Rapid Report pubs.acs.org/crt
Enhanced Toxicity of Cisplatin with Chemosensitizer Phenethyl Isothiocyanate toward Non-Small Cell Lung Cancer Cells When Delivered in Liposomal Nanoparticles Yu-Tsai Yang,† Yi Shi,‡ Michael Jay,† and Anthony J. Di Pasqua*,‡ †
Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States ‡ Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas 76107, United States S Supporting Information *
ABSTRACT: Naturally occurring phenethyl isothiocyanate (PEITC) was previously shown to sensitize human non-small cell lung cancer (NSCLC) cells to the platinum anticancer drug cisplatin (CDDP). Here, CDDP and PEITC were encapsulated in approximately 130 nm liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and L-α-phosphatidylglycerol (EPG). The liposomal formulation enhanced the toxicity of this doublet (1:2 molar ratio of CDDP/PEITC) toward NCI-H596 NSCLC cells; the percent survival of cells was 30.2 ± 6.2% after treatment with the nanoparticle formulation, compared to 50.9 ± 3.5% when administered together free. Thus, such a treatment modality could prove useful in the clinic for the treatment of NSCLC.
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they do in normal tissues, likely due to the enhanced permeation and retention (EPR) effect.5 In a randomized clinical trial, the effectiveness of Lipoplatin with the β-tubulin binding agent paclitaxel was similar to that of CDDP with paclitaxel; however, much less toxicity to the patient was observed.6 To further improve the anticancer potency of CDDP-encapsulated liposomes, phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate (ITC), is utilized here; ITCs are metabolites of glucosinolates, which are contained in cruciferous vegetables such as watercress.7 Previously, we showed that naturally occurring ITCs enhance the efficacy of CDDP and that this is probably due to its ability to bind and degrade β-tubulin.8 In a separate report, a synthetic ITC derivative, ethyl 4-isothiocyantobutanoate, was shown to sensitize ovarian cancer cells to CDDP, and moreover, it allowed for CDDP to overcome CDDP-resistance in cell culture.9 More recently, it was shown that naturally occurring ITCs sensitize cervical and breast cancers to CDDP but not normal human mammary epithelial cells10 and that the naturally occurring ITC benzyl ITC (BITC) sensitizes leukemia cells to CDDP but not normal human lymphocytes.11 The fact that micromolar plasma concentrations of ITCs are safely maintained in humans12 and that they have the ability to sensitize only cancer cells to CDDP is compelling and warrants further investigation.
ung cancer is the leading cause of cancer-related death in the United States, and approximately 85% of all lung cancers are classified as non-small cell lung carcinomas (NSCLC). Unfortunately, NSCLC is extremely difficult to treat, and survival rates are low. After three decades of clinical trials and despite the vast number of new drug combinations studied, the most effective treatments are still those that implement the first-generation platinum anticancer agent cisplatin (cis-diamminedichloroplatinum(II), CDDP) in combination with another drug.1 However, CDDP indiscriminately damages both cancerous and normal tissues after systemic administration and is extremely nephrotoxic. Furthermore, tumors often acquire resistance to CDDP, leading to therapeutic failure. 2 Thus, a treatment modality that aggressively decreases tumor volume but has less toxicity to the patient has been sought. It has been demonstrated that the liposomal CDDP formulation Lipoplatin is less toxic to patients than is free CDDP but has a similar efficacy against NSCLC.3 Liposomes have been investigated as drug delivery vehicles due to their unique structure and properties. Composed of relatively biocompatible and biodegradable materials, liposomes have an aqueous core and at least one bilayer of natural and/or synthetic lipids; the aqueous core is able to accommodate hydrophilic molecules, while hydrophobic molecules can be entrapped in the phospholipid bilayer. Reformulating drugs in liposomes can increase the circulation time of the drug in the bloodstream and alter its tissue distribution.4 Moreover, studies have shown that liposomes accumulate in tumors more than © 2014 American Chemical Society
