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Preparation and Antiproliferative Activity of Liposomes Containing a Combination of Cisplatin and Procainamide Hydrochloride Maurizio Viale,*,† Antonella Fontana,‡ Irena Maric,† Massimiliano Monticone,† Guido Angelini,‡ and Carla Gasbarri‡ †
IRCCS Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, U.O.C. Bioterapie, 16132 Genova, Italy ‡ Dipartimento di Farmacia, Università ‘G. d’Annunzio’di Chieti-Pescara, 66100 Chieti, Italy S Supporting Information *
ABSTRACT: We have previously reported the enhancement of the antiproliferative and apoptotic activities of cisdiamminedichloroplatinum(II) (DDP) when it is coadministered with a class I antiarrhythmic drug procainamide hydrochloride (PA). Here, we determined the antiproliferative activity of DDP, either in solution or loaded in liposomes, in the presence of PA, in the bulk solution, or directly embedded in liposomes together with DDP. Our results show that PA potentiates the activity of DDP-liposomes and that this effect is maintained at least in some of the investigated cell types when both drugs were mixed and loaded together into liposomes.
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The multilamellar liposomes, obtained by hydration of a lipid film, demonstrated to have a mean diameter of 327 ± 3 nm for DDP-liposomes and 465 ± 5 nm for DDP/PA-liposomes with a polydispersity index corresponding to 0.3 ± 0.1 for both the liposomal formulations. As far as the loading capacity is concerned, the not-filtered DDP/PA-liposomes were loaded with 6.5 × 10−4 M PA and 1.0 × 10−4 M DDP, distributed in the internal aqueous compartments and the bulk solution due to their high hydrophilic nature (the LogP values are −2.53 for DDP8 and −1.5 for PA9). The DDP loading spectrophotometrically determined in the filtered liposomes was 2.4 ± 1.4 × 10−5 M, whereas, in the case of DDP/PA-liposomes, the effective DDP and PA contents were 3.0 ± 1.6 × 10−5 M and 3.1 ± 0.3 × 10−4 M, respectively. Electrostatic interactions between the cationic form of PA and the negatively charged liposomes10−13 increase the percentage of PA loading in comparison to that of DDP in filtered DDP/PA-liposomes as confirmed by the change of DDP/PA ratio from 1/6.5 to 1/10. The antiproliferative activity of DDP, either in solution or loaded in liposomes, in the presence of PA in the bulk solution is reported in Figure 1, and it confirms that the antiarrhythmic drug potentiates the activity of DDP in A549 cells. Furthermore, data demonstrate that addition of PA to DDPloaded liposomes does not alter negatively its potentiating activity and that DDP-liposomes have an antiproliferative activity higher than that obtained from DDP in solution (IC50s, mean ± SE: 4.46 ± 0.58 vs 2.23 ± 0.17, for DDP in solution
rocainamide hydrochloride (PA) has been demonstrated to be able to protect the kidney and the liver from cisdiamminedichloroplatinum(II) (DDP)-induced nephrotoxicity1 and hepatotoxicity2 in vivo and also when multiple low doses of this anticancer drug were administered.3 This protection was shown also in pregnant mice without risking embryotoxic or teratogenic effects due to DDP administration but rather protecting newborns.4 This was due to both the change of the pharmacokinetic profile of Pt in the plasma and in the tissues5 and the formation of a coordination complex between DDP and PA that is less toxic than DDP alone.2,6,7 Furthermore, PA is also able to enhance DDP antiproliferative and apoptotic activity in vitro and DDP antitumor and apoptotic activity in vivo.6,7 In the present study, we designed DDP-loaded liposomal formulations for coadministration of DDP with PA either in bulk solution or in liposomes. We aimed to create a delivery system that carries a drug combination more active and less toxic than single DDP. In particular, we prepared unfiltered liposomes containing DDP (i.e., unfiltered DPP liposomes) or DPP and PA (i.e., unfiltered DDP/PA-liposomes) in both internal aqueous compartments and in bulk solution as well as filtered liposomes in which we get rid of drugs in the bulk solution via gel filtration. The liposomal formulations were fully characterized in terms of DDP/PA loading capacity, size, and polidispersity index. Moreover, their antiproliferative activity was evaluated by the MTT assay on the human ovarian carcinoma A2780, lung carcinoma A549, and non-Hodgkin lymphoma DOHH2 cells. © 2016 American Chemical Society
