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Dec 30, 2015 - Academy of Scientific & Innovative Research (AcSIR), 2 Rafi Marg, New Delhi, India. •S Supporting Information. ABSTRACT: Prior studie...
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Systemic Codelivery of a Homoserine Derived Ceramide Analogue and Curcumin to Tumor Vasculature Inhibits Mouse Tumor Growth Sugata Barui,†,‡,§ Soumen Saha,†,‡,∥ Venu Yakati,†,∥ and Arabinda Chaudhuri*,†,∥ †

Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad-500007, Telangana State, India ∥ Academy of Scientific & Innovative Research (AcSIR), 2 Rafi Marg, New Delhi, India S Supporting Information *

ABSTRACT: Prior studies reported significant anticancer activities of ceramides. However, anticancer activities of homoserine based ceramides have not been tested. With a view to compare the anticancer activity of ceramides and homoceramides, in the present study, we have synthesized four serine based and four homoserine based C8-ceramide analogues. Since many cancer cells have shown resistance to ceramides, curcumin is now being used in combination with ceramides because of its ability to reverse multidrug resistance. Aimed at targeting curcumin−ceramide combination to tumor endothelial cells, herein we have used a tumor vasculature targeting liposomes of a newly synthesized pegylated RGDGWK-lipopeptide. Importantly, the liposomal formulations of the homoserine based C8-ceramide analogue containing oleyl chain showed more promising antineoplastic activities under both in vitro and systemic settings than the liposomal formulations of commercially available C8-ceramide. Findings in the mouse tumor growth inhibition study revealed synergistic therapeutic benefit from simultaneous delivery of curcumin and a homoserine based ceramide containing oleyl chain to tumor vasculature. Results in RT-PCR and Western blot experiments suggest that inhibition of solid tumor growth is mediated via inhibition of PI3K-Akt signaling pathway. The present structure−activity study is the first report to demonstrate therapeutic promise of curcumin−homoserine based ceramide combination in antiangiogenic cancer therapy. KEYWORDS: ceramide analogues, pegylated RGDGWK-lipopeptide, tumor vasculature targeting, integrin receptors, antiangiogenic cancer therapy, curcumin−ceramide drug combinations

1. INTRODUCTION In addition to playing important structural roles in cellular membranes, ceramides have also been demonstrated to possess significant tumor suppressor properties including growth arrest, senescence, and apoptosis induction in cancer cells.1,2 The mechanistic origin behind the anticancer properties of ceramides remains incompletely understood. Prior studies have shown that they form large stable ceramide channels in membranes promoting cytoplasmic release of cytochrome c and procaspases from mitochondria.3−5 The size and integrity of ceramide channels depends critically on the local concentration of ceramides.4 Therefore, combination of exogenous ceramides/ceramide analogues with other therapeutics that increase de novo ceramide synthesis is likely to potentiate ceramide induced apoptosis of cancer cells. Curcumin, a dietary polyphenol from Curcuma longa, is an example of such potent inducer of de novo ceramide synthesis,6,7 and use of curcumin in combination with ceramides in combating cancer has begun. Recently, using a xenograft mouse tumor model for human osteosarcoma, Dhule et al. reported that a folate receptor (overexpressed in many cancer cells) targeted pegylated liposome containing curcumin and a commercially available © XXXX American Chemical Society

C6-ceramide encapsulated in its lipid bilayer region induces significant tumor growth inhibition.8 However, in depth structure−activity studies aimed at evaluating the therapeutic promise of the combinations of curcumin and different ceramide analogues in antiangiogenic cancer therapy have not yet been reported. In the antiangiogenic therapeutic modality of combating cancer, tumor cells are deprived of oxygen and essential nutrients by shutting down the sprouting of new blood vessels from existing blood vessels.9,10 The success of such therapeutic strategy crucially depends upon availability of suitable molecular markers expressed on tumor endothelial cell surfaces. Integrins, the transmembrane αβ-heterodimeric glycoprotein receptors, are examples of such unique surface markers of tumor endothelial cells.9 Integrins mediate adhesion of cells to extracellular matrix (ECM) or intercellular adhesion which, through modulation of intracellular signaling processes, regulate Received: August 21, 2015 Revised: December 19, 2015 Accepted: December 30, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00644 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Scheme 1. (A) Structures of Pegylated RGDGWK-Lipopeptide 1 and Pegylated RGELFK-Lipopeptide 2 and (B) Structures of Synthesized C8-Ceramide Analogues

cell survival and proliferation.11−13 Out of the existing 24 integrins, αvβ3, αvβ5, and α5β1 integrins are receiving widespread exploitation as antiangiogenic drug targets. These integrins and their distinguishing extracellular matrix ligands play important roles in mediating tumor angiogenesis.14 In addition to their uses as drug targets, these three integrins are also finding increasing applications for delivering potent cytotoxic drugs/genes/siRNAs selectively to tumor vasculatures.15−24 To this end, aimed at evaluating the promise of integrin receptor mediated delivery of ceramide−curcumin combination to tumor vasculatures, in the present structure− activity study we have synthesized 4 serine based and 4 homoserine based C8-ceramides (Scheme 1B). Using a newly synthesized pegylated analogue of a previously reported RGDGWK-lipopeptide19 (Scheme 1A), we have developed a stable α5β1-integrin receptor selective liposomal formulation for simultaneous delivery of curcumin and ceramides to tumor vasculatures. Herein, we show that intravenous administration of curcumin and homoserine based C8-ceramide (with oleyl chain) cosolubilized within the liposomes of this pegylated RGDGWK-lipopeptide 1 functions synergistically in inhibiting tumor growth in a syngeneic mouse tumor model.

2. MATERIALS AND METHODS 2.1. Reagents, Cell Lines, and Animals. DSPE-(PEG)27NH2 and C8-ceramide were purchased from Avanti Polar lipid. Curcumin, FBS (fetal bovine serum), cell culture medium, Rhodamine PE, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), FITC-labeled annexin V, dimethyl sulfoxide (DMSO), TRIzol, polyethylene glycol 8000, agarose, DOPE, cholesterol, and DOPC were obtained from Sigma, St. Louis, MO, USA. Rabbit polyclonal anti-Akt, anti-p-Akt, anti-cytochrome c, anti-Bad, anti-p-Bad, and anti-β-actin antibodies were obtained from Pierce Biotechnology (USA). Rabbit polyclonal anti-Lass 1−4 antibodies were procured from Abcam (U.K.). VE-cadherin antibody and Texas Red conjugated anti-mouse secondary antibody were purchased from Santa Cruz (USA). BCIP/NBT substrate solution and goat anti-rabbit secondary antibody (alkaline phosphatase conjugate) were procured from Calbiochem (USA). BrdU assay and blood vessel staining kits were purchased from Millipore (USA). CytoSelect 24-well cell migration and invasion assay kits (colorimetric format, 8 μm pore size) were obtained from Cell Biolabs Inc. (USA). Cell culture lysis reagent, first strand cDNA synthesis kit, TUNEL assay kit, and PCR master mix were purchased from Promega Corporation (USA). Antibiotics were purchased from HiMedia (India). Unless otherwise mentioned, all the other B