Received: March 24, 2014 Published: May 16, 2014 946
dx.doi.org/10.1021/tx5001128 | Chem. Res. Toxicol. 2014, 27, 946−948
Chemical Research in Toxicology
Rapid Report
Here, liposomal nanoparticles containing both PEITC and CDDP were prepared, characterized, and then used to improve the efficacy of a chemotherapeutic doublet (1:2 molar ratio of CDDP/PEITC). There are a number of nanomedicine-based cancer therapies described in the literature;13,14 some are in the preclinical and clinical testing stages, while others are approved for use by the FDA. The approach described in this rapid report differs from these in that it uses a naturally occurring ITC to sensitize NSCLC to CDDP and that it delivers both in a single nanocarrier, which can be modified, i.e., the size and surface chemistry can be altered, to optimize for tumor accumulation and retention. During encapsulation, PEITC molecules were likely trapped in the lipid bilayer, as they have a log P value of 3.415 and hydrophilic CDDP encapsulated inside the aqueous core (Figure 1A). The amount of PEITC and CDDP encapsulated
Table 1. Percent Drug Loading of CDDP and PEITC in Liposomes DSPC (μmol)
EPG (μmol)
CDDP (μmol)
PEITC (μmol)
16 16 16 16 16 16 16
4 4 4 4 4 4 4
0 6.7 0.0 6.7 6.7 6.7 6.7
0 0 6.7 3.4 6.7 10.1 13.4
CDDPloading (%)
PEITCloading (%)
0.80 ± 0.25 1.63 0.93 0.87 0.80
± ± ± ±
1.43 0.47 0.35 0.30
1.31 0.66 0.81 1.04 1.37
± ± ± ± ±
1.08 0.15 0.25 0.42 0.85
Table 2. Size and Zeta Potentials of Blank Liposomes and Liposomes Loaded with CDDP and/or PEITC DSPC (μmol)
EPG (μmol)
CDDP (μmol)
PEITC (μmol)
16 16 16 16 16 16 16
4 4 4 4 4 4 4
0 6.7 0.0 6.7 6.7 6.7 6.7
0 0 6.7 3.4 6.7 10.1 13.4
size (nm)
PDI
zeta (mV)
± ± ± ± ± ± ±
0.08 0.09 0.10 0.12 0.11 0.13 0.16
−55 −60 −68 −67 −67 −65 −64
127 117 136 141 138 138 130
47 43 54 60 55 67 77
± ± ± ± ± ± ±
10 10 8 8 9 7 9
loaded with CDDP and/or PEITC ranged from about 120 to 140 nm. The zeta potentials of the empty and loaded liposomes were all highly negative ranging from approximately −60 to −70 mV (Table 2), indicating that the formulation is stable, due to high electrostatic repulsion between the nanoparticles. The human NCI-H596 NSCLC cells were treated with complete growth medium containing free CDDP, PEITC, CDDP and PEITC (CDDP + PEITC), CDDP-entrapped liposomes (Lipo-CDDP), PEITC-entrapped liposomes (LipoPEITC), or both drugs encapsulated in liposomes (Lipo-CDDP + PEITC). The cells were incubated for 24 h, and cytotoxicity was then measured using the MTT assay. As shown in Figure 1C, blank liposomes had no significant effect on cell growth with a percent survival of 95.5 ± 4.4%. In the presence of CDDP (7.5 μM) alone, the percent cell survival was 64.3 ± 7.2%, while with only PEITC (15 μM), the percent cell survival was 84.3 ± 2.8%. When treated with 7.5 μM of CDDP and 15 μM of PEITC (CDDP + PEITC), the cells had a percent survival of 50.9 ± 3.5%. The cytotoxicity associated with the combination of CDDP and PEITC was significantly greater than those associated with CDDP or PEITC alone, with p = 2.5 × 10−2 and 9.3 × 10−6, respectively. Liposomes containing CDDP (7.5 μM) or PEITC (15 μM) showed greater toxicities than the free drugs. The percent cell survival after treatment with liposomes loaded with CDDP (Lipo-CDDP) was 44.8 ± 8.5%, and for liposomes with PEITC (Lipo-PEITC), the percent cell survival was 63.1 ± 2.2%. The cytotoxicities of Lipo-CDDP and Lipo-PEITC were significantly greater than those of free CDDP and PEITC, with p = 1.3 × 10−2 and 3.1 × 10−5, respectively. When treated with liposomes containing 7.5 μM of CDDP and 15 μM of PEITC (Lipo-CDDP + PEITC), the cells had a percent survival of 30.2 ± 6.2%, which is significantly greater than that of CDDP + PEITC (p = 5.4 × 10−4), Lipo-CDDP (p = 3.2 × 10−2), and Lipo-PEITC (p = 8.2 × 10−5). CDDP is a potent anticancer drug which is widely used in chemotherapy to treat various types of cancer. However, CDDP damages cancerous and normal tissues indiscriminately,
Figure 1. (A) Proposed structure and (B) transmission electron microscopy (TEM) image of liposomes loaded with cisplatin (CDDP) and phenethyl isothiocyanate (PEITC). (C) Percent survival of NCIH596 non-small cell lung cancer cells treated with free CDDP and PEITC, and liposome encapsulated compounds, *p < 0.05.
