Turning Ineffective Transplatin into a Highly Potent ... - ACS Publications

Jul 5, 2016 - In summary, we have shown the first example of turning ineffective transplatin into its highly potent Pt(IV) prodrug form. This Pt(IV) d...
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Turning Ineffective Transplatin into a Highly Potent Anticancer Drug via a Prodrug Strategy for Drug Delivery and Inhibiting Cisplatin Drug Resistance Wenliang Li,† Mo Jiang,† Yue Cao,† Lesan Yan,*,‡ Ruogu Qi,§ Yuxin Li,*,† and Xiabin Jing§ †

National Engineering Laboratory for Druggable Gene and Protein Screening, School of Life Science, Northeast Normal University, Changchun 130024, People’s Republic of China ‡ Department of Bioengineering, University of Pennsylvania, 210 South 33rd Street, Philadelphia, Pennsylvania 19104-6321, United States § State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China S Supporting Information *

ABSTRACT: Clinically ineffective transplatin is highly potent against cancer cells when transformed into a transplatin(IV) prodrug nanoparticle. Herein, a hydrophobic transplatin(IV) was synthesized by H2O2oxidization of transplatin and attachment of two hydrophobic aliphatic chains. Transplatin(IV) was subsequently encapsulated by a biodegradable amphiphilic copolymer, MPEG-PLA, forming a well-defined spherical micelles (M(TransPt)). Transplatin(IV) was protected efficiently and could be released under a simulated cancerous intracellular condition. Compared to the cisplatin and transplatin, M(TransPt) showed the highest Pt uptake and a clathrin-dependent endocytosis pathway. Most importantly, M(TransPt) displayed a nanomolar IC50 on A2780 cells and a great potency on cisplatin resistant A2780DDP cell line. Overall, this nanoplatform for delivering trans-geometry platinum(IV) drug exhibits excellent characteristics for enhancing efficacy and overcoming cisplatin drug resistance, and holds a strong promise for clinical use in the near future.

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ince cisplatin was approved for clinical use in 1978, great efforts have been undertaken to improve its anticancer activity and to overcome its adverse side effects, including nephrotoxicity, peripheral neuropathy, and hearing loss. Apart from that, drug resistance, both inherent and acquired, is another drawback and the main reason for the failure of Pt drugs,1,2 which include not only cisplatin but also carboplatin, oxaliplatin, and other platinum(II) (Pt(II)) drugs. These Pt(II) complexes are susceptible to drug resistance because they share a mechanism of action by forming similar Pt-DNA adducts.3,4 To overcome drug resistance, various nonclassical platinum drugs have been synthesized, including multinuclear platinum,5 platinum(IV) (Pt(IV)),6 photosensitive platinum,7 and transplatinum drugs.8 Unfortunately, most platinum drugs in transgeometry are ineffective due to “the structure-activity relationship”.9 Transplatin (Figure 1), an isomer of cisplatin, is ineffective against cancer cells due to its greater reactivity and more rapid deactivation in circulation compared to cisplatin.10 It was also shown that transplatin reacted with glutathione (GSH) at a 16.5 times lower half-time than cisplatin.11 Moreover, mechanistic studies have revealed that cisplatin forms more 1,2-GG intrastrand (∼65%) and 1,2-AG intrastrand adducts (∼25%).12 However, transplatin forms interstand cross-links (∼12%).13 Compared to intrastrand cross-links, © XXXX American Chemical Society

Figure 1. Schematic illustration of the structure of transplatin(IV) prodrug and M(TransPt), and intracellular release and reduction for the active transplatin after endocytosis of M(TransPt).

Received: June 12, 2016 Revised: July 3, 2016

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DOI: 10.1021/acs.bioconjchem.6b00302 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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6.06% (w/w) (Figure 2A), and the mean diameter of the particles increases from 66 to 198 nm (Figure 2B). This size increase may be due to higher loading amounts of the transplatin(IV) prodrug. The PDI and zeta potential varied little (Figure 2C,D). From these data, we choose the optimal formulation to be a drug to polymer ratio of 0.2 in feed (drug/ polymer = 20% in micelles). DLS shows M(TransPt) with a centralized size distribution and an average diameter of 151 nm. TEM shows fine spherical structures (Figure 2E,F). GSH is a tripeptide present at high concentrations (0.5−10 mM) in cells which reacts with Pt(II) agents and is associated with cisplatin resistance by reducing DNA platination and increasing drug sequestration.1 Due to the trans-effect, transplatin reacts >10 times more rapidly with GSH or other thiolated biomolecules than cisplatin does, resulting in its clinical ineffectiveness.13 To show the stability of M(TransPt), transplatin, cisplatin, and M(TransPt) were mixed with GSH. The remaining amount of free thiol at various times was monitored by Ellman’s assay and UV−vis at 412 nm.24 More remaining free thiol indicates less Pt detoxification. Transplatin and cisplatin were rapidly detoxified by GSH (Figure 3A), but