DOI: 10.1021/acs.molpharmaceut.5b00644 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Scheme 2. Synthetic Scheme for Pegylated RGDGWK-Lipopeptide 1a

a Reagents: (i) Boc-NH-(PEG)27-COOH, DCC, dry DCM, 12 h; (ii) dry DCM, TFA, 3 h, 0 °C; (iii) Fmoc-Trp(Boc)-OH (2 equiv), DIPEA (4 equiv), HATU (2 equiv), rt, 1 × 30 min, then 1 × 1 h; (iv) DMF:piperidine (4:1 v/v), rt, 4 min, (×2); (v) Fmoc-Asp(tBu)-Gly(Dmb)-OH (2 equiv), DIPEA (4 equiv), HATU (2 equiv), rt, 1 × 30 min, then 1 × 1 h; (vi) piperidine:DMF (1:4 v/v), rt, 4 min, (×2); (vii) Fmoc-Gly-OH (2 equiv), DIPEA (4 equiv), HATU (2 equiv), rt, 1 × 30 min, then 1 × 1 h; (vii) DMF:piperidine (4:1 v/v), rt, 4 min, (×2); (ix) Fmoc-Arg(Pbf)-OH (2 equiv), DIPEA (4 equiv), HATU (2 equiv), rt, 1 × 30 min then 1 × 1 h; (x) TFA−dry DCM (0.5%), 2 h, 0 °C; (xi) DCC, dry DCM, 12 h; (xii) DCM−DMF−diethylamine (2:1:1), 2 h, 0 °C; (xiii) TFA−thioanisole−TIS (2:2:1), 2 h, 0 °C; (xiv) Amberlyst Cl− ion-exchange resin.

along with essential supplemental kit were obtained from Lonza (USA). Female C57BL/6J mice were supplied by National Institute of Nutrition (Hyderabad, India). All the in vivo experiments were carried out by following standard guidelines and protocols approved by Institutional Biosafety and Ethical Committee. 2.2. Synthesis of Pegylated RGDGWK-Lipopeotide 1. Synthetic steps to prepare the pegylated RGDGWK-lipopeptide

materials were purchased from local commercial suppliers and were used without further purification. B16F10 and RAW264.7 cell lines were procured from National Center for Cell Sciences (Pune, India). Cells were cultured in DMEM medium (Sigma) supplemented with 10% FBS (South American Origin, Gibco) and 1% kanamycin−penicillin−streptomycin in a humidified atmosphere of 5% CO2 (in air) at 37 °C. Human umbilical vein endothelial cells (HUVECs) and its culture medium (EBM-2) C

DOI: 10.1021/acs.molpharmaceut.5b00644 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Scheme 3. Synthesis of C18-Oleyl and HC18-Oleyla

Reagents: (i) TBDPS-Cl, TEA, DMAP, DCM, 12 h, rt; (ii) oleylamine, EDCI, HOBt, DIPEA, DCM, 12 h, rt; (iii) TFA:DCM (1:2), 0 °C, 3 h; (iv) octanoic acid, EDCI, HOBt, DIPEA, 12 h, rt; (v) TBAF, THF, 3 h, rt.

a

vacuum for 4 h. The dried residue was treated with 2 mL of TFA:thioanisole:triisopropylsilane (TIS) (2:2:1, v/v) for 3 h at 0 °C, the reaction mixture was diluted with 8 mL of TFA:DCM (1:9, v/v), and the mixture was concentrated at 30 °C to about 1 mL using a rotary evaporator. The residue was transferred in a 15 mL centrifuge tube and diluted with Et2O (15 mL). The white precipitate was separated by centrifugation at 5000 rpm for 5 min. The supernatant was discarded, and the precipitate was collected, dried, and subjected to chloride ion exchange chromatography over Amberlyst IRA-400 resin (using methanol as eluent) to afford the target pegylated RGDGWKlipopeptide 1 as a white, fluffy solid (36.9 mg, 47% based on estimated intermediate 6, Scheme 2). The 1H NMR spectrum of the pure pegylated RGDGWK-lipopeptide 1 was recorded in CD3OD/CDCl3 (3/1, v/v). Severe line broadening, particularly in the range δ 3−5 ppm, was observed in the 1H NMR spectra (Figure S1A) plausibly due to presence of multiple exchangeable protons and polyoxyethene groups in the headgroup region of the amphiphile. Hence, the final target lipid was characterized by its molecular ion peak observed in MALDI-TOF (Figure S1B). The purity of the target pegylated RGDGWK-lipopeptide 1 was confirmed by reversed phase HPLC using a couple of mobile phases (A, methanol; B, methanol:water (95:5, v/v) (Figure S3). 1H NMR (500 MHz, CDCl3 + CD3OD): δ/ppm 0.9 [t, 6H, CH3(CH2)17−]; 1.1−1.5 [bs, 64H, −(CH2)16−; m, 6H, LysCγH2 + LysCδH2 + ArgCγH2]; 1.5−2.5 [m, 2H, LysCβH2; m, 2H, ArgCβH2; m, 2H, Asp C β H 2 , m, 4H, −N(−CH 2 CH 2 −) 2 ; m, 2H, −NCH2CH2−NH−CO; m, 2H, −NH−CO−CH2CH2−O; m, 2H, LysωCH2]; 2.8−3.4 [m, 2H, ArgCδH2; m, 2H, −N− CH2CH2−NH−CO−; m, 2H, −CO−NH−CH2CH2−O; d, 2H, TrpCβH2]; 3.4−4.8 [m, 4H, GlyCαH2; m, 1H, LysCαH; m, 1H, TrpCαH; m, 1H, AspCαH; m, 1H, ArgCαH + PEG]; 7.0− 7.6 [m, 5H, −CH2−C8H5NH(ind)] (Figure S1A). MALDITOF: m/z = 2568 [M]+ for C128H244N14O37 (Figure S1B). The nontargeting control pegylated RGELFK-lipopeptide 2 was prepared using the same synthetic strategy as used in preparation of pegylated RGDGWK-lipopeptide 1 using appropriately protected glutamic acid. The structure of RGELFK-lipopeptide 2 was confirmed using the MALDI-