Received: June 14, 2016 Published: August 8, 2016 1393
DOI: 10.1021/acs.chemrestox.6b00207 Chem. Res. Toxicol. 2016, 29, 1393−1395
Chemical Research in Toxicology
Rapid Report
Most important, significant differences were observed between the two kinds of preparations. In particular, the solubilization of either single or both drugs in the aqueous core and interlamellar regions of liposomes (i.e., filtered DDP- and DDP/PA-liposomes) significantly favors the antiproliferative activity of the formulation (Figure 2) underlining a promising shuttling effect of the liposomes toward the targeted cells. Finally, with regard to the unfiltered liposomal formulation, the presence of PA together with DDP makes the liposomes significantly more active than those containing DDP alone only when DOHH2 cells were investigated (p < 0.01), while a simple positive trend was found for A549 cells (p = 0.088, Figure 2A and B). In the case of filtered liposomal formulation, we did not observe differences due to the concurrent presence of PA and DDP. Unlike the unfiltered formulation, on DOHH2 cells the activity of the liposomes containing DDP+PA was lower than that of the DDP-loaded ones (Figure 2A). In conclusion, the results reported here demonstrate that liposomes provide an efficient drug delivery system14−22 for the coadministration of DDP and PA by increasing the antiproliferative activity of the Pt compound. Moreover, filtration demonstrated to improve the activity of DDP administered by liposomal formulation by enhancing the effective drug concentration that reaches the target. Nevertheless, the loading of both DDP and PA into liposomes does not generally cause a significant increase of DDP activity except when the unfiltered liposomes are incubated with the DOHH2 cells and, as a trend, with the A549 cells. The absence of a synergistic effect of codelivering both drugs through the filtered liposomal formulations might be essentially due to (i) the actual loading capacity of the drugs and consequently the effective DDP/PA ratio in the internal aqueous core and (ii) reduced cellular uptake21,22 of the larger liposomes in the presence of PA. We indirectly verified the first hypothesis treating A2780 and A549 cells with different concentrations of DDP combined with PA in the ratios of 1/6.5 and 1/10 (that was the ratio verified in filtered liposomes). Concentration−response curves allowed the calculation of mean IC50 in both conditions. The data obtained in both cell lines solved our first hypothesis. Indeed, the antiproliferative activity of DDP/PA co-exposure was
Figure 1. Antiproliferative activity on A549 cells of combinations of PA in water and DDP in saline or loaded into filtered liposomes (applied concentrations: for DDP-liposomes, from 5 to 0.312 μM, dilution factor 1:2; for DDP in solution, from 16 to 1 μM, dilution factor 1:2). Data are shown as the mean ± SE of 6 or 7 experiments. a is indicative of p values (vs 0 μM PA, < 0.05) calculated according to the Wilcoxon signed-rank test for nonparametric data.
and filtered DDP-liposomes, respectively). It is of note that in no cases did PA alone show a significant antiproliferative activity (cell viability of A549 cells (%): 98.0 ± 5.6 (n = 14), 96.2 ± 8.3 (n = 14), and 96.0 ± 12.1 (n = 12) for PA at 10, 40, and 160 μM, respectively). Then, we tested the liposomal systems containing the combination of both drugs in two different ways (i.e., filtered and unfiltered DDP/PA-liposomes; see Supporting Information) in order to verify whether the previously demonstrated6,7 effect of PA on DDP activity was observed also for the drugs embedded in proper delivery systems. Both filtered and unfiltered DDP/PA-liposomes were prepared starting from an initial DDP/PA molar ratio of 1/6.5 which is the most used ratio for in vivo experiments.1,2,4,5,7 In these conditions, all types of liposomes showed a general activity that was significantly higher than that of DDP alone administered in solution (Figure 2), with the only exception of unfiltered DDP-liposomes on DOHH2 cells (Figure 2A).
