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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Reaching Biocompatibility with Nanoclays: Eliminating the Cytotoxicity of Ir(III) Complexes Malte C. Grüner,*,†,§ Kassio P. S. Zanoni,†,§ Camila F. Borgognoni,‡,§ Cristiane C. Melo,‡ Valtencir Zucolotto,‡ and Andrea S. S. de Camargo*,† †
Laboratory of Spectroscopy of Functional Materials (LEMAF), São Carlos Institute of Physics, University of São Paulo, Avenida Trabalhador Sãocarlense 400, 13566-590, São Carlos, Brazil ‡ Group of Nanomedicine and Nanotechnology (GNano), São Carlos Institute of Physics, University of São Paulo, Avenida Trabalhador Sãocarlense 400, 13566-590, São Carlos, Brazil ACS Appl. Mater. Interfaces Downloaded from pubs.acs.org by UNIV OF SUSSEX on 08/04/18. For personal use only.
S Supporting Information *
ABSTRACT: Cyclometalated IrIII complexes are promising candidates for biomedical applications but high cytotoxicity limits their use as imaging and sensing agents. We herein introduce the use of Laponite as carrier for tripletemitting cyclometalated IrIII complexes. Laponite is a versatile nanoplatform because of its biocompatibility, dispersion stability and large surface area that readily adsorbs functional nonpolar and cationic molecules. These inorganic− organic hybrid nanomaterials mask cytotoxicity, show efficient cell uptake and increase luminescent properties and photostability. By camouflaging intrinsic cytotoxicity, this simple method potentially extends the palette of available imaging and sensing dyes to any metal−organic complexes, especially those that are usually cytotoxic. KEYWORDS: iridium(III), Laponite, cytotoxicity, biocompatibility, theranostics
T
he growing importance of cyclometalated IrIII complexes in biological applications lies in their strategical features for cancer treatment, photodynamic therapy (PDT), cellular imaging, theranostics, and biosensors.1−7 While their luminescence is tunable in a wide color range and the complexes are available for two-photon excitation, the intrinsic cytotoxicity hampers their use for bioimaging because of interference in cellular processes, such as S-phase cell arrest, binding to human serum albumin, interacting with the minor groove of the DNA and inducing early apoptotic mechanisms.8−11 Nevertheless, these properties are exploitable, for example, in chemotherapy, in which cyclometalated IrIII complexes have shown an even higher toxicity than cisplatin against cancer cells, while the resistance factor is low.1,11,12 On the other hand, Laponite RD (LAP) is a highly biocompatible synthetic nanoclay produced by BYK Additives, which has numerous applications in pharmaceutical, cosmetic, and food formulations.13 It consists of nanosized discs with a high aspect ratio (25 nm in diameter and 1 nm in height), and it can be considered a twodimensional inorganic polymer. LAP is a proven molecular carrier while offering efficient cell uptake and high biocompatibility; hence, it is considered a key nanoplatform for theranostics including drug delivery, PDT, and imaging.14,15 Moreover, LAP can alter the solubility of molecules and oligomers resulting in decreased biocidal activity against bacteria or increased cell uptake.16,17 In this study, we demonstrate, for the first time, that the direct adsorption of cyclometalated IrIII complexes onto LAP drastically decreases cytotoxicity, enhances photophysical performance and increases photostability as compared to © XXXX American Chemical Society
their water-soluble counterparts. For comparisons, we prepared cationic IrIII complexes (1−3) either as water-soluble chloride (Cl−) or as water-insoluble hexafluorophosphate (PF6−) salts (Figures 1). The cationic complexes bearing PF6− counterions were dissolved in EtOH and adsorbed onto Laponite in a concentration of approximately 10 molecules per nanodisc according to the method of Kynast and co-workers leading to the inorganic−organic nanohybrids LAP1, LAP2, and LAP3, which are readily dispersible in water.18 The complete adsorption was confirmed by UV−vis spectroscopy of the supernatant after the loading procedure (Figure S1). As previous studies have shown, 10 molecules per nanodisc is a good compromise between a sufficient functionalization (to evade high particle concentrations in experiments with cells) and an insignificant molecular aggregation on the particles surface (which would compromise the photophysical performance).15 Since the PF6− salts are insoluble in water, leakage of complexes from the nanomaterial into aqueous media is prevented and a comparison with the freely soluble Cl− salts becomes valid. Solutions of LAP1−LAP3 at higher concentrations (c ≥ 10 mg/mL) forms highly luminescent hydrogels, as depicted in Figure S3. Complexes 1−3 (Cl−) are readily soluble in water and exhibit intense emissions in broad spectra (Figures S2 and S4− S7) and tunable assorted colors, ascribed to the phosphorReceived: June 29, 2018 Accepted: July 31, 2018 Published: July 31, 2018 A
DOI: 10.1021/acsami.8b10842 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 1. (A) Cyclometalated IrIIICl− complexes (1−3) and PF6− complexes adsorbed onto Laponite RD (LAP1−LAP3). (B) Photographs of the emission of aqueous solutions of 1−3 and LAP1−LAP3 under UV irradiation (c = 8.64 × 10−5 M).