1 are shown schematically in Scheme 2. Final lipid was characterized by MALDI-TOF. Purity of the target pegylated RGDGWK-lipopeptide 1 was analyzed by reversed phase HPLC. Steps i and ii: Pegylated amine intermediate 2 was prepared by following the same protocol as described earlier.22 Steps iii−viii: Solid phase peptide synthetic technique (Fmoc strategy based) was exploited for preparing RGDGWK peptide sequence as shown in Scheme 2. 100 mg of H-Lys(Boc)-2ClTrt resin 3 (Nε-Boc-lysine preloaded 2-chloro trityl resin with loading efficiency 0.72−0.77 mmol/g) was first swelled in 10 mL of DMF for 4 h. The swelled resin was coupled with Fmoc-Trp(Boc)-OH (2 equiv) using HATU (2 equiv) and DIPEA (4 equiv) at room temperature in DMF for 1.5 h to afford intermediate 4 (Scheme 2). The resin was first washed with 10 mL of DMF (×2) and then with DMF:piperidine (4:1, v/v, 10 mL, 4 min × 2) at rt to remove Fmoc group. Thereafter the same Fmoc strategy based peptide coupling protocols were applied for sequential coupling of Fmoc-Asp(tBu)-Gly(Dmb)OH, Fmoc-Gly-OH, and Fmoc-Arg(Pbf)-OH (2 equiv each) using HATU (2 equiv) and DIPEA (4 equiv) in DMF solvent for 1.5 h at rt for each amino acid to afford the resin associated hexapeptide intermediate 5 (Scheme 2). The resin-bound intermediate 5 was then washed thoroughly with DCM (10 mL × 5), dried, and reacted with 60 mL of TFA:DCM (0.5%, v/v) for 2 h at 0 °C to obtain protected hexa-peptide intermediate 6 (0.056 g, 56%). Steps ix−xii: The pegylated amine intermediate 2 (0.080 g, 0.04 mmol) was dissolved in dry DCM (3 mL), and the solution was poured into an ice cold solution of DCC (0.007 g, 0.03 mmol) in DCM (which was already kept under stirring condition for 30 min) and protected hexapeptide intermediate 6 (0.038 g, 0.03 mmol) in dry DCM (5 mL). The resulting solution was left stirred at room temperature for 12 h. Then the solvent was evaporated in the rotary evaporator at 30 °C and the residue was completely dried under high vacuum for 1 h. Fmoc group present in the resulting solid compound was removed by stirring with 2 mL of DCM:DMF:diethylamine (2:1:1, v/v) for 2 h at 0 °C. After removing solvent in the rotary evaporator at 30 °C, the residue was kept under high D

DOI: 10.1021/acs.molpharmaceut.5b00644 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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3.6−3.9 [m, 2H, −Si−O−CH2−]; 4.1−4.4 [m, 1H, −NH− CO−CH−NH−]; 5.2−5.4 [m, 2H, CH3(CH2)6CH2CH CHCH2(CH2)6CH2−NH−]; 7.2−7.8 [m, 10H, Si−(C6H5)2] (Figure S4A). ESI-MS of IIIb: m/z = 708 [M + 1]+ for C43H70N2O4Si (Figure S4B). Steps iii and iv: 5 mL of TFA was poured into a solution of intermediate IIIb (1.1 g, 1.6 mmol) in 10 mL of dry DCM at 0 °C. The reaction mixture was stirred for 3 h at 0 °C. Excess TFA was removed by nitrogen flushing. The resulting residue was dissolved in 200 mL of chloroform and washed sequentially with aqueous saturated NaHCO3 (100 mL × 3) and brine (100 mL × 1). The organic layer was collected, dried over sodium sulfate (anhydrous), and filtered. The filtrate was concentrated to afford free amine intermediate (0.9 g, 1.5 mmol, 95% yield). The resulting amine (0.9 g, 1.5 mmol) was dissolved in 10 mL of dry DCM, and the solution was added to an ice cold reaction mixture of solid HOBt (0.35 g, 2.3 mmol), EDCI (0.45 g, 2.3 mmol), and octanoic acid (0.33 g, 2.3 mmol) in 20 mL of dry DCM. Diisopropylethylamine (DIPEA) was then added dropwise to the reaction mixture under stirring condition until it became alkaline to litmus (pH 9). The resulting solution was stirred for 12 h at rt. The reaction mixture was then diluted with 100 mL of chloroform and washed sequentially with icecooled 1 N HCl (100 mL × 2), saturated aqueous sodium bicarbonate (100 mL × 2), and brine (100 mL × 1). The organic layer was collected, dried over sodium sulfate (anhydrous), filtered, and concentrated by rotary evaporator. The resulting residue was subjected to column chromatographic purification (60−120 mesh silica gel). Pure intermediate IVb (0.74 g, 67% yield) was eluted using 10% EtOAc− hexane (v/v). 1H NMR of IVb (300 MHz, CDCl3): δ/ppm =0.9 [t, 3H, CH3(CH2)6CH2CHCH−; t, 3H, CH3(CH2)6− CO−]; 1−1.8 [bs, 24H, CH3(CH2)6CH2CH CHCH2(CH2)6CH2−NH−; bs, 10H, CH3(CH2)5CH2−CO; s, 9H, −Si−C(CH3)3]; 1.8−2.2 [m, 2H, −O−CH2−CH2−; m, 4H, CH3(CH2)6CH2CHCHCH2(CH2)6CH2−NH−; m, 2H, CH3(CH2)5CH2−CO]; 3.1−3.3 [m, 2H, CH 3 (CH 2 ) 6 CH 2 CHCHCH 2 (CH 2 ) 6 CH 2 −NH−]; 3.6−4 [m, 2H, −Si−O−CH2−]; 4.5−4.7 [m, 1H, −NH−CO−CH− NH−]; 5.3−5.5 [m, 2H, CH3(CH2)6CH2CH CHCH2(CH2)6CH2−NH−]; 7.2−7.8 [m, 10H, Si−(C6H5)2] (Figure S5A). ESIMS of IVb: m/z = 734 [M + 2]+ for C46H76N2O3Si (Figure S5B). Step v: Intermediate IVb (150 mg, 0.2 mmol) obtained in the above step was dissolved in a mixture of 10 mL of dry THF and 1 mL of tetra-n-butylammonium fluoride (TBAF) at 0 °C for 3 h to ensure complete deprotection of tert-butyldiphenylsilyl (TBDPS) group. THF was removed by rotary evaporator, and the concentrated residue was dissolved in 50 mL of chloroform and washed with aqueous saturated NaHCO3 (50 mL × 3) and brine (50 mL). The organic layer was dried over sodium sulfate (anhydrous), filtered, and concentrated. The residue upon recrystallization from methanol−ether afforded pure ceramide analogue HC18-oleyl (78 mg, 78%). 1H NMR of HC18-oleyl (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3(CH2)6CH2CHCH−; t, 3H, CH3(CH2)6CO−]; 1−1.8 [bs, 24H, CH3(CH2)6CH2CH CHCH2(CH2)6CH2−NH−; bs, 10H, CH3(CH2)5CH2−CO; s, 9H, −Si−C(CH3)3]; 1.8−2.2 [m, 2H, −O−CH2CH2−; m, 4H, CH3(CH2)6CH2CHCHCH2(CH2)6CH2−NH−; m, 2H, CH3(CH2)5CH2−CO]; 3.1−3.3 [m, 2H, CH 3 (CH 2 ) 6 CH 2 CHCHCH 2 (CH 2 ) 6 CH 2 −NH−]; 3.6−4 [m, 2H, −Si−O−CH2−]; 4.5−4.7 [m, 1H, −NH−CO−CH−