Figure 2. Values of IC50 calculated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 72 h after exposure of cells either to DDP or the liposomal formulations containing DDP, with or without PA, in saline. Each bar represents the mean ± SE of 9−26 experiments. The kind of treatment for each bar is shown in panel C. p values were calculated according to the Mann−Whitney test. Panel A, DOHH2 cells; differences between the IC50 values for treatments with liposome preparations are statistically significant from DDP in solution treatment (p < 0.01), except in the case of unfiltered DDP-liposomes. a, p < 0.01, vs unfiltered DDP-liposomes; b, p < 0.05, vs unfiltered DDP/PAliposomes; c, p < 0.01, vs filtered DDP-liposomes. Panel B, A549 cells; all treatments with liposome preparations were significantly more active than DDP in solution treatment (p < 0.01). a, p < 0.10, vs unfiltered DDP-liposomes; b, p < 0.01, vs unfiltered DDP-liposomes; c, p < 0.01, vs unfiltered DDP/PA-liposomes. Panel C, A2780 cells; all treatments with liposome preparations were significantly more active than treatments with solutions of DDP (p < 0.01, and p < 0.05 for unfiltered DDP-liposomes). a, p < 0.01, vs unfiltered DDP-liposomes; b, p < 0.01, vs unfiltered DDP/PA-liposomes. 1394
DOI: 10.1021/acs.chemrestox.6b00207 Chem. Res. Toxicol. 2016, 29, 1393−1395
Chemical Research in Toxicology
Rapid Report
(5) Vannozzi, M. O., Ottone, M., Mariggiò, M. A., Cafaggi, S., Parodi, B., Cilli, M., Lindup, E., and Viale, M. (2003) Pharmacokinetic and pharmacodynamic analysis of platinum after combined treatment of cisplatin and procainamide hydrochloride in mice bearing P388 leukemia. Anticancer Res. 23, 1509−1516. (6) Viale, M., Fenoglio, C., de Totero, D., Prigione, I., Cassano, A., Vincenti, A., Bocca, P., and Mariggiò, M. A. (2016) Potentiation of cisplatin-induced antiproliferative and apoptotic activities by the antiarrhythmic drug procainamide hydrochloride. Pharmacol. Rep. 68, 654−61. (7) Esposito, M., Viale, M., Vannozzi, M. O., Zicca, A., Cadoni, A., Merlo, F., and Gogioso, L. (1996) Effect of the antiarrhythmic drug procainamide on the toxicity and antitumour activity of cisdiamminedichloroplatinum(II). Toxicol. Appl. Pharmacol. 140, 370− 377. (8) Screnci, D., McKeage, M. J., Galettis, P., Hambley, T. W., Palmer, B. D., and Baguley, B. C. (2000) Relationship between hydrophobicity, reactivity, accumulation and peripheral nerve toxicity of a series of platinum drugs. Br. J. Cancer 82, 966−972. (9) Mian, M. S., El-Obeid, H. A., and Al-Badr, A. A. (2001) Analytical Profiles of Procainamide Hydrochloride, in Analytical Profiles of Drug Substances and Excipients (Brittain, H. G., Ed.), Vol. 28, pp 251−332, Academic Press, New York. (10) Feinstein, M. B. (1964) Reaction of local anesthetics with phospholipids. J. Gen. Physiol. 48, 357−374. (11) Wang, H. H., Earnest, J., and Limbacher, H. P. (1983) Local anesthetic-membrane interaction: a multiequilibrium model. Proc. Natl. Acad. Sci. U. S. A. 80, 5297−5301. (12) Gasbarri, C., and Angelini, G. (2014) Spectroscopic investigation of fluorinated phenols as pH-sensitive probes in mixed liposomal systems. RSC Adv. 4, 17840−17845. (13) Zappacosta, R., Semeraro, M., Baroncini, M., Silvi, S., Aschi, M., Credi, A., and Fontana (2010) Liposome destabilization by a 2,7diazapyrenium derivative through formation of transient pores in the lipid bilayer. Small 6, 952−959. (14) Chaudhury, A., and Das, S. (2015) Folate receptor targeted liposomes encapsulating anti-cancer drugs. Curr. Pharm. Biotechnol. 