interstrand cross-links have a higher level of toxicity because fundamental cellular processes depend on DNA chain separation including DNA replication and transcription.14 Specifically, transplatin is ineffective, which could be due to its high reactivity. Therefore, we hypothesize that keeping transplatin intact before its entry into cancer cells may allow it to behave as a qualified antineoplastic agent. The development of Pt(IV) drugs has been the subject of much recent research.15 Octahedral Pt(IV) drugs are relatively kinetically inert and nontoxic but can be triggered to release their toxic Pt(II) counterparts.15 This characteristic makes Pt(IV) drugs ideal prodrugs for cancer therapy. In addition to that, nanoparticle-mediated drug delivery also offers a possible solution by loading prodrugs for drug delivery.16−19 Here, a novel transplatin(IV) prodrug was synthesized by H2O2-oxidization of transplatin and attachment of two hydrophobic aliphatic chains (Transplatin(IV)). The axial aliphatic chains protect the active pharmacophore transplatin, increase its stability as a Pt(IV) prodrug, increase its hydrophobicity for polymer encapsulation and drug delivery, and increase its lipophilicity for cell uptake and maximal drug efficacy.6 This Pt(IV) complex was encapsulated by the amphiphilic biodegradable carrier polymer methoxyl poly(ethylene glycol)-block-poly(lactic acid) (MPEG-b-PLA) to form nanosized micelles (M(TransPt)) that enter cancer cells via endocytosis.20,21 The intracellular acidity and excessive reductive agents such as ascorbic acid/GSH trigger reduction of the transplatin(IV)22,23 to release transplatin(II). Finally, transPt-DNA adducts form to induce cell death (Figure 1). Transplatin(IV) synthesis via t,t,t,-[Pt(NH3)2Cl2(OH)2] is described in the Supporting Information. The structure of transplatin(IV) was verified by 1H NMR, ESI-MS, and IR spectra (Figure S1−S3 in Supporting Information). Transplatin(IV) has two aliphatic chains, making it hydrophobic for encapsulation. MPEG-PLA, an FDA approved polymer, was used to load transplatin(IV) by the nanoprecipitation method. Drug loading by ICP-MS, size and zeta potential by DLS, and TEM images of M(TransPt) are shown in Figure 2. The drug to polymer ratio (w/w) varied from 0.01 to 0.4, and the ratio greater than 0.4 results in precipitates of transplatin(IV) and the polymer. As the drug to polymer ratio increases from 0.01 to 0.4, Pt loading increases from 0.46% to

Figure 3. (A) GSH mediated rapid detoxification of transplatin and cisplatin, but not M(TransPt). Free thiol remaining after incubating Pt drugs with GSH was tested by Ellman’s assay. (B) Drug release of M(TransPt) at pH 5.0, pH 7.4, and 10 mM VC aqueous solution.

M(TransPt) showed little GSH detoxification, indicating that the Pt(IV) prodrug strategy successfully protects transplatin because Pt(IV) molecules are inert to GSH and protected in the core of micelles. Drug release from micelles was evaluated by dialysis in buffered solutions (pH 7.4 and pH 5.0) and 10 mM VC (ascorbic acid) aqueous solution at 37 °C to simulate the neutral environment in blood circulation and the acidic and reductive conditions in cancer cells, respectively.25−27 Platinum release was significantly faster at pH 5.0 than at pH 7.4, with the fastest release in 10 mM VC (Figure 3B), indicating that M(TransPt) is more susceptible to reducing agents than to acid hydrolysis. As discussed above, Pt(IV) are inert to GSH. This makes its structures relatively stable after being released. Because the release media (pH 5.0 and VC 10 mM) simulates intracellular conditions, M(TransPt) is expected to undergo similar release behavior in cancer cells. A2780 (cisplatin sensitive ovarian cancer), A2780DDP (cisplatin resistant ovarian cancer), and LNCaP (human prostate cancer) were chosen to evaluate the in vitro cellular cytotoxicity of M(TransPt) compared to cisplatin and transplatin. Transplatin(IV) was found to be insoluble in 1% DMSO, making cell testing impossible. Dose dependent viability curves of cisplatin, transplatin(II), and M(TransPt)treated cells are shown in Figure 4A−C, and derived IC50 values are shown in Figure 4D. We find that transplatin(II) is nearly nontoxic, in agreement with previous reports.9 Cisplatin was