TOF mass spectral technique (Figure S2B) since, as in case of RGDGWK-lipopeptide 1, extensive line broadening in the range 3−5 ppm was observed in 1H NMR spectrum (Figure S2A). The purity of the pegylated RGELFK-lipopeptide 2 was confirmed by reverse phase HPLC using couple of mobile phases (A, methanol; B, methanol:water (95:5, v/v) (Figure S3). 1H NMR (500 MHz, CDCl3): δ/ppm 0.6−0.9 [t, 6H, CH3(CH2)17−; m, 6H, −CH(CH3)2]; 1.1−1.8 [m, 1H, −CH(CH 3 ) 2 ; m, 2H, −CH 2 CH(CH 3 ) 2 ; bs, 64H, −(CH2)16−; m, 6H, LysCγH2 + LysCδH2 + ArgCγH2]; 1.8− 2.2 [m, 2H, LysCβH2; m, 2H, ArgCβH2; m, 2H, GluCβH2]; 2.4−2.7 [m, 4H, −N(−CH2CH2−)2; m, 2H, −N−CH2CH2− NH−CO; m, 2H, −NH−CO−CH2CH2−O]; 2.8−3.3 [m, 2H, PheCαH−CH2−Ph; m, 2H, GluCγH2; m, 2H, LysωCH2; m, 2H, ArgCδH2; m, 2H, −N−CH2CH2−NH−CO−; m, 2H, −CO− NH−CH2CH2−O]; 3.4−4.5 [m, 2H, GlyCαH2; m, 1H, PheCαH; m, 1H, LeuCαH; m, 1H, LysCαH; m, 1H, GluCαH; m, 1H, ArgCαH + PEG]; 7.1−7.4 [m, 5H, −CH2−C6H5] (Figure S2A). MALDI-TOF: m/z = 2601 [M + 1]+ for C131H253N13O37 (Figure S2B). 2.3. Synthesis of C8-Ceramide Analogues. The synthetic strategies adopted for preparing the presently described serine and the homoserine based C8-ceramide analogues are shown in Scheme 3 using HC18-oleyl as a representative example. Step i: NαBoc-L-homoserine (Ib) (10.7 g, 48.7 mmol) was dissolved in 100 mL of dry DCM, and DMAP (catalytic amount) was added to it at 0 °C. Triethylamine (5.92 g, 58.5 mmol) was added to the reaction mixture, and the reaction mixture was kept under stirring at 0 °C for 10 min. Finally tertbutyl(chloro)diphenylsilane (TBDPS-Cl) (16.1 g, 58.5 mmol) was added at 0 °C and the reaction mixture was stirred at rt for 12 h under nitrogen atmosphere. The reaction mixture was diluted with 250 mL of chloroform and washed sequentially with ice-cold 1 N HCl (150 mL × 2) and brine (150 × 2 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and filtered, and the solvent was removed by rotary evaporator. The residue was purified by column chromatography with 60−120 mesh silica gel using 5% methanol− chloroform (v/v) as eluent to afford intermediate IIb (13.2 g, 60% yield). Step ii: Solid HOBt (0.9 g, 5.8 mmol) and EDCI (1.14 g, 5.8 mmol) were added sequentially to an ice cold solution of acid intermediate IIb (2.6 g, 5.8 mmol) in 15 mL of dry DCM. After 30 min, olyelamine (1.3 g, 4.8 mmol) dissolved in 12 mL of dry DCM was added to the mixture. Diisopropylethylamine (DIPEA) was added dropwise to the stirred reaction mixture to make it alkaline to litmus (pH 9). The reaction mixture was stirred for 12 h at rt under nitrogen atmosphere, transferred in 250 mL of chloroform, and washed sequentially with ice-cold 1 N HCl (150 × 2 mL), aqueous saturated sodium bicarbonate (150 × 2 mL), and brine (150 × 1 mL). The organic layer was collected, dried over sodium sulfate (anhydrous), filtered, and concentrated by rotary evaporator. The obtained residue upon column chromatographic purification (60−120 mesh silica gel) using 12% EtOAc−hexane (v/v) as eluent resulted in intermediate IIIb (2.3 g, 57% yield). 1H NMR of IIIb (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3(CH2)6CH2CH CH−]; 1−1.6 [bs, 24H, CH3(CH2)6CH2CH CHCH2(CH2)6CH2−NH−; s, 9H, −O−C(CH3)3; s, 9H, −Si−C(CH3)3]; 1.8−2.2 [m, 2H, O−CH2−CH2−; m, 4H, CH3(CH2)6CH2CHCHCH2(CH2)6CH2−NH−], 3.1−3.3 [m, 2H, CH3(CH2)6CH2CHCHCH2(CH2)6CH2−NH−]; E

