed sparsely distributed. Contrarily DTPP-TPGS at the same time point continued to produce signal from the tumor region (encircled).

contrary, DTPP-TPGS produced similar G2M phase arrest (70−75%) in all three cell lines, implying comprehensive anticancer activity. NTPP-TPGS caused predominantly G1S arrest which might be due to its TPGS content.36 DOX inhibits topoisomerase II, which damages DNA during the Sphase.37 Damaged DNA leads to G2M check point activation and explains greater localization of cell arrest in the G2M phase for DOX treated cells. Since DTPP-TPGS is active against all the three cell lines, it causes arrest of all the three cells in the G2M phase. DTPP-TPGS, DOX solution, NTPP-TPGS, caused necroapoptosis. DTPP-TPGS caused consistently greater necroapoptosis than DOX solution in all the cells. These results were in coherence with Western blotting results for apoptotic proteins as observed before (Figure 7B). Apart from DNA damage, generation of ROS is also a significant cytotoxic mechanism of DOX.38 Generated semiquinones, play a key role in depolarizing mitochondrial membrane potential, which results in extrinsic cell death. We had already established the ability of TPP-TPGS to act via CII, producing ROS, and consequently, we investigated whether DTPP-TPGS could also amplify the ROS load produced by DOX using probe H2DCFDA and confocal microscopy (Figure 7C). Expectedly, upon treatment of cells with DTPP-TPGS, DOX solution, and NTPP-TPGS, H2DCFDA was reduced to green fluorescent dye in cytosol. However, the relative fluorescence in DTPP-TPGS treated cells was greater than that in DOX solution treated cells, suggesting greater ROS stress. NTPP-TPGS also had prooxidant activity, which was responsible for greater stress produced by DTPP-TPGS. 3.7. In Vivo Antitumor Activity and Toxicity Study. Figure 8A demonstrates discernible reduction in tumor size in animals treated with DTPP-TPGS when compared to those treated with DOX solution. DTPP-TPGS (Figure 8B) significantly (p < 0.001) reduced tumor volume (102.36 ± 6.23 to 39.74 ± 10.2 mm3) in comparison to DOX solution (from 100.43 ± 5.20 to 76.38 ± 6.24 mm3). DTPP-TPGS caused approximately 4-fold and 1.5-fold greater reduction in tumor burden when compared to PBS or DOX solution, respectively (Figure 8C). This enhancement in antitumor activity is attributed to mitochondrially targeted oxidative stress exerted by the carrier material, greater relative uptake offered by DTPP-TPGS, improved tumoral localization due to EPR effect (refer to pharmacokinetic data), and specific targeting due to positively charged carrier material. We also conducted histological examination of major organs harvested from animals subjected to acute toxicity study (Figure 8E). Unsurprisingly, animals subjected to PBS had perfectly conserved tissue architecture. Whereas, animals treated with NTPP-TPGS had mild histological damage, suggesting low toxicity burden of carrier material. In contrast, the cardiac myofibrils, hepatic parenchyma, and glomerular spaces of animals treated with DOX solution were severely damaged. However, DTPP-TPGS only induced mild cardiac fibrillary thinning, and portal vein congestion. Body weight measurements were similarly favorable (Figure 8D) as animals treated with DTPP-TPGS (85.8 ± 3.02%) had significantly less (p < 0.001) change in body weight than those treated with DOX solution (72.2 ± 1.30%). Critical loss in body weight due to chemotherapy induced cytotoxicity of normal cells, anemia, GI shedding, etc. is a severe hindrance in sustained treatment of a patient, and any reduction in the

4. CONCLUSIONS We developed a novel nanocarrier composed of triphenylphosphonium cation (TPP) tagged tocopheryl polyethylene glycol succinate (TPP-TPGS) for improving the efficacy of doxorubicin hydrochloride in drug resistant breast cancer cells in vitro and 4T1 cell induced breast cancer in vivo. In future prospects DTPP-TPGS needs to be validated in multidrugresistant cancer cell expressing solid tumors and assessed for its long-term stability. Further we wish to utilize TPP-TPGS as a stabilizer to develop a robust and novel multifunctional Vitamin E nanoemulsion carrying multiple cytotoxic drugs. It is anticipated that the obtained delivery system will not only elicit a trifaceted assistive mechanism to reassert the efficacy of N


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popularly used P-glycoprotein substrates such as doxorubicin, paclitaxel, and docetaxel in solid tumors expressing a sizable population of multidrug-resistant cells but will also simultaneously instigate an immunomodulatory response which will prime the body toward impeding further tumor propagation on its own.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.9b00177. Confirmatory NMR spectra, protocols for characterization (electron microscopy, in vitro drug release of DTPP-TPGS), LC-MS method for detection of DOX, and in vivo real-time imaging (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone No.: +91 522-2772450, 2772550. Fax: +91 522 2623405. E-mail: [email protected]. ORCID

Manish Kumar Chourasia: 0000-0002-8318-9325 Present Address #

(K.K.D.R.V.) Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS DBT has supported this study by sanctioning grant BT/ PR14769/NNT/28/996/2015. Contributions of the flow cytometry and fluorescence microscope operators from SAIF Division of CDRI are duly acknowledged. This is CSIR CDRI Communication 9871.

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REFERENCES

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