Water-Soluble Pd8L4 Self-assembled Molecular Barrel as an

Apr 10, 2017 - (16) However, to the best of our knowledge, aqueous coordination architectures have not been explored as carriers of curcumin drug. Her...
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Water-Soluble Pd8L4 Self-assembled Molecular Barrel as an Aqueous Carrier for Hydrophobic Curcumin Imtiyaz Ahmad Bhat,† Ruchi Jain,‡ Mujahuddin M. Siddiqui,† Deepak K. Saini,*,‡ and Partha Sarathi Mukherjee*,† †

Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India Department of Molecular Reproduction, Development and Genetics and Centre for Biosystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India

‡

S Supporting Information *

ABSTRACT: A tetrafacial water-soluble molecular barrel (1) was synthesized by coordination driven self-assembly of a symmetrical tetrapyridyl donor (L) with a cis-blocked 90° acceptor [cis-(en)Pd(NO3)2] (en = ethane-1,2-diamine). The open barrel structure of (1) was confirmed by single crystal X-ray diffraction. The presence of a hydrophobic cavity with large windows makes it an ideal candidate for encapsulation and carrying hydrophobic drug like curcumin in an aqueous medium. The barrel (1) encapsulates curcumin inside its molecular cavity and protects highly photosensitive curcumin from photodegradation. The photostability of encapsulated curcumin is due to the absorption of a high proportion of the incident photons by the aromatic walls of 1 with a high absorption cross-sectional area, which helps the walls to shield the guest even against sunlight/UV radiations. As compared to free curcumin in water, we noticed a significant increase in solubility as well as cellular uptake of curcumin upon encapsulation inside the watersoluble molecular barrel (1) in aqueous medium. Fluorescence imaging confirmed that curcumin was delivered into HeLa cancer cells by the aqueous barrel (1) with the retention of its potential anticancer activity. While free curcumin is inactive toward cancer cells in aqueous medium at room temperature due to negligible solubility, the determined IC50 value of ∼14 μM for curcumin in aqueous medium in the presence of the barrel (1) reflects the efficiency of the barrel as a potential curcumin carrier in aqueous medium without any other additives. Thus, two major challenges of increasing the bioavailability and stability of curcumin in aqueous medium even in the presence of UV light have been addressed by using a new supramolecular water-soluble barrel (1) as a drug carrier.

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and egress of the bigger guest molecules.9 In this respect, cylindrical and barrel-shaped architectures are very promising, as they have windows similar to their cavity size. Barrel-shaped molecules have immense importance in biological systems, as βbarrel proteins allow the diffusion of small molecules and ions through cell membranes.10 Despite the fact that there has been mounting interest in barrel-shaped molecules with large cavities due to various applications, the synthesis of discrete watersoluble barrel-shaped architectures by metal−ligand selfassembly remains very challenging.11 On the other hand, turmeric has been a favorite choice as a spice, topical household remedy for treatment of sprains and swellings, as well as in Ayurveda for various ailments.12 The main active component of turmeric is curcumin, which is widely known for its pharmacological activities, including antiinflammatory, antitumor, antioxidant, and antiamyloid properties.13 Phase I clinical trials in humans have shown that curcumin is safe even at high doses (12g/day).14 Despite its

INTRODUCTION Coordination driven self-assembly has emerged as a convincing tool to design and construct a wide variety of discrete assemblies ranging from two-dimensional (2D) to threedimensional (3D) architectures.1,2 The unique interior environment of these 3D architectures has been exploited for myriad applications, such as host−guest chemistry,3 stabilization of reactive species,4 drug delivery,5 supramolecular catalysis,6 and as sensors.7 Various design strategies, such as edge- and facedirected self-assembly, symmetry interaction model, and molecular library approach have been utilized to synthesize coordination cages with varying cavity size.8 The final fate of a self-assembly is not only controlled by the direction and predictable nature of the metal−ligand coordination sphere but also by the reaction conditions. Symmetrical ligands owing to their predictable coordination ability and the ease of convergence have always been the choice to construct discrete closed-shell architectures. Most of the reported 3D discrete architectures of Pd(II)/Pt(II) acceptors have closed-shell topology with smaller windows in comparison to their large internal cavity space, which in turn restricts both the ingress © 2017 American Chemical Society

