Glutathione boosting the cytotoxicity of a magnetic platinum(iv) nano-prodrug in tumor cells

Fe3O4 nanoparticles with Pt-prodrug payloads display MRI capability and tumor-specific cytotoxicity correlating positively with glutathione-mediated DNA cleavage and reduction.


Introduction
Cisplatin is a rst-line chemotherapeutic drug against a variety of cancers. 1 However, its application has been heavily conditioned by severe systemic toxicities like nephrotoxicity and neurotoxicity. 2 In addition, the efficacy of cisplatin is limited because of inherent or acquired drug resistance. 3 These defects mainly result from its indiscriminate body distribution and insufficient tumor accumulation, and also from its detoxication by sulfur-containing biomolecules. 4 Targeted prodrug systems have been proven effective in minimizing the systemic toxicity and in maximizing the tumor accumulation of Pt drugs. 5 SPIONs could guide drugs preferentially to the biological target through an external magnet and provide a strong negative contrast effect in T 2 -weighted MRI; 6 and polymer-modied SPIONs possess some excellent properties, such as hydrophilicity, nontoxicity, and nonimmunogenicity, for drug delivery. 7 Therefore, in the past few years we endeavoured to load Pt II moieties onto SPIONs as theranostic agents for simultaneous therapy and diagnosis. 8 However, Pt II moieties are capable of reacting with bionucleophiles during the delivery, and hence could be toxic to normal tissues.
Octahedrally coordinated Pt IV complexes are substantially more inert than Pt II complexes and thus can avoid the undesirable side reactions in the blood plasma. 9 Likewise, they are inactive towards nuclear DNA, so the reduction of Pt IV complexes to kinetically labile Pt II species is necessary to exert their cytotoxic effects. GSH is one of the intracellular reductants that activate Pt IV complexes. 10 Many studies have shown that cisplatin-based Pt IV complexes can be reduced to reactive Pt II species and bind DNA to form 1,2-d(GpG) DNA-Pt crosslinks akin to those formed by cisplatin. 11 Meanwhile, the level of cellular GSH may decrease during the reduction, which is benecial for attenuating its detoxifying effect on Pt II species. For these potential merits, we decide to change the previous Pt II pharmacophore to a Pt IV complex.
In this study, we continue to use SPIONs (Fe 3 O 4 ) to design the targeted Pt prodrug system. In order to increase the stability and stealthiness in vivo, SPIONs are coated with PEG, which has been proven to improve the biocompatibility, blood circulation time, and immunotherapeutic efficacy of nanoparticles. 12 A Pt IV complex, c,t,c-[PtCl 2 (OH)(O 2 CCH 2 CH 2 CO 2 H)(NH 3 ) 2 ] (HSPt), is synthesized as the pharmacophore, which is an asymmetrically functionalized prodrug of cisplatin. HSPt is loaded onto the surface of the PEGylated SPIONs, forming HSPt-PEG-SPIONs (Fig. 1). This nanocomposite exhibits some unique properties in vitro, such as GSH-promoted cytotoxicity against tumor cells and nontoxicity towards normal cells. Its mechanism of action also differs from that of cisplatin. Moreover, it produces a signicant negative contrast in MRI, and thus could be a potential theranostic agent for chemotherapy.