in liposomes was characterized via UV−visible spectroscopy and the lipid concentration determined using the Bartlett assay.16−18 Using eq 1 (Supporting Information), the percent drug loading of CDDP and PEITC were each approximately 1% w/w (Table 1). Percent CDDP drug loading was independent of PEITC concentration, and percent loading of PEITC increased with PEITC concentration; however, the encapsulation efficiency was the highest when the lowest amount of PEITC (3.4 μmol) was used in the formulation. The PEITC and CDDP loaded liposomes were observed by TEM (Figure 1B). Particle size distributions and zeta potentials were measured using a Zetasizer Nano-ZS. As shown in Table 2, the average diameter of empty liposomes and liposomes 947
dx.doi.org/10.1021/tx5001128 | Chem. Res. Toxicol. 2014, 27, 946−948
Chemical Research in Toxicology
Rapid Report
(4) Al-Jamal, W. T., and Kostarelos, K. (2011) Liposomes: From a clinically established drug delivery system to a nanoparticle platform for theranostic nanomedicine. Acc. Chem. Res. 44, 1094−1104. (5) Gabizon, A. A., Shmeeda, H., and Zalipsky, S. (2006) Pros and cons of the liposome platform in cancer drug targeting. J. Liposome Res. 16, 175−183. (6) Bastas, A., Marosis, K., Stathopoulos, J., Provata, A., Yiamboudakis, P., Veldekis, D., Lolis, N., Georgatou, N., Toubis, M., Pappas, C., and Tsoukalas, G. (2010) Liposomal cisplatin combined with paclitaxel versus cisplatin and paclitaxel in non-small cell lung cancer: a randomized phase III multicenter trial. Ann. Oncol. 21, 2227− 2232. (7) WHO (2004) IARC Handbook of Cancer Prevention, Vol. 9, IARC Press, Lyon, France. (8) Di Pasqua, A. J., Hong, C., Wu, M. Y., McCracken, E., Wang, X., Mi, L., and Chung, F.-L. (2010) Sensitization of non-small cell lung cancer cells to cisplatin by naturally occurring isothiocyanates. Chem. Res. Toxicol. 23, 1307−1309. (9) Bodo, J., Chovancova, J., Hunakova, L., and Sedlak, J. (2005) Enhanced sensitivity of human ovarian carcinoma cell lines A2780 and A2780/CP to the combination of cisplatin and synthetic isothiocyanate ethyl 4-isothiocyanatobutanoate. Neoplasma 52, 510−516. (10) Wang, X., Govind, S., Sajankila, S. P., Mi, L., Roy, R., and Chung, F.-L. (2011) Phenethyl isothiocyanate sensitizes human cervical cancer cells to apoptosis induced by cisplatin. Mol. Nutr. Food Res. 55, 1−10. (11) Lee, Y., Kim, Y. J., Choi, Y. J., Lee, J. W., Lee, S., and Chung, H. W. (2012) Enhancement of cisplatin cytotoxicity by benzyl isothiocyanate in HL-60 cells. Food Chem. Toxicol. 50, 2397−2406. (12) Liebes, L., Conaway, C. C., Hochster, H., Mendoza, S., Hecht, S. S., Crowell, J., and Chung, F.-L. (2001) High-performance liquid chromatography-based determination of total isothiocyanate levels in human plasma: Application to studies with 2-phenethyl isothiocyanate. Anal. Biochem. 291, 279−289. (13) Prabhu, V., Uzzaman, S., Grace, V. M. B., and Guruvayoorappan, C. (2011) Nanoparticles in drug delivery and cancer therapy: The giant rats tail. J. Cancer. Ther. 2, 325−334. (14) Huynh, N. T., Passirani, C., Saulnier, P., and Benoit, J. P. (2009) Lipid nanocapsules: A new platform for nanomedicine. Int. J. Pharmaceut. 379, 201−209. (15) Morse, M. A., Eklind, K. I., Hecht, S. S., Jordan, K. G., Choi, C.I., Desai, D. H., Amin, S. G., and Chung, F.-L. (1991) Structureactivity relationships for inhibition of 4-(methylnitrosamino)-l-(3pyridyl)-l-butanone lung tumorigenesis by arylalkyl isothiocyanates in A/J mice. Cancer Res. 57, 1846−1850. (16) Zhang, Y., Cho, C.-G., Posner, G. H., and Talalay, P. (1992) Spectroscopic quantitation of organic isothiocyanates by cyclocondensation with vicinal dithiols. Anal. Biochem. 