16, 333−343. (15) de Souza, T. P., Fahr, A., Luisi, P. L., and Stano, P. (2014) Spontaneous encapsulation and concentration of biological macromolecules in liposomes: an intriguing phenomenon and its relevance in origins of life. J. Mol. Evol. 79, 179−192. (16) Tahover, E., Patil, Y. P., and Gabizon, A. A. (2015) Emerging delivery systems to reduce doxorubicin cardiotoxicity and improve therapeutic index: focus on liposomes. Anti-Cancer Drugs 26, 241−258. (17) Monteiro, N., Martins, A., Reis, R. L., and Neves, N. M. (2014) Liposomes in tissue engineering and regenerative medicine. J. R. Soc., Interface 11, 20140459. (18) Kieler-Ferguson, H. M., Fréchet, J. M., and Szoka, F. C., Jr. (2013) Clinical developments of chemotherapeutic nanomedicines: polymers and liposomes for delivery of camptothecins and platinum (II) drugs. WIREs Nanomed. Nanobiotechnol. l5, 130−138. (19) Harrington, K. J., Lewanski, C. R., and Stewart, J. S. (2000) Liposomes as vehicles for targeted therapy of cancer. Part 2: clinical development. Clin. Oncol. 12, 16−24. (20) Fontana, A., Viale, M., Guernelli, S., Gasbarri, C., Rizzato, E., Maccagno, M., Petrillo, G., Aiello, C., Ferrini, S., and Spinelli, D. (2010) Strategies for improving the water solubility of new antitumor nitronaphthylbutadiene derivatives. Org. Biomol. Chem. 8, 5674−5681. (21) Ito, J., Kato, T., Kamio, Y., Kato, H., Kishikawa, T., Toda, T., Sasaki, S., and Tanaka, R. (1991) A cellular uptake of cis-Platinum encapsulating liposome through endocytosis by human neuroblastoma cell. Neurochem. Int. 18, 257−264. (22) Andar, A. U., Hood, R. R., Vreeland, W. N., DeVoe, D. L., and Swaan, P. W. (2014) Microfluidic preparation of liposomes to determine particle size influence on cellular uptake mechanisms. Pharm. Res. 31, 401−413.
significantly higher for DDP/PA 1/6.5 with respect to 1/10 (A2780, 0.60 ± 0.23 μM vs 0.81 ± 0.36 μM, p < 0.05; A549, 3.27 ± 0.93 μM vs 4.47 ± 0.85 μM, p < 0.05). To the best of our knowledge, the coadministration of DDP and PA into the same liposomal formulation is unprecedented. Although preliminary, the promising results obtained for notfiltered DDP/PA liposomes with respect to unfiltered DDP liposomes and for DDP liposomes compared to DDP in solutions shed light on the effective benefit of the entrapment of the investigated drugs into the liposome system. Overall, the present study improves the design of new formulations that aim to further increase the efficacy of the combined administration of DDP and PA.
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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.chemrestox.6b00207. Structures of DDP and PA, and materials and methods for the preparation and analysis of different samples (PDF)
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AUTHOR INFORMATION
Corresponding Author
*IRCCS Azienda Ospedaliera Universitaria San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, U.O.C. Bioterapie, L.go R. Benzi, 10, 16132, Genova, Italy. E-mail: maurizio.viale@ hsanmartino.it. Funding
This work was carried out with financial support from the University “G. d’Annunzio” of Chieti-Pescara. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Dr. Diana Velluto for the helpful proof reading and editing of the manuscript. ABBREVIATIONS DDP, cisplatin; PA, procainamide hydrochloride; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide REFERENCES
(1) Viale, M., Vannozzi, M. O., Pastrone, I., Mariggiò, M. A., Zicca, A., Cadoni, A., Cafaggi, S., Tolino, G., Lunardi, G., Civalleri, D., Lindup, W. E., and Esposito, M. (2000) Reduction of cisplatin nephrotoxicity by procainamide: does the formation of a cisplatinprocainamide complex play a role? J. Pharmacol. Exp. Ther. 293, 829− 836. (2) Zicca, A., Cafaggi, S., Mariggiò, M. A., Vannozzi, M. O., Ottone, M., Bocchini, V., Caviglioli, G., and Viale, M. (2002) Reduction of cisplatin hepatotoxicity by procainamide in rats. Eur. J. Pharmacol. 442, 265−272. (3) Fenoglio, C., Boncompagni, E., Chiavarina, B., Cafaggi, S., Cilli, M., and Viale, M. (2005) Morphological and histochemical evidence of the protective effect of procainamide hydrochloride on tissue damage induced by repeated administration of low doses of cisplatin. Anticancer Res. 25, 4019−4024. (4) Ognio, E., Chiavarina, B., Peterka, M., Mariggiò, M. A., and Viale, M. (2006) Study of feasibility of the tretment with procainamide hydrochloride and cisplatin in pregnant mice. Chem.-Biol. Interact. 164, 232−240. 1395
DOI: 10.1021/acs.chemrestox.6b00207 Chem. Res. Toxicol. 2016, 29, 1393−1395
