A Luminescent Manganese PhotoCORM for CO Delivery to Cellular

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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

A Luminescent Manganese PhotoCORM for CO Delivery to Cellular Targets under the Control of Visible Light Jorge Jimenez, Indranil Chakraborty, Annmarie Dominguez, Jorge Martinez-Gonzalez, W. M. Chamil Sameera, and Pradip K. Mascharak* Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, United States S Supporting Information *

ABSTRACT: A photoactive manganese carbonyl complex derived from dansylimidazole (Imdansyl), namely, [Mn(Imdansyl)(CO)3(phen)](CF3SO3) (1), has been synthesized and structurally characterized. This is the first luminescent manganese carbonyl-based photoCORM reported in the literature. This complex exhibits CO release under the exclusive control of lowpower broadband visible light. The corresponding rhenium carbonyl complex, namely, [Re(Imdansyl)(CO)3(phen)](CF3SO3) (2), has also been reported, which is luminescent but sensitive only to UV-B (λ 400 nm). The apparent CO release rate (kCO) of 1 has been determined by recording the electronic absorption spectra in MeCN solution

Figure 3. Electronic absorption spectra of complex 1 (green trace), dansylimidazole ligand (blue trace), and 1,10-phenanthroline (red trace) in MeCN solutions at 298 K.

TDDFT calculations provide insight into the origin of the electronic transitions giving rise to these bands. Ground state of 1 is the closed-shell singlet, and the key structural parameters of the calculated singlet state optimized structure are in good agreement with the X-ray structure (Table S2, Supporting Information). The triplet state optimized structure is 22.2 kcal/ mol above the ground state. TDDFT calculations also reveal that for 1, the HOMO is mostly composed of π orbital of the Imdansyl ligand, while the LUMO is a π* orbital of the phen ligand with contribution from the π* orbital of Imdansyl (Figure 4). The HOMO−LUMO gap of Mn complex is 0.14 AU (3.80 eV) corresponding to the ∼370 nm band. According to our TDDFT calculations on the Mn complex 1 and the natural transition orbitals (Supporting Information), the key excitation at 350 nm ( f = 0.28) corresponds to π D

DOI: 10.1021/acs.inorgchem.7b02480 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Nature of the Photoproducts. Time-dependent IR spectroscopic study has been performed to evaluate the number of CO molecules liberated from 1 upon illumination. In such study, MeCN solution of complex 1 was taken in a glass Petri dish and placed on top of a low-power visible light source (10 mW/cm2) for 30 min. The photolyzed solution was then dried under vacuum, and the IR spectrum of the solid was recorded in KBr matrix. The results clearly indicated loss of all three CO molecules from 1 upon visible light illumination (Figure 7). Figure 5. UV−vis traces from the Mb assay for 1 in PBS/MeCN solution at 298 K; black trace, oxidized Mb; green trace, reduced Mb; and red trace, COMb.

and monitoring the spectral changes upon exposure to visible light at 4 s intervals. The kCO value calculated from the ln(C) vs time (t) plot at 298 K is 10.5 ± 0.02 min−1 (conc = 3.16 × 10−5 M). The quantum yield value (ϕ380 = 0.35 ± 0.03) was determined by standard actinometry using potassium ferrioxalate.36 Unlike other Mn-based photoCORMs reported so far, MeCN solution of 1 exhibits strong luminescence and a broad emission band (excitation at 370 nm) with maximum around 600 nm (Figure 6). When compared to the strong

Figure 7. IR spectra of 1 (black trace) and the solid mixture obtained from the photolyzed solution (red trace).