Received: February 18, 2017 Published: April 10, 2017 5352

DOI: 10.1021/acs.inorgchem.7b00449 Inorg. Chem. 2017, 56, 5352−5360

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Inorganic Chemistry Scheme 1. Schematic Representation of the Synthesis of Molecular Barrel 1

to Me4Si as internal standard (0.0 ppm) or proton resonance resulting from incomplete deuteration of D2O at 4.79 ppm and CDCl3 at 7.26 ppm. UV−vis and emission spectra were recorded using PerkinElmer Lambda-750 and Horiba Jobin Yvon fluoromax4 spectrophotometers, respectively. A 400 W UV lamp was used for UV irradiation studies. ESI-MS experiments were done in an Agilent 6538 Ultra-High Definition (UHD) Accurate Mass Q-TOF spectrometer. A Bruker D8 Quest diffractometer was used for collecting single crystal X-ray data. Synthesis of L. Ligand L was synthesized following the reported procedure.17 Isolated yield: 1.21 g, 46%. IR: υ (cm−1) = 1573, 1485, 1331, 1302, 1274, 1214, 991, 810, 623, 539. 1H NMR (400 MHz, (CDCl3): δ(ppm) = 8.47 (d, J = 6.0 Hz, 8H), 7.21 (s, 4H), 7.00 (d, J = 6.0 Hz, 8H). 13C NMR (100 MHz, CDCl3): δ (ppm) = 153.57, 152.27, 143.61, 129.56, 117.33. Synthesis of 1. Ligand L (0.0143 g, 0.034 mmol) and cis[(en)Pd(NO3)2] (0.020 g, 0.068 mmol) were taken in a 4 mL vial, and 1.5 mL of Millipore water was added to it. The suspension was kept at constant stirring at 60 °C for 12 h. The resulting faint bluish solution was centrifuged, and the clear supernatant solution was diffused with methanol to obtain shining single crystals after 15 days. However, diffusion of the acetone to the resulting clear solution afforded an offwhite precipitate of the tetrafacial M8L4 molecular barrel (1). Isolated yield: 0.019 g, 55%. IR: υ (cm−1) = 3101, 1599, 1495, 1314, 1215, 1059, 824, 593. 1H NMR (400 MHz, D2O): δ (ppm) = 8.51 (d, J = 5.23 Hz, 32H), 7.85 (s, 8H), 7.46 (d, J = 5.23 Hz, 32H), 7.24 (s, 8H) and 3.18(s, 32H). ESI-MS (m/z) = 1268.0125 [1 − 3NO3−]+3, 936.3440 [1 − 4NO3−]+4 and 603.9027 [1 − 6NO3−]+6. Curcumin Encapsulation (1⊂curcumin). 1 (10 mg) was taken in D2O (pH = 6.3), and 2 mg of curcumin was added to it. The suspension was stirred for 24 h at room temperature. The undissolved curcumin was removed by centrifugation, and NMR was recorded with the clear solution obtained. The solvent was removed under vacuum to obtain the inclusion complex. υ (cm−1) = 3101.16, 1599.11, 1495.38, 1314.15, 1215.22, 1059.77, 824.88, 593.55. 1H NMR (400 MHz, D2O): δ (ppm) = 8.43 (brs, 32H), 7.77 (s, 8H), 7.35 (brs, 32H), 7.13 (s, 8H), 6.79 (d, J = 16.0 Hz, 4H), 6.75 (s, 4H), 6.60 (d, J = 8.0 Hz, 4H), 6.25 (d, J = 16.0 Hz, 5H), 5.39 (s, 5H), 3.43 (s, 5H). UV−Visible Spectroscopy. Curcumin (1 mg) was taken in six different 4 mL vials. A stock solution (10−3 M) of 1 was prepared separately, and pH was recorded as 6.3. To the vial labeled as (A), 50 μL of a stock solution of 1 was added. To vial (B), 100 μL of a stock solution of 1 was added. Similarly, to the vials labeled as (C), (D), and (E); 150 μL, 200 μL, and 250 μL stock solutions of 1 were added, respectively. Millipore water was added to adjust 2 mL as the total volume in each vial. All six vials were kept for constant stirring at room temperature for 24 h. After centrifugation, clear yellow solutions were