DOI: 10.1021/acs.molpharmaceut.5b00644 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics NH−]; 5.3−5.5 [m, 2H, CH3(CH2)6CH2CH CHCH2(CH2)6CH2−NH−]; 7.2−7.8 [m, 10H, Si−(C6H5)2] (Figure S7A). ESIMS of HC18-oleyl: m/z = 496 [M + 2]+, 520 [M + 1 + Na]+ for C30H58N2O3 (Figure S7B). All the other seven ceramide analogues were synthesized using the same synthetic scheme as described above for preparing HC18-oleyl using appropriate aliphatic amines in step ii. The 1H NMR and ESIMS data for all these seven analogues are provided below. 1 H NMR of C18-oleyl (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3(CH2)6CH2CHCH−; t, 3H, CH3(CH2)6CO−]; 1− 1.8 [bs, 24H, CH3(CH2)6CH2CHCHCH2(CH2)6CH2− NH−; bs, 10H, CH3(CH2)5CH2−CO]; 1.8−2.4 [m, 4H, CH3(CH2)6CH2CHCHCH2(CH2)6CH2−NH−; m, 2H, CH3(CH2)5CH2−CO]; 3.1−3.3 [m, 2H, CH3(CH2)6CH2CHCHCH2(CH2)6CH2−NH−]; 3.4−4.2 [m, 2H, −Si−O−CH2−]; 4.3−4.5 [m, 1H, −NH−CO−CH− NH−]; 5.3−5.5 [m, 2H, CH3(CH2)6CH2CH CHCH2(CH2)6CH2−NH−]; 7.2−7.8 [m, 10H, Si−(C6H5)2] (Figure S6A). ESIMS of C18-oleyl: m/z = 481 [M + 1]+, 503 [M + Na]+ for C29H56N2O3 (Figure S6B). 1 H NMR of C8 (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3(CH2)7−NH−; t, 3H, CH3(CH2)6−CO−]; 1.1−1.6 [bs, 12H, CH3(CH2)6CH2−NH; bs, 10H, CH3(CH2)5CH2−CO]; 2.1−2.3 [m, 2H, CH3(CH2)5CH2−CO]; 3.1−3.3 [m, 2H, CH3(CH2)6CH2−NH]; 3.4−3.8 [m, 2H, −Si−O-CH2−]; 4.3− 4.5 [m, 1H −CO−CH−NH−] (Figure S8A). ESIMS of C8: m/z = 343 [M + 1]+, 365 [M + Na]+ for C19H38N2O3 (Figure S8B). 1 H NMR of HC8 (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3(CH2)7−NH−; t, 3H, CH3(CH2)6CO−]; 1.1−2.1 [bs, 12H, CH3(CH2)6CH2−NH; bs, 10H, CH3(CH2)5CH2−CO; m, 2H, O−CH2CH2−]; 2.2−2.4 [m, 2H, CH3(CH2)5CH2− CO]; 3.1−3.3 [m, 2H, CH3(CH2)6CH2−NH]; 3.4−3.8 [m, 2H, −Si−O−CH2−]; 4.4−4.8 [m, 1H, −CO−CH−NH−] (Figure S9A). ESIMS of HC8: m/z = 357 [M + 1]+ for C20H40N2O3 (Figure S9B). 1 H NMR of C14 (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3(CH2)13−NH−; t, 3H, CH3(CH2)6−CO−]; 1.1−1.6 [bs, 24H, CH3(CH2)12CH2−NH; bs, 10H, CH3(CH2)5CH2−CO]; 2.1−2.3 [m, 2H, CH3(CH2)5CH2−CO]; 3.1−3.3 [m, 2H, CH3(CH2)12CH2−NH]; 3.4−3.7 [m, 2H, −Si−O−CH2−]; 4.3−4.5 [m, 1H, −CO−CH−NH−] (Figure S10A). ESIMS of C14: m/z = 427 [M + 1]+ for C25H50N2O3 (Figure S10B). 1 H NMR of HC14 (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3(CH2)13−NH−; t, 3H, CH3(CH2)6−CO−]; 1.1−2.1 [bs, 24H, CH3(CH2)12CH2−NH; bs, 10H, CH3(CH2)5CH2−CO; m, 2H, O−CH2CH2−]; 2.2−2.4 [m, 2H, CH3(CH2)5CH2− CO]; 3.1−3.3 [m, 2H, CH3(CH2)12CH2−NH]; 3.4−3.8 [m, 2H, −Si−O−CH2−]; 4.4−4.8 [m, 1H, −CO−CH−NH−] (Figure S11A). ESIMS of HC14: m/z = 442 [M + 2]+ for C26H52N2O3 (Figure S11B). 1 H NMR of C18 (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3-(CH2)17−NH−; t, 3H, CH3(CH2)6−CO−]; 1.1−1.6 [bs, 24H, CH3(CH2)16CH2−NH; bs, 10H, CH3(CH2)5CH2−CO]; 2.1−2.3 [m, 2H, CH3(CH2)5CH2−CO]; 3.1−3.3 [m, 2H, CH3(CH2)16CH2−NH]; 3.4−3.7 [m, 2H, −Si−O−CH2−]; 4.2−4.4 [m, 1H, −CO−CH−NH−] (Figure S12A). ESIMS of C18: m/z = 483 [M + 1]+ for C29H58N2O3 (Figure S12B). 1 H NMR of HC18 (300 MHz, CDCl3): δ/ppm 0.9 [t, 3H, CH3(CH2)17−NH−; t, 3H, CH3(CH2)6−CO−]; 1.2−2.1 [bs, 24H, CH3(CH2)16CH2−NH; bs, 10H, CH3(CH2)5CH2−CO; m, 2H, O−CH2CH2−]; 2.2−2.4 [m, 2H, CH3(CH2)5CH2−

CO]; 3.6−3.8 [m, 2H, CH3(CH2)16CH2−NH]; 3.4−3.8 [m, 2H, −Si−O−CH2−]; 4.5−4.7 [m, 1H, −CO−CH−NH−] (Figure S13A). ESIMS of HC18: m/z = 498 [M + 2]+, 520 [M + Na]+ for C30H60N2O3 (Figure S13B). The purities of final synthesized ceramide analogues were confirmed by reverse phase HPLC using two different mobile phases (A, methanol; B, methanol:water (95:5, v/v) (Figure S14). 2.4. Preparation of Liposomes. Mixture of DOPC, DOPE, cholesterol, the PEGylated lipopeptides, DSPE(PEG)27-NH2, synthesized C8-ceramide analogue (or commercially available C8-ceramide), and STPP at a mole ratio of 1:0.4:0.25:0.05:0.01:0.5:0.02 were dissolved in 100 μL of 3:1, v/v, chloroform:methanol in a glass vial. For preparing liposomal formulations of both curcumin and ceramide, curcumin stock solution (10 mg/mL in 1:1 chloroform:methanol, v/v) was added to the lipid mixture such that the final lipid/curcumin ratio became 15:1 (w/w). The lipid mixture was first concentrated as a thin layer by a steady and thin flow of moisture free nitrogen gas, and it was kept under high vacuum for 8 h. The lipid mixture was then incubated with sterile deionized water overnight at rt. It was then vortexed (2−3 min) at rt and sonicated in an ice bath until clarity using a Branson 450 sonifier (at 100% duty cycle and 25 W output power) to get small unilamellar vesicles (SLVs). Liposomes containing curcumin were centrifuged for 45 min at 5000 rpm to remove unencapsulated curcumin. Twenty microliters of liposomes was lysed with 1% Triton X-100 and diluted with methanol to a final volume of 200 μL. The amount of curcumin entrapped in supernatant liposomal solution was quantified by measuring absorbance at 425 nm (λmax) using absorbance of liposome without curcumin as a blank. The concentrations (μg/mL) of the liposomally entrapped curcumin were then calculated from a standard calibration curve constructed using known concentrations of curcumin. 2.5. Hydrodynamic Diameter and Zeta Potential (ξ) Measurements. The sizes and the surface charges (zeta potentials) of liposomal ceramide, liposomal curcumin, and liposome containing both curcumin and ceramide were measured by a Zeta sizer, 3000HSA (Malvern UK) in deionized water. 2.6. Integrin Receptor Selectivity Studies. Rh-PE labeled liposomes (0.1 mol % with respect to lipopeptide) containing DOPC, DOPE, cholesterol, RGDGWK-lipopeptide 1, DSPE-(PEG)27-NH2, and STPP in 1:0.4:0.25:0.05:0.01:0.02 mol ratios were used for cellular uptake study. HUVEC and B16F10 cells were seeded at a density of ∼10,000 cells/well in a 96-well plate, and the cells were cultured for 18−24 h before the treatment. Cells were then separately preincubated with each of the three monoclonal antibodies against αvβ3/αvβ5/ α5β1 integrins (50 μL at a dilution of 1:25 in 10% complete medium) at rt. After 45 min a fresh 50 μL of these three different monoclonal antibodies was added to the cell at a dilution of 1:25 (v/v) in 10% complete medium. 50 μL of medium containing 10 μL of Rh-PE labeled liposome of pegylated RGDGWK-lipopeptide 1 was added to each well. After 3 h cells were washed with PBS (100 μL × 2) and viewed with an epifluorescence microscope. Uptake of Rh-PE labeled liposome was further quantified by flow cytometry in B16F10 cell line. Briefly cells were seeded in 6-well plate at a density of ∼2 × 105 cells per well, 18 h prior to treatment. Cells were similarly preincubated with 1 mL of three monoclonal antibodies against αvβ3/αvβ5/α5β1 integrins at a dilution of F