Table 1. Emission Properties for Aqueous Solutions of 1−3 and LAP1−LAP3 at 298 K ϕb sample
λmax (nm)
N2-saturated (ϕ0)
air-equilibrated
O2-saturated
τ0 (μs)c
kq (109 L mol−1 s−1)d
1 2 3 LAP1 LAP2 LAP3
578 582 555 580 573 523
0.67 0.07 0.77 0.12 0.51 ∼1.00
0.32 0.03 0.47 0.08 0.45 0.92
0.05 0.02 0.14 0.07 0.40 0.70
2.4 2.0 1.0 2.0 0.9 1.5
2.3 0.58 2.1 0.20 0.15 0.13
a
a
Emission maximum. bAbsolute emission quantum yield, measured using an integrating sphere. cemission lifetime in N2-saturated solution; Estimated quenching rate constant. See data treatment in the Supporting Information
d
Figure 2. Confocal images of HTC cells treated with 1.7 μM of (A) 1−3 and (B) LAP1−3 including control and unloaded LAP after 3 h of incubation. Scale bar 20 μm; λexc,two‑photon = 800 nm.
escent deactivation from the lowest-lying triplet state.19−21 As summarized in Table 1, the emission quantum yields in N2saturated (O2-free) solutions (ϕ0) are very high, and emission lifetimes (τ0) are in the microsecond range, typical of IrIII emitters. The origins of such remarkable emissions for 1−3 were previously addressed in depth (in acetonitrile) in preceding publications.19,20 In summary, the radiative proper-
ties of these complexes are tunable according to the electronwithdrawing strength of the substituent groups in the phenylpyridinic ligand, with the emissive triplet state mainly assigned to spin−orbit-coupling-mixings of charge transfer transitions. As a drawback, however, aqueous solutions of 1−3 undergo a rapid quenching by molecular triplet oxygen (3O2), especially in O2-enriched media, forming reactive singlet B
DOI: 10.1021/acsami.8b10842 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces
Figure 3. (A−C) MTT and (D−F) CVS cytotoxicity assays for three different IrIII complex concentrations (c = 2.16, 4.32, and 8.64 μM) after 24h incubation. (G−H) Bright field microscopy of the highest concentration treatment (c = 8.64 μM).
oxygen (1O2) in the process and practically extinguishing emission, with drastic losses in ϕ0 in comparison to O2-free solutions. The magnitude of their quenching rate constants (kq) in water (∼2 × 109 L mol−1 s−1, Table 1 and Figures S8−S10), is similar to the ones observed for other RuII, OsII, and IrIII phosphors.20 The adsorption of the complexes onto the Laponite nanodiscs leads to improved photophysical properties for LAP2 and LAP3, mainly by an increased rigidity surrounding the adsorbed cations that destabilizes charge transfer state’s energies and diminishes nonradiative vibrational deactivations.21 As a result, the emission maxima are blueshifted and ϕ0 are increased, with an impressive unitary ϕ0 for LAP3. On the other hand, LAP1 exhibits a slight redshift, lower ϕ0 and shorter τ0, which all together are symptomatic of intermolecular π-stackings of the large quinoline rings due to the spatial confinement on the nanodiscs. Moreover, the emission quenching by 3O2 is remarkably prevented for LAP1−LAP3, with losses in quantum yield of only ∼34% (average) in O2saturated solutions. Since the oxygen quenching process is diffusion controlled, and the diffusion constant is inversely proportional to the radius of the nanomaterial (Stokes− Einstein equation), the larger LAP conjugates have a smaller chance to react with 3O2 in a quenching event. Additionally, the LAP nanodiscs hamper the diffusion of dissolved 3O2 by forcing access to the adsorbed complexes from only one suitable direction. Their high quantum yields even in O2saturated solutions (e.g., as high as 0.70 for LAP3) make these inorganic−organic hybrid nanomaterials particularly interesting for imaging applications in O2-rich cellular media. Also, strategically advantageous for practical imaging uses, photobleachings of adsorbed complexes by long exposures to intense
UV irradiations are practically null in comparison to their free soluble forms. As followed by changes in UV−vis spectra (Figures S11−S13), continuous UV irradiation for 1 h (with a Hg(Xe) lamp; incident light: 35 mW) results in absorption losses of up to 15% for IrIII cations in solution, while the absorption for LAP-adsorbed species is essentially constant. Hepatoma tissue culture (HTC) cells were used as a model for investigating the uptake and toxicity of free and LAPconjugated IrIII compounds. Cell uptake was assessed via confocal microscopy after 3 h of incubation with 1.7 μM solutions of 1−3, corresponding to 200 μg/mL dispersions of LAP1−LAP3, proving the internalization of the tested compounds during a comparable time frame (Figure 2). While 1−3 are distributed equally throughout the cytosol of HTC cells, the LAP1−LAP3 nanodiscs locate more around the nucleus, similarly observed before for rhodamine-loaded laponites dispersed in human adipose derived stem cells.22 Endocytosis, as well as interactions between the membrane’s phospholipids and the nanodisc’s cationic edges, can facilitate the efficient cellular uptake.23,24 The confocal microscopy results (obtained under λexc,two‑photon = 800 nm) also demonstrate the investigated complexes are two-photon active, an important characteristic for theranostic applications. Because the compounds could possibly interfere with the HTC cell metabolism, three different cytotoxicity tests were applied: (i) measurement of the mitochondrial activity using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide); (ii) measurement of the amount of adherent cells via CVS (crystal violet staining); (iii) cell observation under bright field microscopy.25 The concentrations tested for cytotoxicity were 2.16, 4.32, and 8.64 μM corresponding to the amount of IrIII cations either adsorbed onto LAP or as C