efficacy and safety, the major limitations deterring the approval of curcumin as a therapeutic agent are (i) low bioavailability due to poor aqueous solubility, (ii) lack of stability under physiological pH, and (iii) photodegradation.15 An elegant strategy employed to overcome these fundamental barriers of transport and stabilization of curcumin is its encapsulation within the hydrophobic cavity of water-soluble carriers. The encapsulation of a hydrophobic guest can not only increase the water solubility of the guest molecule, but also increase its uptake by cancer cells for selective and improved anticancer activity. Recently, efforts have been given to increase the aqueous solubility and hence the bioavailability of curcumin by encapsulating it in various host molecules such as liposomes, hydrogels, phospholipid vesicles, and lipid based nanoparticles (NPs).16 However, to the best of our knowledge, aqueous coordination architectures have not been explored as carriers of curcumin drug. Herein, we report the template free synthesis of a watersoluble tetrafacial molecular-barrel 1 via [4 + 8] self-assembly of a symmetric tetrapyridine donor L with a 90° acceptor M [where M = cis-(en)Pd(NO3)2; en = ethane-1,2-diamine] (Scheme 1). The barrel (1) has an intramolecular hydrophobic pocket surrounded by four aromatic panels (L). The hydrophobic pocket of the barrel is tested to have high potential for encapsulation of curcumin in aqueous medium. Such encapsulation not only helps curcumin to be soluble in water, but also protects photosensitive curcumin from photodegradation under sunlight/UV-radiations. Further investigations revealed the potential of this barrel as a safe carrier for the transportation of curcumin with good anticancer activity of the encapsulated curcumin. The determined IC50 value of ∼14 μM for curcumin in aqueous medium in the presence of the barrel (1) is as effective as is curcumin in toxic high-boiling organic solvents, such as dimethyl sulfoxide (DMSO).

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EXPERIMENTAL SECTION

General Methods. Unless otherwise mentioned, the chemicals used in this study were purchased from reputed commercial sources. N1,N1,N4,N4-Tetra(pyridin-4-yl)benzene-1,4-diamine17 and di(4pyridyl)amine were synthesized by slight modification of the known procedure.18 NMR spectra were recorded using a Bruker 400 MHz instrument, and the chemical shifts (δ) are accounted in ppm relative 5353