205, 100−107. (17) Anilanmert, B., Yalcin, G., Arioz, F., and Dolen, E. (2001) The spectrophotometric determination of cisplatin in urine using ophenylenediamine as derivatizing agent. Anal. Lett. 34, 113−123. (18) Bartlett, G. R. (1958) Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466−468. (19) Anderson, M., and Omri, A. (2004) The effect of different lipid components on the in vivo stability and release of liposome formulations. Drug Delivery 11, 33−39.
and tumors often gain resistance to CDDP, leading to therapeutic failure. To address these issues, we used liposomes as a delivery vehicle for CDDP and PEITC, which were previously shown to act synergistically.8 Studies have shown that drugs reformulated in liposomes have an increased circulation time in the bloodstream and, furthermore, increased accumulation in tumors, which is due to the enhanced permeability and retention (EPR) effect.4 In this study, liposomes containing the relatively hydrophilic CDDP and hydrophobic PEITC were prepared and characterized. DSPC was chosen because of its high phase transition temperature (Tc = 55 °C); liposomes composed of DSPC were previously shown to have greater drug retention over 48 h at 4 and 37 °C than those composed of phospholipids with lower Tc values.19 The typical long circulation time associated with liposomes of this size may allow for the synergistic CDDP and PEITC treatment modality to more effectively reduce the NSCLC tumor burden with fewer side effects, and the highly negative zeta potential of the nanoparticles indicate high stability of the formulation.
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ASSOCIATED CONTENT
S Supporting Information *
Experimental details. This material is available free of charge via the Internet at fhttp://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*3500 Camp Bowie Blvd., Fort Worth, TX 76107. Tel: 817735-2144. E-mail:
[email protected]. Funding
This work was supported by the University Research Council of The University of North Carolina at Chapel Hill (to A.J.D.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Victoria J. Madden (Microscopy Services Laboratory at The University of North Carolina at Chapel Hill) for her help with TEM. We also thank Dr. Fung-Lung Chung (Professor of Oncology at Georgetown University) for his helpful discussions at this project’s inception and Mr. Chintan H. Kapadia (The University of North Carolina at Chapel Hill) for his help early in the liposomal formulation process.
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ABBREVIATIONS PEITC, phenethyl isothiocyanate; NSCLC, non-small cell lung cancer; CDDP, cisplatin; DSPC, 1,2-distearoyl-sn-glycero-3phosphocholine; EPG, L-α-phosphatidylglycerol; ITC, isothiocyanate; BITC, benzyl isothiocyanate
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REFERENCES
(1) Wheate, N. J., Walker, S., Craig, G. E., and Oun, R. (2010) The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Trans. 39, 8113−8127. (2) A. Shen, D.-W., Pouliot, L. M., Hall, M. D., and Gottesman, M. M. (2012) Cisplatin resistance: A cellular self-defense mechanism resulting from multiple epigenetic and genetic changes. Pharmacol. Rev. 64, 706−721. (3) Stathopoulos, G. P., Antoniou, D., Dimitroulis, J., Stathopoulos, J., Marosis, K., and Michalopoulou, P. (2011) Comparison of liposomal cisplatin versus cisplatin in non-squamous cell non-smallcell lung cancer. Cancer Chemother. Pharmacol. 68, 945−950. 948
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