A luminescent europium(III)–platinum(II) heterometallic complex as a theranostic agent: a proof-of-concept study

Abstract

A luminescent heterometallic multifunctional theranostic Eu–Pt₂ complex [{cis-PtCl₂(DMSO)}₂Eu(L)(H₂O)] has been synthesized, possessing two therapeutic Pt-centers as covalent DNA binders and one emissive Eu³⁺-center which is sensitized by platinum-based metal-to-ligand charge-transfer excited states.

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Multifunctional cancer theranostic agents with multiple therapeutic and diagnostic centers in a single platform have gained popularity in recent years.^1–3 Recently, there are a few reports on heterometallic hairpin-shaped lanthanide–Pt₂ complexes for DNA recognition and magnetic resonance imaging (MRI) based theranostic agents or for selectively delivering gadolinium to tumor cell nuclei.⁴ In this regard, theranostic Gd³⁺-based platinum complexes have been reported^4c in which Gd³⁺ and conjugated cis-[Pt(NH₃)₂Cl]⁺-moieties act as MRI contrast and therapeutic centers, respectively. These complexes showed therapeutic efficiency along with improved MR imaging capability. However, their DNA-binding ability is limited because there are only two available DNA-crosslinking sites. In addition, the clinical use of platinum drugs is severely affected by drug resistance mediated by inadequate levels of platinum reaching critical target DNA.⁵ Notwithstanding this progress, it is highly desirable to increase the DNA binding sites that significantly enhance platinum content at target sites along with the tagging of a bright luminescent center as a diagnostic probe. To this end, we report the design of a multimodal targeted theranostic Eu–Pt₂ conjugate possessing four DNA binding sites which can effectively target nuclear DNA along with a highly luminescent Eu³⁺ center to enable interference-free live-tracking of the drug using fluorescence microscopy. Luminescent lanthanide complexes were widely exploited in various bioassays since they offer unique photophysical properties like narrow emission bands, a large Stokes’ shift and long-lived excited state lifetimes.^6,7 Since direct excitation of a Ln³⁺ f–f transition is very inefficient, chemists have designed a variety of chelating agents conjugated to a sensitizing organic chromophore called an antenna, which can transfer its excited state energy efficiently to the emissive Ln³⁺ ion leading to bright luminescence compared to the direct excitation of Ln³⁺ ions.^8,9 Our approach exploits the advantages of transition metal complexes as they exhibit many desirable properties as a sensitizer than as an organic chromophore, such as tunable absorption bands, long-lived excited states which maximize the ET to Ln³⁺, excellent photochemical stability and kinetic inertness.^10,11

Herein, we demonstrate a multifunctional Eu–Pt₂ complex, [{cis-PtCl₂(DMSO)}₂Eu(L)(H₂O)] (1) (Scheme 1), which has cytotoxic cis-[PtCl₂(DMSO)] moieties that enable DNA binding, whereas the EuL unit acts as a luminescent reporter. Thus, a combination of a luminescent imaging probe and a conjugated therapeutic agent in a single hybrid 5d–4f complex can provide real-time feedback on drug delivery, distribution and target site localization in a non-invasive manner using fluorescence microscopy. Another key design feature of this complex is having four potential DNA cross-linking sites due to the presence of labile Pt–Cl bonds, thus a higher level of activated platinum will reach DNA, which is a possible way to lower drug resistance. Eu–Pt₂ complex 1 was prepared in a sequential manner (Scheme S1, ESI‡) starting with a multidentate DTPA-bisamide ligand, H₃L = N,N′′-bis(3-amidoquinolyl)diethylenetriamine-N,N′,N′′-triacetic acid, derived from the acylation of 3-aminoquinoline by DTPA-bis(anhydride). [Eu(L)(H₂O)] was synthesized by reacting a 1 : 1 molar ratio of the deprotonated ligand and Eu(NO₃)₃·6H₂O in water. [{cis-PtCl₂(DMSO)}₂Eu(L)(H₂O)] (1) was isolated after the reaction of EuL with freshly prepared cis-[Pt(DMSO)₂Cl₂] in a 1 : 2 molar ratio. Detailed ESI-MS studies of the Eu–Pt₂ complex reveal an m/z of 714.52 corresponding to {M − 2Cl}²⁺ with a matching isotopic distribution profile, which can be unequivocally attributed to the formation of Eu–Pt₂ along with other physicochemical data (Fig. S1, ESI‡). We observed dissociable chloride ligands in aqueous solution which is crucial for forming cross-links with base pairs in nuclear DNA.