DOI: 10.1021/acs.inorgchem.7b00449 Inorg. Chem. 2017, 56, 5352−5360

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Inorganic Chemistry obtained. From each of these solutions, 400 μL was taken and diluted to 3 mL with Millipore water. The final solutions obtained were taken for UV-measurement. Cell Culture. HeLa cells used in this study were cultured in the modified Dulbecco’s medium DMEM containing 3.7 g/L sodium bicarbonate, 110 mg sodium pyruvate, antibiotics (100 units/mL penicillin-streptomycin), and 10% fetal bovine serum (FBS). Cells were maintained at 37 °C in 5% CO2 humidified incubator. Loading of Curcumin in 1. The amount of curcumin trapped inside 1 was determined by spectrophotometric quantitation. To determine this, curcumin powder was dissolved in DMSO to prepare a stock solution of 3 mM, which was further diluted with water to prepare solutions of different concentrations, viz. 2, 4, 6, 10, and 20 μM, in aqueous DMSO mixture. The dilutions with water were made in such a way that the final volume of DMSO does not exceed 10% of the total volume, when used in the final cellular assays. The absorbance of each dilute solution was then recorded at 430 nm using a Tecan M1000 plate reader and was plotted as a function of concentration of curcumin in DMSO to obtain a standard linear plot. To estimate the amount of curcumin entrapped in 1⊂curcumin, initially five different stock solutions of 1 were prepared, viz 16.6 μM, 50.0 μM, 83.3 μM, 116.6 μM, and 150.0 μM in 3 mL of Millipore water. To each of the above-mentioned solutions of 1, 2 mg of curcumin was added and stirred to prepare 1⊂curcumin solutions. From each stock solution of 1⊂curcumin, 10 μL was taken and diluted with 90 μL of water. The absorbance of the resulting solutions was recorded at 430 nm. Separately, stock solutions of 16.6 μM, 50.0 μM, 83.3 μM, 116.6 μM, and 150.0 μM concentrations of 1 were prepared. Dilute solutions of 1 (100 μL) were prepared using 10 μL of these solutions of 1, and absorbance was recorded at 430 nm (used as blank). The recorded absorbances for 1⊂curcumin complexes were fitted to a linear equation derived from the standard plot to determine the amount of curcumin loaded in each cage. MTT Cell Cytotoxicity Assay. Five ×103 cells were seeded on a 96 well flat transparent bottom plate for 16−20 h in the growth media. The cells were loaded with 10 μL of 1⊂curcumin solutions with 16.6 μM, 50.0 μM, 83.3 μM, 116.6 μM, and 150.0 μM concentrations diluted with 90 μL of growth media for 48 h. The culture media was then replaced with 100 μL of DMEM containing 25 mM MTT reagent for 2 h in a CO2 incubator at 37 °C until the color in the wells turned to light brown. The medium containing MTT was then removed and replaced with 100 μL of DMSO to terminate the reaction and dissolve the formazan crystals. The plate was incubated for an additional 5 min at 37 °C. The absorbance was then measured at 540 nm using a Tecan M1000 plate reader. Cytotoxicity was recorded as a fold change in cell survival percentage in comparison to the control untreated cells and calculated as (OD of control cells/OD of treated) × 100 for IC50 value determination. Fluorescence Microscopy Analysis for Cellular Uptake of Curcumin. 1 ×105 HeLa cells were seeded on 18 mm acid treated glass coverslips for 16−20 h. The cells were then treated with curcumin in water, and 15 μM solutions of 1, 1⊂curcumin, and curcumin in DMSO, respectively, for 6 h. The intrinsic green fluorescence of curcumin was recorded in the GFP channel (excitation filter 480/20 nm and emission filter 525/30 nm) using an Olympus IX83 inverted fluorescence microscope controlled with Slidebook 6.0 software. All the images were captured and processed using the same acquisition settings. X-ray Data Collection and Structure Refinements. Single crystal X-ray diffraction data of 1 were collected using a Bruker D8 QUEST CMOS diffractometer.19 Intensity data were collected using graphite-monochromatized Mo Kα radiation (0.71073 Å) at 110 K. The structure was solved by direct methods and Fourier analysis and refined by the full-matrix least-squares method based on F2 with all reflections20,21 using the SHELX-9722 incorporated in WinGX.23 Nonhydrogen atoms were refined with anisotropic displacement coefficients. Crystal Data for 1. C120H144N48O24Pd8; MW = 3494.02, tetragonal I4/m, a = 19.054(7) Å, b = 19.054(7) Å, c = 32.418(12) Å, α = 90°, β = 90°, γ = 90°, V = 11769(9) Å3, Z = 2, Mo Kα radiation

(λ = 0.71073 Å), T = 100(2) K, R1 = 0.0668, wR2 = 0.2495 (I > 2σ(I)).