DOI: 10.1021/acs.molpharmaceut.5b00644 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

μM curcumin, 5 μM curcumin and 5 μM HC18-oleyl, 10 μM C8-cer, and 5 μM curcumin and 5 μM C8-cer in 3 mL of complete medium. After 24 h cells were trypsinized, centrifuged, and washed with PBS. The washed cells were fixed with 2% paraformaldehyde in PBS, permeabilized with 0.1% Triton-X 100 in PBS, and centrifuged at 3,000 for 2 min. One microgram of primary antibody against Cyclin B1 (marker for G2/M phase of cell cycle) was dissolved in 250 μL of PBS, and the solution was added to the pelleted cells, mixed, vortexed, and incubated in a covered ice bucket for 20 min. The supernatant was discarded after centrifugation, and 1 μg of FITC-conjugated secondary antibody in 250 μL of PBS was added to each pellet, vortexed, and incubated in a covered ice bucket for 30 min. Supernatant was aspirated, and the pellets were resuspended in 500 μL of PBS to be analyzed by flow cytometer (BD FACS Canto II). 2.10. BrdU Incorporation Assay. HUVEC and B16F10 cells were seeded at a density of ∼15,000 per well in a 96-well plate and allowed to grow for 18−24 h. Cells were separately treated with liposomal formulations of pegylated RGDGWKlipopeptide 1 containing 10 μM curcumin, 10 μM HC18-oleyl, 5 μM curcumin and 5 μM HC18-oleyl, 10 μM C8-cer, and 5 μM curcumin and 5 μM C8-cer in complete DMEM medium for B16F10 cells and in complete EBM2 medium in the case of HUVEC cells. After 12 h the medium was discarded and the cells in each well were incubated with 100 μL of complete medium containing 1× BrdU reagent (20 μL) for an additional 12 h. ELISA based BrdU Cell Proliferation Assay Kit (Millipore) was used to assess the incorporated BrdU following the manufacturer’s instructions. Results were expressed as percent viability = [A450(treated cells) − background/ A450(untreated cells) − background] × 100. 2.11. Invasion and Migration Study. The chamber plates supplied with the CytoSelect 24-well cell migration assay kit (Cell Biolabs Inc. USA) were warmed up and rehydrated using 300 μL of warm serum free medium. Cells were seeded at ∼1 × 105 cells per well and exposed to liposomal formulations of pegylated RGDGWK-lipopeptide 1 containing 10 μM curcumin, 10 μM HC18-oleyl, 5 μM curcumin and 5 μM HC18-oleyl, 10 μM C8-cer, and 5 μM curcumin and 5 μM C8cer in complete DMEM medium for B16F10 cells and in complete EBM2 medium in the case of HUVEC cells. Cells were then allowed to invade through a polycarbonate membrane coated with a uniform layer of dried solution of basement membrane matrix toward the lower chamber containing 500 μL of serum containing medium for 24 h. Invasive cells degrade the matrix protein and pass through the pores of the polycarbonate membrane. For migration assays, cells were seeded (either untreated or pretreated with the above-mentioned liposomal drugs) at ∼1 × 105 cells per well and allowed to migrate toward serum containing medium in the lower chamber for 24 h. Migratory cells can pass through the pores of the polycarbonate membrane due to its ability to extend protrusions toward chemoattractants (via actin cytoskeleton reorganization). The polycarbonate inserts were placed in a clean well containing 400 μL of cell staining solution and washed several times in a beaker of water, and the stained inserts were dried in air. Each insert was then transferred to an empty well containing 200 μL of extraction solution. 100 μL of extracted solutions from each sample were placed in a 96-well microtiter plate, and the optical density of the extracted solutions was measured at 550 nm with a plate reader.

1:25 (v/v) in 10% complete medium at rt. After 45 min a fresh 1 mL of these three different monoclonal antibodies at a dilution of 1:25 (v/v) in 10% complete medium was added to the cell. One milliliter of medium containing 100 μL of Rh-PE labeled liposome of pegylated RGDGWK-lipopeptide 1 was also added to each plate. After 3 h cells were trypsinised and washed with PBS, and the cellular uptake of Rh-PE was analyzed by flow cytometry. 2.7. In Vitro Cell-Growth Inhibition Study. Cellular cytotoxicity of curcumin and ceramides coencapsulated in liposomes of the targeted pegylated RGDGWK-lipopeptide 1 was measured in various cells by the standard MTT assay. HUVEC, B16F10, and CHO cells were seeded at a density of ∼5000 cells per well in 96-well plates 18−24 h before treament. Cells were treated with various concentrations of liposomally encapsulated curcumin and ceramide using DMEM medium supplemented with 10% FBS for B16F10 and CHO cells and complete EBM2 medium in the case of HUVEC cells. After 24 and 48 h, 10 μL of MTT solution (stock 5 mg/mL in PBS) was added and incubated for 4 h at 37 °C. After that medium was removed, the formazan crystals (formed within the living cells) dissolved in 1:1 (v/v) DMSO/methanol (50 μL) and the absorbances of the resulting solutions were determined at 550 nm. Results were expressed as percent viability = [A550(treated cells) − background/A550(untreated cells) − background] × 100. To draw an isobologram the required individual concentrations of HC18-oleyl and curcumin to produce a defined single-agent effect (50% toxicity) are plotted on the X and Y axes in a two-dimensional graph. A line of additivity was drawn by connecting these two points. Then the concentration of each liposomal drug in combination to produce a similar effect was placed in the same graph. Combination index was calculated as CI = Ccur,x/ICx,cur + CHC18‑oleyl,x/ICx,HC18‑oleyl, where ICx,cur and ICx,HC18‑oleyl are the concentration required for corresponding individual drug encapsulated in liposomal formulation to produce x% effect. Ccur,x and CHC18‑oleyl,x denotes concentration of each drug entrapped in combination in the liposomal formulation to provide same x% effect. 2.8. Flow Cytometric Apoptosis Analysis. 18−24 h before treatment, B16F10 and HUVEC cells were seeded in a 6-well plate at a density of ∼3 × 105 cells per well. Cells were separately treated with liposomal formulations of pegylated RGDGWK-lipopeptide 1 containing 10 μM curcumin, 10 μM HC18-oleyl, 5 μM curcumin and 5 μM HC18-oleyl, 10 μM C8cer (commercially available C8-ceramide), and 5 μM curcumin and 5 μM C8-cer in 1.5 mL of complete EBM2 medium for HUVEC and in 1.5 mL of DMEM medium supplemented with 10% FBS for B16F10 cells. After 24 h cells were trypsinized, washed with 500 μL of PBS, and centrifuged. The collected cells were suspended in 500 μL of binding buffer. 5 μL of annexin-V FITC and 10 μL of PI were added. The mixture was incubated for 15 min in the dark and then was analyzed by flow cytometer (BD FACS Canto II). 2.9. Flow Cytometric Cell Cycle Analysis. B16F10 cells were cultured in T25 cell culture flask in 2 mM thymidine in DMEM medium (supplemented with 10% FBS) for 18 h. Cells were then washed with PBS, subcultured in a new T25 flask, and grown in fresh complete medium without thymidine for 8 h. Cells were again incubated in the presence of 2 mM thymidine in complete medium for 18 h and then in fresh medium for 2 h. Cells (∼1 × 106 cells per flask) were then separately treated with liposomal formulations of pegylated RGDGWK-lipopeptide 1 containing 10 μM HC18-oleyl, 10 G