Figure 2. Nanoparticle formulation of transplatin(IV) prodrugs and characterization of M(TransPt). (A) Drug loading based on Pt, (B) mean size, (C) PDI, and (D) zeta potential of M(TransPt) as a function of drug to polymer ratio. Representative (E) DLS and (F) TEM of M(TransPt). Scale bar: 500 nm. Data (A, B, D) are means ± SD (n = 3). B

DOI: 10.1021/acs.bioconjchem.6b00302 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 4. In vitro evaluation of M(TransPt) on human cancer cell lines. Cell viability of (A) A2780, (B) A2780DDP, and (C) LNCaP cells after 48 h incubation with cisplatin, transplatin(II), and M(TransPt) micelles at various platinum concentrations. (D) IC50 values of cisplatin and M(TransPt). Data are means ± SD (n = 3).

Figure 5. M(TransPt) maximizes Pt drug uptake via endocytosis and induces cell cycle arrest and apoptosis. (A) Uptake of Pt drugs by A2780 and A2780DDP cells over 4 and 8 h. Drugs were dissolved in RPMI 1640 medium at an equal Pt concentration of 40 μM. (B) Uptake percentage of M(TransPt) by A2780 cells in the presence of endocytosis inhibitors. Cells were cotreated with 10 mM sodium azide, genistein (200 μg/mL), chlorpromazine (50 μg/mL), wortmannin (200 nM), and methyl-b-cyclodextrin (200 nM) at 37 °C for 4 h with M(TransPt) at 40 μM Pt. Uptake at 37 °C without inhibitors was set as 100% and cells treated at 4 °C were set as a control. (C) Proportion of A2780 cells in each phase of the cell cycle. Cells were treated with M(TransPt) at 0.1 μM Pt for 48 h. Data shown as mean ± S.D., n = 3. (D) Apoptosis was induced by M(TransPt) at 0.1 μM Pt. Flow cytometry analysis was conducted by Annexin V/PI staining after 72 h.

effective in A2780 cells but less effective in resistant A2780DDP cells and LNCaP cells. However, M(TransPt) is highly potent on all cell lines tested, indicating that M(TransPt) is a more effective formulation for cancer therapy. M(TransPt) is more efficient than cisplatin for all tested cell lines (Figure 4D). Cisplatin had IC50 values of 1.14 μM and 14.0 μM on A2780 and A2780DDP, respectively, yielding a resistance factor of 12.2. However, M(TransPt) had IC50 values of 0.067 μM and 0.30 μM on A2780 and A2780DDP, respectively, giving a resistance factor of only 4.48 (Figure 4D). M(TransPt) was 46.2 and 5.9 times more potent than cisplatin on A2780DDP and LNCaP cell lines, respectively. We have found that turning transplatin, which by itself is ineffective, into a Pt(IV) prodrug with polymer encapsulation can improve potency as well as overcome cisplatin resistance. Platinum drugs bind cell nuclear DNA,28 and higher uptake of these drugs improves their efficacy. Therefore, we determined the intracellular uptake of cisplatin, transplatin(II) and M(TransPt) by A2780 and A2780DDP after 4 and 8 h of drug incubation at equal Pt concentration of 40 μM. Uptake of these drugs showed time dependence (Figure 5A): as incubation time increased from 4 to 8 h, uptake increased, with the most drastic change in M(TransPt) treated cells. Further, both A2780 and A2780DDP cells treated with M(TransPt) internalized more drug than those treated with cisplatin and transplatin(II). Pt uptake by A2780 cells treated with M(TransPt) at 4 h was 8.5-fold and 17.5-fold that of cells treated with cisplatin and transplatin(II), respectively. The greater uptake of M(TransPt) corresponds to the increased proliferation inhibition efficacy in cell lines shown in Figure 4. Visualization of uptake by confocal laser scanning (CLSM) of Rhodamine B labeled nanoparticles (M(TransPt/RhB)) (Figure S4 in Supporting Information) shows M(TransPt/ RhB) distributed primarily in the cytosol. To study the uptake pathway of M(TransPt), various endocytosis inhibitors were used.29 Pt uptake of M(TransPt) at 40 μM Pt by A2780 cells (Figure 5B) indicates intracellular uptake inhibition is in this order: low temperature (4 °C) > chlorpromazine (clathrin-dependent endocytosis) > genistein (clathrin-independent endocytosis) > sodium azide (electron transport chain inhibitor). However, methyl-β-cyclodextrin