DOI: 10.1021/acsami.8b10842 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Letter
ACS Applied Materials & Interfaces water-soluble counterpart. The final LAP particle concentrations were 0.25, 0.5, and 1.0 mg/mL. In all cytotoxicity assays, free 1−3 complexes are highly toxic for the given concentrations (except for lowest concentrations of 2 and 3 in MTT assay), with average viabilities of only 10% for the highest concentrations in the MTT assays (Figure 3, Figures S14−S17). On the other hand, conjugated LAP1−LAP3 samples are remarkably harmless, exhibiting high viabilities in every assay. In fact, the CVS assays and bright field microscopy show an increase in cell proliferation of ca. 150−220% in viability, a phenomenon that had also been observed for other nanoparticles including mesoporous silica and TiO2, partially ascribed to a nanoparticle-induced aggregation of growth factor receptors on the cell surface.26−29 This effect can be assigned to the LAP nanoparticles alone as demonstrated in control experiments with unloaded LAP resulted in the same enhanced cell proliferation (Figure S18). These results strongly indicate that adsorption onto LAP remarkably alters the interactions between IrIII cations and cells, including uptake mechanisms and cytosol distribution, ultimately decreasing toxicity. Therefore, adsorbed IrIII complexes inherit the biocompatibility of their LAP carriers, standing as a smart and strategical characteristic viably exploitable to access the whole range of otherwise toxic [IrIII PF6] complexes for bioimaging and sensing in assorted colors without harming the host cells. Furthermore, the functionalization of the nanodisc’s rim via silanization and introduction of suitable targeting molecules can make subcellular organelle targeting feasible.30 For example, PEGylation of LAP leads to their accumulation in tumor tissues via the enhanced permeability and retention (EPR) effect.17 Therefore, the future development of new hybrid IrIII−LAP nanomaterials through the association between molecular and surface engineering can open new possibilities for in vitro and in vivo investigations of cellular processes. In summary, we have demonstrated that LAP is responsible for masking cytotoxicity of IrIII complexes as well as improving photophysical properties and photostability, which makes LAP nanodiscs a versatile nanoplatform for a wide range of otherwise cell-harmful substances not suitable for in vitro and in vivo studies so far. Apart from their use as imaging agents, the emission of IrIII complexes is quenched by molecular 3O2 to produce 1O2 depending on the molecular design, which can be exploitable for PDT as well, especially in combination with passive LAP targeting of tumor tissues. Hence, the presented novel hybrid organic−inorganic nanomaterials bear potential for in vitro and in vivo bioimaging, as well as for theranostic applications, leading to a wide field of future investigations toward the next generation of biocompatible triplet emitters.
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or LAP3, UV−vis spectral changes after photobleaching via continuous UV irradiation for complex 1 or LAP1, complex 2 or LAP2, and complex 3 or LAP3, optical microscopy images of HTC cells, and cytotoxicity tests with unloaded Laponite RD (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Malte C. Grüner: 0000-0002-3900-0265 Kassio P. S. Zanoni: 0000-0003-4586-6126 Valtencir Zucolotto: 0000-0003-4307-3077 Author Contributions §
Co-first authors.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Center for Research, Technology and Education in Vitreous Materials (CeRTEV), Project 2013/07793-6, funded by Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP), which also granted postdoctorate fellowships to M.C.G. (Grant 2015/ 24118-6) and K.P.S.Z. (Grant 2016/07706-4).
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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/acsami.8b10842. Experimental procedures, absorption spectra of complexes 1−3, cyclometallated IrIIICl− and PF6− complexes adsorbed onto Laponite RD, luminescent hydrogels of LAP1−LAP3, emission spectra at 298 K of complex 1 or LAP1, complex 2 or LAP2, and complex 3 or LAP3, Stern−Volmer plots for the emission quenching of complex 1 or LAP1, complex 2 or LAP2, and complex 3 D
DOI: 10.1021/acsami.8b10842 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsami.8b10842 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