Preparation and characterization
The initial SPIONs were synthesized referring to a reported method. 13 PEG-SPIONs were obtained by modifying the SPIONs with silane-diethyltriamine-methoxy PEG, which contains an amine group in the chain. The TEM image shows that the size of the SPIONs is around 11 nm ( Fig. 2A). The XRD spectrum of the SPIONs matches well with that from the JCPDS card (no. 01-1111) for the cubic-phase magnetite (Fig. 2B). The saturation magnetization (M s ) of the SPIONs is 70.05 emu g À1 at room temperature ( Fig. 2C), which is lower than that of the bulk Fe 3 O 4 (89 emu g À1 ) but is higher than that of the modied SPIONs. 14 The absence of coercivity and remanence indicates that the SPIONs exhibit a superparamagnetic property at room temperature. The existence of the polymer on the surface of the SPIONs is conrmed by IR spectroscopy (Fig. 2D). Thermal gravitational analysis shows that the weight of the PEG-SPIONs decreases ca. 80% at 300-400 C due to the evaporation of the surface polymer (Fig. 2E). In the XPS spectrum, the PEG-SPIONs exhibit Fe2p 3/2 and Fe2p 1/2 photoelectron peaks at 711.68 and 725.38 eV, but no obvious charge transfer satellite is observed near the Fe2p 3/2 peak (Fig. 2F), suggesting that Fe is basically at a mixed oxidation state of +II and +III. 15 The ICP-MS and ninhydrin assay show that the molar ratio of free amino groups to Fe in the PEG-SPIONs is 0.146.
HSPt was derived from cisplatin by oxidation with hydrogen peroxide and substitution with succinate, successively. Owing to the existence of an amino group in the polymer and a carboxyl group in HSPt, HSPt-PEG-SPIONs were readily formed via EDC/NHS coupling chemistry. Aer conjugation, the average hydrodynamic diameter of the PEG-SPIONs increased from 31.60 AE 3.50 to 47.80 AE 5.20 nm ( Fig. 3A and B), whereas the zeta potential decreased from +18.10 AE 1.30 to +1.77 AE 0.50 mV ( Fig. 3C and D) due to the shielding of amine groups. These changes suggest that HSPt has bonded with the PEG-SPIONs. The weak positive potential may help the HSPt-PEG-SPIONs pass through the cell membrane (negative inside). Actually, the cellular uptake of Fe and Pt is indeed enhanced aer the conjugation (see Table S1 †). In the XPS spectrum of the HSPt-PEG-SPIONs (Fig. 3E), two new peaks are observed at 73.68 and 77.08 eV as compared with that of the PEG-SPIONs (Fig. 2F), which are assignable to the photoelectron peaks of Pt4f 7/2 and Pt4f 5/2 , respectively, and are consistent with the reported binding energies for Pt IV species. 16 The maximum loading ratio of Pt to Fe (w/w) for the HSPt-PEG-SPIONs is 0.20 (see Table S2 †).

Transverse relaxivity (r 2 ) and in vitro MRI
Transverse relaxivity (r 2 ) represents the efficiency of a contrast agent in shortening the proton relaxation time. 17 As Fig. 4A shows, the T 2 relaxation rate (1/T 2 ) of the HSPt-PEG-SPIONs increases linearly with the Fe concentration and the r 2 value is calculated to be 228.96 mM À1 s À1 , which is signicantly higher than that of the T 2 -weighted MRI contrast agent Feridex (SPION, r 2 z 100 mM À1 s À1 ) used in clinics, 18 and is also higher than that of similar nanocomposites. 19 Fig. 4B reveals an explicit Fe concentration-dependent darkening effect of HSPt-PEG-SPION aqueous suspensions. The in vitro imaging potential was examined by testing the contrast effect in HeLa cells. As Fig. 4C shows, the MR images of the cells aer incubation with HSPt-PEG-SPIONs present an enhanced contrast in comparison with that of the control. The signal intensity is negatively correlated with the concentration of Fe. These results indicate that HSPt-PEG-SPIONs can effectively shorten the T 2 relaxation time, enter into tumor cells and produce negative contrast in MRI.

In vivo MRI
The in vivo tumor-specic accumulation and imaging potential of the HSPt-PEG-SPIONs were evaluated under an external magnetic eld in B6 mice bearing implanted RM1 murine prostate cancer. Fig. 5 shows the T 2 -weighted MR images acquired before and aer the intravenous injection of the HSPt-PEG-SPIONs. A noticeable darkening effect is observed in the circled tumor area aer the injection, indicating that a substantial amount of HSPt-PEG-SPIONs has accumulated within the cancer tissue. Evidently, the negative contrast enhancement is attributed to the high relaxivity (r 2 ) of the HSPt-PEG-SPIONs. The results show that HSPt-PEG-SPIONs can target tumor tissues with the help of external magnet and achieve real-time imaging of tumor tissues during the therapy.