Scheme 1 Molecular structure of Eu–Pt₂ complex 1 showing a Pt → Eu energy transfer pathway upon MLCT photo-excitation leading to f–f emission from Eu³⁺ and dissociable Pt–Cl bonds as DNA-cross linking sites.

The UV-vis spectrum of 1 exhibits a high energy band at 273 nm due to ligand centered π → π* transitions and a broad band ranging from 332–350 nm corresponding to π → π* MLCT transitions of the quinoline bound Pt²⁺ moiety (Fig. 1a). Here we have judiciously utilized this ³MLCT excited state as a means to populate the ⁵D₀ emissive states of europium through efficient energy transfer. The ³MLCT → f energy transfer through such photosensitization is shown in few Eu–Pt complexes.¹² The addition of cis-[Pt(DMSO)₂Cl₂] to EuL resulted in the appearance of a new band at 350 nm with an isobestic point at ∼270 nm, indicative of the formation of Eu–Pt₂ complex 1 during titration (Fig. S2 in ESI‡). Upon excitation of the MLCT band, the Eu–Pt₂ complex under time-gated mode displayed narrow emission bands spanning from 575–700 nm characteristic of the ⁵D₀ → ⁷F_J (J = 0–4) f–f transitions of Eu³⁺ (Fig. 1b). The luminescence spectra demonstrate the efficient photosensitized energy transfer from the MLCT excited state of the quinoline bound Pt²⁺ moiety to the emissive ⁵D₀ excited state of Eu³⁺ with an overall quantum yield (ϕ_overall) of 0.04. Spectrophotometric titration of cis-[Pt(DMSO)₂Cl₂] with [Eu(L)(H₂O)] at λ_ex = 330 nm showed the formation of [{cis-PtCl₂(DMSO)}₂Eu(L)(H₂O)] (1) with a gradual increase in europium centered emission until it reached a plateau at Eu/Pt = 0.5 (Fig. 1c). The decay rate of the emissive ⁵D₀ excited state measured at 616 nm results in a monoexponential decay curve with a lifetime (τ_obs) of 0.65(±10%) ms in aqueous buffer medium, indicating the presence of a single chemical environment. The enhancement of the τ_obs in the presence of DNA (τ_obs = 0.89 (±10%) ms) indicates minimization of the nonradiative relaxation pathways in the DNA bound form of the Eu–Pt₂ complex (Fig. 1d).

Fig. 1 (a) UV-vis and (b) emission spectrum (λ_ex = 330 nm) of Eu–Pt₂ complex 1 in Tris-HCl buffer. (c) Evolution of the emission spectra with increasing [EuL] added to a solution of [PtCl₂(DMSO)₂] in DMF (λ_ex = 330 nm), Inset: the plot of relative emission intensity at 615 nm vs. [Eu]/[Pt] ratio. (d) Luminescence decay profile from the ⁵D₀ state of Eu³⁺ in complex 1 at λ_em = 616 nm (λ_ex = 330 nm) (black) and in the presence of CT-DNA (blue) in Tris-HCl buffer (pH = 7.2). [1] = 90 μM, [DNA] = 450 μM, delay time and gate time = 0.1 ms. The solid lines are the best fits considering the single-exponential behaviour of the decay.