(clathrin-independent endocytosis) has little effect on uptake of M(TransPt), indicating that M(TransPt) is more dependent on clathrin-dependent endocytosis. Platinum drugs are thought to induce cell cycle arrest and apoptosis. The ability of M(TransPt) to induce these effects is shown in Figure 5C,D and Figure S5−6 (Supporting Information). A PBS treated control group shows 79.0%, 14.6%, and 5.8% of cells in G0/G1, S, and G2/M, respectively. M(TransPt) decreased the percentage of cells in G0/G1, and increased the proportion of cells in S and G2/M to 31.1% and 9.5%, respectively. The relatively large increase of cells in S phase indicates that M(TransPt) induces cell cycle arrest in it. S phase arrest may lead to cell apoptosis;30,31 therefore, we tested the apoptotic action of M(TransPt) by flow cytometry. The apoptosis rates were 25.3% and 36.9% for M(TransPt) treated A2780 and A2780DDP cell lines (Figure 5D). The dramatic difference in apoptosis between M(TransPt) and control indicates that M(TransPt) is able to induce cell apoptosis. The apoptosis rates on A2780DDP was higher than A2780, which may be one of the reasons why M(TransPt) can inhibit cisplatin drug resistance. In summary, we have shown the first example of turning ineffective transplatin into its highly potent Pt(IV) prodrug form. This Pt(IV) drug was further formulated into nanoparticles as M(TransPt) at a drug to polymer ratio of 0.2. Though transplatin is rapidly detoxified by GSH by Pt−S bond formation, M(TransPt) is not readily inactivated by GSH. Unlike transplatin, M(TransPt) showed great potency even on cisplatin resistant cell lines in vitro. M(TransPt) displayed a nanomolar IC50 on A2780 cells. Moreover, M(TransPt) could overcome cisplatin drug resistance. This is likely due to higher Pt uptake with M(TransPt) compared to that of other C