DOI: 10.1021/acs.molpharmaceut.5b00644 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics 2.12. Semiquantitative RT-PCR. HUVEC and B16F10 cells were seeded at a density of ∼1 × 106 cells in a T25 flask for 18−24 h before treatment. Cells were separately treated with liposomal formulations of pegylated RGDGWK-lipopeptide 1 containing 10 μM HC18-oleyl, 10 μM curcumin, 5 μM curcumin and 5 μM HC18-oleyl, 10 μM C8-cer, and 5 μM curcumin and 5 μM C8-cer in 3 mL of DMEM containing 10% FBS for B16F10 and in complete EBM2 medium in the case of HUVEC cells for 24 h. Total RNA was extracted with TRIzol reagent (Invitrogen). First-strand cDNAs were synthesized and amplified from the corresponding mRNAs using Reverse Transcription System, Promega (USA), according to the manufacturer’s instructions. The amplified sequences were electrophoresed in 2% agarose gel and visualized using 0.1% ethidium bromide under UV light. 2.13. Western Blot Analysis. B16F10 and HUVEC cells were seeded at a density of ∼1 × 106 cells in T-25 flasks 18−24 h prior to treatmet. Cells were separately treated with liposomal formulations of pegylated RGDGWK-lipopeptide 1 containing 10 μM HC18-oleyl, 10 μM curcumin, 5 μM curcumin and 5 μM HC18-oleyl, 10 μM C8-cer, and 5 μM curcumin and 5 μM C8-cer in 3 mL of complete EBM2 medium for HUVEC and in 3 mL of complete DMEM medium for B16F10 cells. Twentyfour hours after treatment cells were detached and collected using a cell scraper and lysed with lysis reagent, Promega (USA), at 4 °C. Total protein in the cell lysates was estimated by BCA assay according to the manufacturer’s instructions. 80 μg of total proteins was electrophoretically fractioned in 12% SDS−PAGE, transferred to nitrocellulose membrane (Amersham Biosciences, NJ), and separately immunoblotted with rabbit polyclonal PhosphoDetect anti-Bad, anti-Bad, anticytochrome c, PhosphoDetect anti-Akt, anti-Akt, PI3K, protein phosphatase 2A, protein phosphatase 1, sphingosine kinase, and anti-β actin (as control) at 1:1000 dilutions (v/v) overnight at 4 °C. After washing with PBS-T (10 mL × 3, 10 min each), the membranes were incubated with alkaline phosphatase conjugated goat anti-rabbit secondary antibody (in 10 mL of 0.05% TBS-T with 1:5000 dilution) for 2 h. Protein bands were detected using BCIP/NBT chromogenic solution (Calbiochem), and expression levels were normalized to β-actin. Cell lysate collected from similarly treated B16F10 cells was also immunoblotted for rabbit polyclonal anti-Lass 1, Lass-2, Lass-4, anti-BAX, and anti-Bcl-2. 2.14. Biodistribution Studies. ∼1.5 × 105 B16F10 cells in 100 μL of Hanks buffer salt solution (HBSS) were injected into 6−8 week old C57BL/6J mice in the right flank, n = 2. Eighteen days after tumor cell inoculation mice were intravenously injected with curcumin (12 mg/kg bw of mouse) formulated in liposomes of pegylated RGDGWKlipopeptide 1 in 5% aqueous glucose. Twenty-four hours post iv injection, tumors and organelles (heart, lung, spleen, liver, and kidney) were collected, washed with ice-cold saline, and homogenized (with 0.8 mL of saline). Citrate buffer (pH 3.0, 50 μL, and 50 mM) was added in each homogenized sample and vortexed for 30 s. Curcumin was then extracted with ethyl acetate (2 mL × 2) from aqueous layer. Organic layer was collected in fresh centrifuge tubes, evaporated, and redissolved in 100 μL of methanol. The amount of curcumin in each sample was measured by quantitative reverse phase HPLC (using 5 μm particle size C18e analytical RP HPLC column, Merck-Millipore; methanol as mobile phase with 0.5 mL/min flow rate and a UV detection wavelength of 425 nm) using a

standard graph constructed with known concentrations of curcumin. 2.15. Liposomes of Pegylated RGDGWK-Lipopeptide 1 Are Targeted to Tumor Vasculatures. ∼1.5 × 105 B16F10 cells in 100 μL of HBSS buffer were injected in the right flank of C57BL/6J mice (6−8 weeks old), n = 4. On day 18 post tumor cell inoculation, mice were intravenously administered with a single dose of 60 μL of Rh-PE labeled liposomes of either pegylated RGDGWK-lipopeptide 1 or nontargeting pegylated RGELFK-lipopeptide 2 in a total volume of 250 μL in 5% aqueous glucose. After 24 h, mice were sacrificed, and tumors were processed and cryosectioned (10 μm) using a cryostat instrument, Leica. The cryosections were fixed (with 4% formaldehyde in PBS) and stained for blood vessel marker vWF (von Willebrand factor) with a blood vessel staining kit (Chemicon, USA) following the manufacturer’s protocol. The micrographs were taken in bright field for blood vessel and in red field for Rhodamine. 2.16. Tumor Growth Inhibition Study. Tumors were inoculated similarly as described in section 2.14. On day 14, mice were randomly sorted into nine groups. Each group (n = 5) was administered intravenously either with 5% aqueous glucose or with liposomal formulation of pegylated RGDGWKlipopeptide 1 encapsulating the ceramide analogues (Scheme 1) described herein (12 mg/kg bw of mouse) on days 14, 16, 18, 20, and 22 post tumor cell innoculation. In a second set of experiments B16F10 melanoma tumor bearing mice (n = 5) were treated (from day 14) with liposomal formulation of RGDGWK-lipopeptide 1 containing HC18-oleyl (12 mg/kg bw of mouse), commercially available C8-cer (12 mg/kg bw of mouse), curcumin (12 mg/kg bw of mouse), curcumin (6 mg/ kg bw of mouse) and C8-cer (6 mg/kg bw of mouse), curcumin (6 mg/kg bw of mouse) and HC18-oleyl (6 mg/kg bw of mouse), and 5% aqueous glucose as vehicle for five injections every alternate day. Tumor volumes (V = (1/2)ab2, where a = maximum length of the tumor and b = minimum length of the tumor determined perpendicular to each other) were measured with slide calipers for up to 24 days. Results represent the means ± SD (for n = 5) (*P < 0.01 compared to control). 2.17. Immunohistochemical Studies. Tumor bearing C57/6J black mice were treated similarly as described in section 2.16. Twenty-four hours after the last injected dose, mice were sacrificed, and tumors were excised, cryosectioned, fixed (with 4% formaldehyde in PBS), and immunostained for VE-cadherin (red marker for endothelial cells) and TUNEL (green marker for apoptosis positive cells) following the manufacturer’s protocol. The stained tumor slides were observed under a fluorescent microscope using green and red filters. 2.18. Statistical Analysis. The data were represented as mean ± SD. Error bars represent mean values ± SEM. Twotailed Student’s test was used to determine the statistical significance of the experimental data. *P < 0.05 were accepted as statistically significant.