In the present work we have also undertaken the task of identifying the photoproducts more precisely with the aid of spectroscopy and X-ray crystallography. Interestingly the relative intensity of the emission band of the photolyzed solution resembles closely to that of the solution of the 1 under identical experimental conditions (Figure 6). Emission spectrum of the photolyzed solution of 1 is devoid of any band around 400 nm, which suggests the absence of free phen ligand. This solution exhibits a six-line EPR spectrum indicative of the presence of Mn(II) species (Figure 8) and resembles closely the EPR spectrum of the photolyzed solution obtained from the photoCORM [Mn(PTA)(phen)(CO)3](CF3SO3) reported by us in a previous account.12 These observations cumulatively prompted us to further scrutinize the nature of the photodecomposition product(s) in such solution. Careful crystallization of product(s) from the photolyzed solution led

Figure 6. Emission spectra of complex 1 (green trace), the photolyzed solution (blue trace), and free Imdansyl ligand (red trace) in MeCN solution at 298 K.

luminescence of the Imdansyl ligand (λem = 600 nm), this band is ∼10% quenched. In a very recent account, Schiller and coworkers have reported a Mn-based photoCORM derived from a tridentate aminopyridine ligand with a dansyl chromophore directly attached to the amino-N.37 Interestingly, this nonluminescent photoCORM exhibits the strong 600 nm luminescence of the dansyl chromophore only after CO photorelease. In this photoCORM, the PET arising from excitation of the N electron(s) to the dansyl π system is quenched due to the strong electron-withdrawing effect of the three coordinated CO ligands from the Mn(I) center which in turn draws electron density from the N atom directly connected to it. In contrast, the luminescence in 1 is not quenched because of the intervening imidazole moiety which employs two different N atoms for binding the dansyl chromophore and the Mn(I) center, respectively (Figure 1). This luminescent property of 1 provides a unique opportunity to track cellular uptake of this photoCORM with the aid of fluorescence microscopy (vide infra). As expected, complex 2 exhibits a strong fluorescence band at 530 nm (λex = 360 nm) much like that of [Re(MeIm)(CO)3(phen)](CF3SO3).20

Figure 8. X-band EPR spectrum (at 123 K) of the photolyzed solution of complex 1 in MeCN solution (50 μM). Microwave frequency, 9.44 GHz, modulation amplitude, 2.00 G, and modulation frequency, 100 kHz. E

DOI: 10.1021/acs.inorgchem.7b02480 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Figure 9. Fluorescence microscopy images of HT-29 colon cancer cells: (a) fluorescence image, (b) bright-field image, and (c) overlaid image.

to isolation of colorless single crystals of two distinct morphologies. X-ray crystallographic study on each of these two types revealed the identity of one of the products as a Mn(II) complex, namely, [Mn(phen)3](CF3SO3)2, and the other as a second Mn(II) species, namely, [Mn(phen)2(H2O)2](CF3SO3)2 (see Supporting Information for details). Fluorescent spectral studies confirmed that both these Mn species are devoid of luminescence. Because the photolyzed solution exhibits noticeable luminescence, it is clear that the third photodecomposed product is the free Imdansyl ligand. In a separate experiment, a solution of free Imdansyl exhibited no diminution of its florescence upon addition of the two Mn photoproducts, confirming that the fluorescence of the photolyzed solution does arise from free Imdansyl ligand. Finally in order to evaluate the cellular uptake properties of complex 1, human colorectal cancer cells (HT-29) were treated with 1 for 1 h in McCoy’s 5A medium (devoid of phenol red and fetal bovine serum). An independent experiment showed that 1 is stable in such medium for at least 24 h (Supporting Information, Figure S9). After 1 h of incubation, the medium was aspirated, and the cells were washed carefully with PBS. The cellular images were then captured with the aid of a fluorescence confocal microscope. The images shown in Figure 9 clearly demonstrate efficient internalization of complex 1 within the cancer cells. Internalization of a Mn-based photoCORM within cancer cells has not been visualized before due to ready quenching of fluorescence of ligands upon ligation to Mn center. In our previous work, we had to first establish internalization of the photoCORM within cancer cells with analogous Re-based photoCORMs and then demonstrate COinduced death of the cells using the corresponding Mn complex under visible light illumination.12 Complex 1, for the first time, allows one to confirm the presence of the photoCORM within the target cells because of its inherent luminescence. Complete loss of the three CO ligands results in modest enhancement of this luminescence as expected on the basis of Figure 6. The facile cellular uptake of 1 and excellent CO delivery capacity as demonstrated by the myoglobin assay prompted us to investigate its CO-induced ability to eradicate HT-29 cells. The cell viability assay shown on Figure 10 clearly indicates a dose-dependent eradication of HT-29 cells with 100 μM of 1, resulting in a reduction to 47% upon exposure to low-power visible light. In addition, incubation with exhaustively photolyzed solutions of 1 exhibited minimal cell damage. Furthermore, dark experiments revealed minimum toxicity