Drug release
The release of Pt II species from HSPt-PEG-SPIONs in the presence of GSH was monitored by ICP-MS (see Fig. S1 †), HPLC and identied by ESI-MS. Peaks assignable to [Pt(NH 3 ) 2 Cl(OH) + Na] + and [Pt(NH 3 ) 2 Cl 2 (OH)(O 2 CCH 2 CH 2 CO 2 )] À are observed in the HPLC at 6 h aer the reaction (see Fig. S2 and Table S3 †), indicating that GSH can facilitate the dissociation of HSPt-PEG-SPIONs and reduce the Pt IV prodrug into Pt II species. As compared with the spectrum recorded at the start point, the intensity of HSPt and polymer peaks increased, while that of the GSH peak decreased markedly. It is known that intracellular GSH can react with cisplatin to form a Pt-GS or Pt-GS-Pt adduct, which can diminish the amount of reactive Pt II species available for DNA and hence undermine the cytotoxicity of cisplatin. 20 In this case, we only detected the [Pt(NH 3 ) 2 (GS)H 2 O] + adduct, which still maintains some reactivity towards DNA. The results imply that the depletion of GSH was mainly caused by the dissociation and reduction of HSPt-PEG-SPIONs, and the detoxication of Pt II species by GSH is limited in this system.
The dissociation of the HSPt-PEG-SPIONs in HeLa cells was monitored by confocal uorescence microscopy. Due to the existence of surface amine groups, uorescein isothiocyanate  (FITC) could readily be conjugated onto PEG-SPIONs along with HSPt, forming the hybrid composite HSPt(FITC)-PEG-SPION; meanwhile, the uorescence of FITC was quenched due to the Förster resonance energy transfer mechanism (see Fig. S3 †). 19 However, the uorescence recovered as the composite was internalized by the cells (Fig. 6 versus S4 †), showing that FITCor HSPt-polymer moieties can become detached from the SPIONs in the presence of cellular GSH. The co-localization image aer nucleolar staining with Hoechst 33342 revealed that the uorescent FITC-polymer species or HSPt-polymer species appear only outside the nuclei, suggesting that further dissociation is required if the Pt unit is aimed at nuclear DNA.

Interaction with DNA
The impact of HSPt-PEG-SPIONs on DNA was studied by agarose gel electrophoresis. As Fig. 7 shows, without GSH, HSPt-PEG-SPIONs only slightly damage the supercoiled DNA (form I), producing a nite amount of nicked DNA (form II); while cisplatin retards the migration rate of form I due to the unwinding of the DNA duplex induced by Pt binding. 21 With GSH, HSPt-PEG-SPIONs effectively cleave form I into form II and further into linear DNA (form III); and the cleavage efficiency is positively related to the concentration of GSH (see Fig. S5 †). The appearance of linear DNA demonstrates that some double-strand breaks (DSBs) have occurred. By contrast, cisplatin and HSPt only cause mild damage to DNA in the presence of GSH (see Fig. S6 †). The results again suggest that the reduction product is not cisplatin, which is in accordance with the above HPLC results. It is believed that the cytotoxicity of cisplatin primarily originates from its covalent binding to DNA and subsequent inhibiting of DNA transcription. 9 Since the reduced Pt II species here do not induce DNA inter-and intra-strand crosslinks as cisplatin does, we suppose the DNA strand breaks, especially the DSBs, play a key role in the    bioactivity of HSPt-PEG-SPIONs. In fact, the formation of DSBs represents the most lethal form of DNA damage, because unrepaired or misrepaired DSBs can lead to cell death and genomic instability. 22 In current chemotherapeutic agents, only bleomycin induces direct DSBs, whereas other agents such as cisplatin induce DSBs by indirect routes. 23 Direct DNA DSBs induced by Pt anticancer drugs were never reported before. We presumed that some reactive oxygen species (ROS) might be responsible for the cleavage and hence conducted a verication using common radical scavengers such as DMSO for cOH, KI for H 2 O 2 , and NaN 3 for 1 O 2 (see Fig. S7 †). Contrary to our assumption, the cleavage activity of the HSPt-PEG-SPIONs did not reduce markedly, suggesting that ROS may not be a determinant in the process. Thus the cleavage mechanism is not yet clear.