3. RESULTS 3.1. Synthesis of Pegylated Lipopeptides and Ceramide Analogues. Structures of the lipopeptides and ceramide analogues have been shown in Scheme 1. The solid phase peptide synthesis (SPPS) strategy (Fmoc based) was adopted for preparing integrin receptor selective tumor vasculature targeting pegylated RGDGWK-lipopeptide 1 (Scheme 2). The similar Fmoc based SPPS strategy was also used for preparing H

DOI: 10.1021/acs.molpharmaceut.5b00644 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics the control nontargeting RGELFK-lipopeptide 2. Ceramide analogues were prepared by using conventional protection− deprotection chemistry. Representative detailed synthetic procedures for C18-oleyl and HC18-oleyol ceramides have been described in Scheme 3. Briefly, the alcohol group of BOCprotected serine or homoserine was protected using TBDPSCl. The TBDPS-protected intermediates (IIa/IIb) were then coupled with a C18H35-oleyl alkyl amine using EDCI, HOBt at pH 8−9. The BOC groups of the resulting intermediates (IIIa/ IIIb) were removed with TFA:DCM (1:2, v/v). The free amine intermediates were immediately (without isolation) subjected to EDCI/HOBt coupling with n-octanoic acid. The fluoride ion mediated deprotection of the resulting coupled products (IVa/ IVb) finally afforded the target ceramides (Va/Vb). All the other six ceramides shown in Scheme 1B were prepared applying the same strategy by using appropriate alkyl amines containing appropriate chain lengths. All the final molecules were characterized by ESI mass spectrometry and 1H NMR (Figures S1, S2, S4−S13). Purities of the lipopeptides and all the newly synthesized ceramide analogues were confirmed by reversed phase HPLC using two different mobile phases (Figure S3 and Figure S14). 3.2. Sizes and Zeta Potentials (ξ). Hydrodynamic diameters and zeta potentials of the liposomal formulations of pegylated RGDGWK-lipopeptide 1 containing only curcumin and only ceramide (either commercially available C8-ceramide or newly synthesized C8-ceramide analogues) as well as liposomal formulations of both curcumin and ceramide were measured by dynamic light scattering in deionized water. The liposomal sizes and surface potentials for all these formulations were found to be within the range of 190−220 nm and 3−5 mV, respectively (Table S1). 3.3. Cellular Uptake and Integrin Receptor Selectivity of the Liposomal Formulation of RGDGWK-Lipopeptide 1. The cellular uptake efficiencies of the Rh-PE labeled liposomes of pegylated RGDGWK-lipopeptide 1 (containing DOPC, DOPE, cholesterol, the pegylated lipopeptides, DSPE(PEG)27-NH2, and STPP in 1:0.4:0.25:0.05:0.01:0.02 mol ratios) were measured by epifluorescence microscopy. The uptake efficiencies in both tumor (B16F10) and endothelial (HUVEC) cells were reduced when cells were presaturated with monoclonal antibody against α5β1, αvβ3, and αvβ5 integrins (Figure 1 and Figure S15, part I). As expected, cellular uptake efficiency was most adversely affected for cells presaturated with α5β1-mAb (Figure 1 and Figure S15, part I). Toward further confirming α5β1-intergin receptor selective cellular uptake event, we performed an additional flow cytometry based cellular uptake study using Rh-PE labeled liposomal formulations. The findings summarized in Figure S15, part II, clearly show that the degree of cellular uptake of the Rh-PE-labeled liposomes gets most significantly compromised when cells are preincubated with mAb against α5β1 integrin thereby providing further support for the α5β1-intergin selective cellular uptake event. 3.4. In Vitro Cell Growth Inhibition Studies. To begin with, we measured the relative cytotoxic effects of the various liposomal formulations containing only curcumin and only ceramide as well as formulations containing both curcumin and ceramide in tumor (B16F10), endothelial (HUVEC), and noncancer (RAW 264.7) cells using MTT assay across the concentration range 5−20 μM. The findings summarized in Figure S16 revealed the homoserine based HC18-oleyl ceramide analogue to be the most potent cytotoxic ceramide

Figure 1. Liposomes of pegylated RGDGWK-lipopeptide 1 enter endothelial cells mainly via α5β1 integrin receptor. HUVEC cells, presaturated with monoclonal antibodies against α5β1, αvβ3, or αvβ5 integrins, were treated with Rh-PE (red) labeled liposomes of pegylated RGDGWK-lipopeptide 1. After 3 h of liposome addition, epifluorescence microscopic images were taken for cells devoid of antibody treatment (A−C) as well as for cells preincubated with mAbs against α5β1 integrins (D−F), αvβ3 integrins (G−I), and αvβ5 integrins (J−L). Data shown here are representative of two separate experiments. Scale bar equals 50 μm.

described herein for use in combination with curcumin in liposomal formulations. Importantly, liposomally coencapsulated HC18-oleyl and curcumin was found to be more cytotoxic (in both tumor and endothelial cells) than liposomally coencapsulated commercially available C8-ceramide (labeled as C8-cer) and curcumin (Figure S17). Contrastingly, the percentage of cell viabilities of liposomally treated noncancerous (RAW264.7) cells was found to be significantly high (Figure S18). With a view to confirm synergistic action of curcumin and HC18-oleyl encapsulated in the presently described liposomes of pegylated RGDGWK-lipopeptide 1, the widely used isobologram was constructed in HUVEC cells as described in Materials and Methods. The combination index (CI) of the present formulation was calculated for HUVEC cells after 24 h of treatment. The IC50 values (50% cell killing event) of curcumin and HC18-oleyl entrapped individually in the present liposomal system were found to be 9 μM and 9.5 μM respectively. Similar 50% cell killing effect was shown by combined liposome of curcumin and HC18-oleyl at a concentration of 2.2 μM for each drug, which fell well below (synergistic effect) the line of additivity and not on (additive effect) or above (antagonistic effect) the line of additivity in the isobologram (Figure S17). The combination index (calculated as described in Materials and Methods) value 0.47 (