Since DNA is the most important target for therapeutic platinum drugs, we attempted detailed binding studies of 1 with DNA. The hairpin-shaped complex 1 is activated by aquation through the substitution of the chloride ligands by water which generates a potent cation, [Eu(L)(H₂O){cis-Pt(OH₂)_n/2(DMSO)}₂]ⁿ⁺ (n = 1–4), which can readily cross-link with the nucleobases of ds-DNA (Scheme S2, ESI‡). The binding interaction of the Eu–Pt₂ complex with calf-thymus DNA (CT-DNA) was studied using UV-vis titration, competitive displacement of ethidium bromide by fluorescence (K_app = 4.9 × 10⁵ M⁻¹), circular dichroism (CD) and isothermal titration calorimetry (ITC) (Fig. S6–S8, ESI‡). The ITC titration plot suggests a biphasic sequential binding interaction¹³ of Eu–Pt₂ with CT-DNA with an initial favorable exothermic binding event (K₁ = 1.7 × 10⁵ M⁻¹, ΔH₁ = −26.0 kcal mol⁻¹), followed by a second exothermic event (K₂ = 9.1 × 10⁵ M⁻¹, ΔH₂ = −77.6 kcal mol⁻¹) presumably due to successive sequential covalent cross-link formation with base pairs of duplex-DNA (Fig. 2a). The intrinsic binding constant (K_b = 1.5(±0.3) × 10⁵ M⁻¹) along with a hypochromism and bathochromic shift of the electronic spectral bands suggests a favorable binding interaction of the complex 1 with DNA due to EuPt₂–DNA adduct formation, strong electrostatic interaction with activated {Eu–Pt₂}ⁿ⁺ and favorable stacking interactions of the two planar –Pt(L) chromophores with the planar base pairs of ds-DNA as observed in similar hairpin shaped Eu–Pt₂ complexes.^4a,b Formation of such bis(bifunctional) platinum-DNA cross-links should induce major unrepairable structural distortion of the DNA double-helix. The significant decrease in ellipticity in the CD spectra of CT-DNA in the presence of 1 (Fig. 2b) also suggests unwinding of the DNA helix and major structural deformation of DNA induced by the Pt–DNA cross-links.¹⁴ This structural distortion of DNA could be beyond cellular DNA repair machinery and thereby inhibit transcription and replication, triggering cell-death pathways.⁵ The decrease in DNA migration rate in the presence of Eu–Pt₂ compared to EuL in the gel electrophoretic mobility assay using SC pUC19 DNA also suggests unwinding of the supercoiled DNA helix by 1 (Fig. 2c).¹⁵ We have observed significant enhancement of the Eu-based luminescence intensity originating from the ⁵D₀ → ⁷F_J transitions of Eu³⁺ upon the addition of DNA due to efficient energy transfer to Eu³⁺ and enhanced excited state lifetime in a hydrophobic environment created due to binding of the Eu–Pt₂ complex with DNA (Fig. 2d). Serum albumin proteins constitute a major component in blood plasma proteins and play important roles in drug transport and metabolism. The interaction of the Eu–Pt₂ complex with bovine serum albumin (BSA) studied using a tryptophan emission quenching experiment showed a high binding propensity (K_BSA = 1.50 ± 0.03 × 10⁵ M⁻¹) desirable for efficient transport to the pathological site (Fig. S9, ESI‡).