Cytotoxicity
The cytotoxicity of the HSPt-PEG-SPIONs against human nonsmall lung cancer A549, human cervical cancer HeLa, and human gastric cancer SGC-7901 cell lines was tested by MTT assays. Meanwhile, the concentration of GSH in these cell lines before and aer the drug treatment was measured by a GSH assay kit. The inhibition efficiency of the HSPt-PEG-SPIONs is comparable to that of cisplatin towards A549 and HeLa cell lines, but is inferior to that of cisplatin towards the SGC-7901 cell line (see Fig. S8 and Table S4 †). The half-maximal inhibitory concentrations (IC 50 ) of the HSPt-PEG-SPIONs and the cellular GSH concentrations are listed in Table 1. The results indicate that the IC 50 values decrease with the concentration of GSH, that is, intracellular GSH can boost the cytotoxicity. In order to further conrm the promotive effect of GSH, the A549 cell line was pre-treated with buthionine sulfoximine (BSO), which is an inhibitor of intracellular GSH synthesis. 24 Aer the treatment, the cytotoxicity of the HSPt-PEG-SPIONs was retested against this cell line. As Fig. 8 shows, the cytotoxicity was dramatically reduced because of the decrease in GSH, thus verifying the promotive effect of GSH on the cytotoxicity of the HSPt-PEG-SPIONs.
It is known that GSH negatively inuences the therapeutic efficacy of Pt II drugs through detoxication due to its chelation with Pt II centers. 20 For Pt IV prodrugs, GSH usually shows a biphasic action involving reduction of Pt IV and sequent chelation with Pt II . However, as we demonstrated above, chelation of Pt II was not observed except a limited coordination with the reduced Pt II species. Therefore, the cytotoxicity of the HSPt-PEG-SPIONs is positively related to the concentration of intracellular GSH or to the depletion of GSH, as high levels of GSH are benecial to the reduction of HSPt and the cleavage of DNA. These results imply that HSPt-PEG-SPIONs may have an advantage for overcoming the tumor resistance to cisplatin induced by GSH. It should be claried that the depletion of cellular GSH not only resulted from the reduction of the Pt IV prodrug, but also resulted from the consumption for scavenging the ROS generated through DNA damage 25 and for forming the Pt-GS complex with Pt II species. Therefore, the percentage of GSH depletion in Table 1 appears to be quite signicant.  Finally, the biocompatibility of the HSPt-PEG-SPIONs was examined on HL-7702 normal liver cells by an MTT assay. More than 80% of the cells are alive even when the concentration reaches 100 mM, while only fractional cells are alive at this concentration for cisplatin (Fig. 9). Therefore, HSPt-PEG-SPIONs are almost nontoxic to normal liver cells.

Conclusions
We have prepared a SPION-based nanocomposite loaded with a Pt IV prodrug as a potential targeted theranostic agent for cancer treatment. In addition to magnetic-targeting and antiproliferative properties, the composite also showed low toxicity to normal cells. The foremost ndings are the signicant MR contrast enhancement, unique DNA damaging mode and increased cytotoxicity in the presence of GSH. The former guarantees the applicability of the nanocomposite as an MR contrast agent for real-time imaging, and the latter two reveal a novel mechanism of action for Pt IV prodrugs loaded on nanoparticles. The role of GSH in cisplatin resistance has been studied extensively in both cell lines and cancer tissues, and an increase in the GSH level has been correlated with cisplatin resistance in ovarian, cervical, lung, embryonal and bladder cancer cell lines due to increased inactivation of the drug. 3 The results of this study indicate that the basic function of intracellular GSH for nano-based Pt IV prodrugs is different from that for Pt II drugs, which has never been perceived so far. The exceptional cleavage mode of DNA and distinct action of GSH may suggest that the cisplatin resistance related to DNA repair and GSH detoxication could be conquered by functionalized Pt IV prodrugs.