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(9) Shaw, J. L., Dockery, C. R., Lewis, S. E., Harris, L., and Bettis, R. (2009) The trans effect: A guided-inquiry experiment for upperdivision inorganic chemistry. J. Chem. Educ. 86, 1416−1418. (10) Jung, Y., and Lippard, S. J. (2007) Direct cellular responses to platinum-induced DNA damage. Chem. Rev. 107, 1387−1407. (11) Suchánková, T., Vojtíšková, M., Reedijk, J., Brabec, V., and Kašpárková, J. (2009) DNA and glutathione interactions in cell-free media of asymmetric platinum (II) complexes cis-and trans-[PtCl2(isopropylamine) (1-methylimidazole)]: relations to their different antitumor effects. JBIC, J. Biol. Inorg. Chem. 14, 75−87. (12) Eastman, A. (1987) The formation, isolation and characterization of DNA adducts produced by anticancer platinum complexes. Pharmacol. Ther. 34, 155−166. (13) Brabec, V., and Leng, M. (1993) DNA interstrand cross-links of trans-diamminedichloroplatinum(II) are preferentially formed between guanine and complementary cytosine residues. Proc. Natl. Acad. Sci. U. S. A. 90, 5345−5349. (14) Enoiu, M., Jiricny, J., and Schärer, O. D. (2012) Repair of cisplatin-induced DNA interstrand crosslinks by a replicationindependent pathway involving transcription-coupled repair and translesion synthesis. Nucleic Acids Res. 40, 8953−8964. (15) Johnstone, T. C., Wilson, J. J., and Lippard, S. J. (2013) Monofunctional and higher-valent platinum anticancer agents. Inorg. Chem. 52, 12234−12249. (16) Nguyen, M. M., Carlini, A. S., Chien, M. P., Sonnenberg, S., Luo, C., Braden, R. L., Osborn, K. G., Li, Y., Gianneschi, N. C., and Christman, K. L. (2015) Enzyme-Responsive Nanoparticles for Targeted Accumulation and Prolonged Retention in Heart Tissue after Myocardial Infarction. Adv. Mater. 27, 5547−5552. (17) Wang, H., Xie, H., Wu, J., Wei, X., Zhou, L., Xu, X., and Zheng, S. (2014) Structure-based rational design of prodrugs to enable their combination with polymeric nanoparticle delivery platforms for enhanced antitumor efficacy. Angew. Chem., Int. Ed. 53, 11532−11537. (18) Li, W. (2015) The era of nanotechnology and omics sciences. European Journal of BioMedical Research 1, 1−2. (19) Li, Y., Huang, Y., Wang, Z., Carniato, F., Xie, Y., Patterson, J. P., Thompson, M. P., Andolina, C. M., Ditri, T. B., Millstone, J. E., Figueroa, J. S., Rinehart, J. D., Scadeng, M., Botta, M., and Gianneschi, N. C. (2016) Polycatechol Nanoparticle MRI Contrast Agents. Small 12, 668−677. (20) Xiao, H., Li, W., Qi, R., Yan, L., Wang, R., Liu, S., Zheng, Y., Xie, Z., and Jing, X. (2012) Co-delivery of daunomycin and oxaliplatin by biodegradable polymers for safer and more efficacious combination therapy. J. Controlled Release 163, 304−314. (21) Iversen, T. G., Skotland, T., and Sandvig, K. (2011) Endocytosis and intracellular transport of nanoparticles: present knowledge and need for future studies. Nano Today 6, 176−185. (22) Reiber, H., Ruff, M., and Uhr, M. (1993) Ascorbate concentration in human cerebrospinal fluid (CSF) and serum. Intrathecal accumulation and CSF flow rate. Clin. Chim. Acta 217, 163−173. (23) Michelet, F., Gueguen, R., Leroy, P., Wellman, A., Nicolas, A., and Siest, G. (1995) Blood and plasma glutathione measured in healthy subjects by HPLC: relation to sex, aging, biological variables, and life habits. Clin. Chem. 41, 1509−1517. (24) Kratochwil, N. A., and Bednarski, P. J. (1999) Effect of thiols exported by cancer cells on the stability and growth-inhibitory activity of Pt(IV) complexes. J. Cancer Res. Clin. Oncol. 125, 690−696. (25) Washko, P., Rotrosen, D., and Levine, M. (1991) Ascorbic acid in human neutrophils. Am. J. Clin. Nutr. 54, 1221S−1227S. (26) Shi, Y., Liu, S., Kerwood, D. J., and Dabrowiak, J. C. (2012) Pt(IV) complexes as prodrugs for cisplatin. J. Inorg. Biochem. 107, 6− 14. (27) Li, Z., Qiu, L., Chen, Q., Hao, T., Qiao, M., Zhao, H., Zhang, J., Hu, H., Zhao, X., Chen, D., and Mei, L. (2015) pH-sensitive nanoparticles of poly(l-histidine)-poly(lactide-co-glycolide)-to copheryl polyethylene glycol succinate for anti-tumor drug delivery. Acta Biomater. 11, 137−150.

treatments. M(TranPt) can induce apoptosis with S phase cell cycle arrest. This new strategy of delivering trans-geometry platinum(IV) drugs could gain potential use for enhancing efficacy and overcoming cisplatin drug resistance.

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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.bioconjchem.6b00302. Experimental details, characterization, and supporting figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We give our sincere thanks to Jessica F. Liu from the School of Medicine at the University of Pennsylvania for reviewing and editing the manuscript. This work is supported by the National Natural Science Foundation of China (No. 51403031), by the China Postdoctoral Science Foundation (Nos. 2014M550163 and 2015T80281), by the National Key New Drug Creation and Manufacturing Program of Ministry of Science and Technology (No. 2013ZX09103003004), by the Grant of Jilin Province Science & Technology Committee (Nos. 20110711, 20140520049JH, 20150204038YY, YYZX201121, 20130201008ZY, and 20150309003YY) and the Grant of Changchun Science & Technology Committee (Nos. 2014070 and 2013314).

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DOI: 10.1021/acs.bioconjchem.6b00302 Bioconjugate Chem. XXXX, XXX, XXX−XXX