Figure 10. Dose-dependent cell viability of HT-29 cells upon treatment with 1 in the dark (blue bars) and under low-power visible light (λ > 460 nm) (red bars). Controls with trace of MeCN and 100 μM of photolyzed 1 are also shown.

toward the HT-29 cells even at 100 μM, indicating cell viability reduction exclusively upon CO release from 1 (Figure 10). In order to verify that such doses of CO do not induce cell apoptosis in normal cells, we have also evaluated the effects of CO on human embryonic kidney cells (HEK 293). In this experiment, the viability of HEK-293 cells was only marginally reduced upon exposure to different concentrations of 1 under dark or upon exposure to visible light (Figure 11). Similarly, exhaustively photolyzed solutions of 1 did not affect the viability of HEK-293 cells under any condition.

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CONCLUSIONS Although Mn-based photoCORMs have been successfully employed to eradicate human cancer cells in a dose-dependent way under the control of visible light,5,10,12,13 entry of these CO donors into target cells cannot be tracked because in general they lack luminescence even if they are derived from fluorescent ligands. In some cases, photorelease of the CO molecules under illumination results in deligation of the fluorescent ligand leading to a rise in luminescence in target cells undergoing CO-induced apoptosis.13,36 Such “turn-ON” of luminescence indicates the end of the CO delivery process. Complex 1 is notable in this regard due to its inherent luminescence, a property that allows one to detect its entry into F

DOI: 10.1021/acs.inorgchem.7b02480 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from a National Science Foundation grant (DMR-1409335) and an UCSC COR-SRG grant is gratefully acknowledged. J.J. acknowledges support from the National Institute of Health grant no. 2R25GM058903.

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Figure 11. Results of MTT assay with HEK-293 cells upon treatment with 1 in the dark (blue bars) and under illumination (red bars) along with the controls.

the target cell quite readily. Comparison of these results now suggests that if the fluorescent ligand is not directly connected to the Mn(I) center of the photoCORM (as in 1), then the fluorescence of the remote fluorophore will not be quenched and the photoCORM will be trackable within cellular targets. More work along this line is in progress in our laboratory, and the results will be reported in due time.

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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.inorgchem.7b02480. Experimental and computational details, crystal data and refinement parameters of the photoproducts (Table S1) and key structural parameters (Table S2), packing patterns of complex 1 and 2 (Figures S1 and S2), crystal structures and packing diagrams of the two photoproducts (Figures S3−S6), fluorescence spectrum of complex 2 in MeCN (Figure S7), fluorescence spectrum of complex 1 in PBS (with 2% MeCN) (Figure S8), absorption spectrum of 1 in McCoy’s 5A medium (Figure S9), and the results of DFT and TDDFT calculations (PDF) Accession Codes

CCDC 1567033−1567034 and 1571118−1571119 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data_request/cif, or by emailing [email protected]. uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: (831) 459-2935. ORCID

W. M. Chamil Sameera: 0000-0003-0213-0688 Pradip K. Mascharak: 0000-0002-7044-944X G

DOI: 10.1021/acs.inorgchem.7b02480 Inorg. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.inorgchem.7b02480 Inorg. Chem. XXXX, XXX, XXX−XXX