Fig. 2 (a) ITC plot profile for the interaction of Eu–Pt₂ (0.1 mM) with CT-DNA (0.01 mM) in 5 mM Tris-HCl/NaCl buffer (pH 8.5) at 30 °C. The top panel represents the raw data for 20 injections of 2 μL each. The bottom panel shows the amounts of heat evolved on interaction of the Eu–Pt₂ complex with CT-DNA against the molar ratio of Eu–Pt₂ complex to CT-DNA. Data were fitted by a sequential binding site model. (b) CD spectra of CT-DNA in the presence of EuL and Eu–Pt₂ in Tris-HCl buffer medium. (c) Agarose gel electrophoresis of SC pUC19 DNA after incubation with EuL and Eu–Pt₂ in 50 mM Tris-HCl buffer at 37 °C for 16 h. Lane 1: DNA control, lanes 2–8 (10, 15, 20, 40, 60, 80 and 100 μM complexes respectively). (d) Luminescence spectra of Eu–Pt₂ (λ_ex = 330 nm) with the addition of CT-DNA in 5 mM Tris-HCl buffer. Inset: the relative emission intensity (I/I₀) of 1 at 615 nm vs. [DNA]/[Eu–Pt₂] ratio. (e) Cell viability plots showing the cytotoxicity of the Eu–Pt₂ complex with HeLa and H460 cells on 16 h incubation by MTT assay.

To test our original ‘theranostic’ design, we performed an in vitro cytotoxicity assay using MTT on human cervical carcinoma HeLa and lung carcinoma H460 cell lines. The IC₅₀ values for Eu–Pt₂ complex 1 are 51.0 ± 1.05 μM in HeLa cells and 30.0 ± 1.27 μM in H460 cells (Fig. 2d). It exerts anticancer activity via extensive DNA-adduct formation through conjugated cis-[PtCl₂(DMSO)] moieties with a similar mechanism to cisplatin.

The cellular internalization of the Eu–Pt₂ complex was investigated to probe the diagnostic aspect of [Eu(L)(H₂O)] utilizing the long luminescence lifetime and intrinsic luminescence from Eu³⁺ using confocal fluorescence microscopy (panel A, Fig. 3). The theranostic Eu–Pt₂ complex showed significant cellular uptake within 4 h of incubation with the HeLa cells. Staining with nuclear staining dye Hoechst 33258 demonstrates both nuclear and cytosolic distribution of the complex (panel C, Fig. 3). The red spots observed in some nuclei originate from the luminescence of the Eu³⁺ reporter tag in the Eu–Pt₂ theranostic conjugate (Fig. S11, ESI‡).

Fig. 3 Confocal laser scanning fluorescence microscopic images of the HeLa cells treated with the Eu–Pt₂ complex (25 μM) after 4 h of incubation and Hoechst 33258 dye (5 μg mL⁻¹): (A) cells incubated with the Eu–Pt₂ complex; (B) incubated with the Hoechst 33258 dye for staining nuclei, which show blue emission; (C) merged images showing nuclear and cytosolic localization of the complex; (D) DIC images. Scale bar = 20 μm.

In conclusion, we have developed a luminescent multimodal heterometallic Eu–Pt₂ theranostic system using sensitization and energy transfer from a conjugated Pt²⁺ based chromophore to Eu³⁺. The complex shows a strong binding propensity for DNA via the formation of Pt–DNA cross-links through four potential DNA binding sites. The complex exhibits cytotoxicity through DNA damage, and nuclear localization due to an Eu-based f–f transition is observed using fluorescence imaging. Thus, such systems will have great potential towards designing theranostic agents and delivery vehicles for cancer chemotherapy. Further studies are ongoing towards designing potent lanthanide based theranostic agents as efficient drug delivery platforms and understanding the mechanism of their action.

A.K.P. acknowledges SERB, Govt. of India and IIT Kanpur for funding. A.C. and K.S. acknowledge UGC and CSIR for their research fellowships.

Notes and references

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Footnotes

† Dedicated to Professor Animesh Chakravorty on the occasion of his 80^th birthday.

‡ Electronic supplementary information (ESI) available: Experimental details; synthesis and characterization of compounds; ESI-MS analysis; DNA and BSA binding experiments; cytotoxicity and cellular internalization studies. See DOI: 10.1039/c5dt04470g

§ A. C. and K. S. contributed equally to this work.

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