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RESULTS AND DISCUSSION Synthesis and Characterization of Molecular Barrel 1. Ligand L was synthesized following a modified procedure by copper catalyzed Ullmann condensation reaction of 4,4′dipyridylamine with 1,4-dibromobenzene at 170 °C in diphenyl ether. The reaction of L with [cis-(en)Pd(NO3)2] in 1:2 molar ratio in H2O at 60 °C for 12 h resulted in a clear solution, which upon treatment with an excess of acetone afforded an offwhite precipitate of tetrafacial M8L4 molecular barrel (1). Despite the possibility of formation of several discrete cages, we exclusively obtained a single M8L4 tetrafacial molecular barrel in good yield. The ionic barrel is completely soluble in water due to the presence of several nitrate counterions. The 1H NMR spectrum of 1 in D2O showed the presence of four distinct peaks in the aromatic region 8.51−7.25 ppm (Figure 1). The

Figure 1. Partial 1H NMR spectra of the ligand (L) in CDCl3 and of 1 in D2O.

free ligand (L) showed only three peaks in the NMR spectrum due to pyridyl and phenyl protons. The ligand showed a single peak at 7.48 ppm due to the phenyl protons. However, upon complex formation, two separate peaks of equal intensity due to the phenyl protons were observed in the 1H NMR spectrum. Splitting of the phenyl protons into two different sets indicates that the phenyl ring of the ligand in the barrel is oriented in such a way that two protons have different chemical environments compared to the other two protons. The formation of a single molecular barrel was further confirmed by diffusion-ordered NMR spectroscopy with a clear single band at D = 11.6 × 10−10 m2/s (log D = −9.75) with a hydrodynamic radius of ∼1.16 nm (Supporting Information, Figure S4). The composition of the assembly was accurately determined by ESI-MS spectrometry. The presence of several prominent peaks at m/z = 1268.0125, 936.3440, and 603.9027 corresponding to [1-3NO3−]3+, [1−4NO3−]4+, and [1− 6NO3−]6+ fragments, respectively, confirmed the formation of a M8L4 species (Supporting Information, Figure S5). The isotopic distribution pattern of the fragment [1-3NO3−]3+ matched very well with the theoretical pattern (Figure 2), which further echoes in support of the [4 + 8] composition of L and M in the resulting complex. Though the formation of a 5354

DOI: 10.1021/acs.inorgchem.7b00449 Inorg. Chem. 2017, 56, 5352−5360

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Inorganic Chemistry

the presence of Pd(II) ions at the corners with nitrate groups as counteranions makes it water-soluble. The hydrophobic nature of the cavity is expected to facilitate the entrapment of hydrophobic guest like curcumin to enhance water solubility. Curcumin Encapsulation. Curcumin’s bioavailability is poor after oral administration because it is practically insoluble in water at room temperature. For the development of curcumin into a potential drug, a carrier vehicle is needed to transport it to the desired targets and increase its cellular uptake. We envisaged the use of water-soluble barrel 1 as a potential carrier for curcumin, owing to the presence of hydrophobic cavity. Curcumin was found to be efficiently encapsulated inside 1 upon stirring at room temperature in water for 24 h; and the formation of host−guest system 1⊂curcumin was confirmed by 1H NMR and UV−vis spectroscopic studies. The 1H NMR spectrum of 1⊂curcumin confirmed encapsulation of curcumin molecule in the hydrophobic pocket of barrel 1 (Figure 4). The appearance of a

Figure 2. Calculated (blue) and experimental (red) ESI-MS isotopic distribution patterns of the fragment [1-3NO3−]3+.

single and symmetrical tetrafacial barrel was quite evident from the DOSY-NMR and ESI-MS analyses, the actual reason for two sets of peaks appearing due to the phenyl protons in the 1H NMR spectrum of 1 remained inconclusive. Suitable single crystals of 1 for X-ray diffraction were obtained by diffusion of methanol vapor into an aqueous solution of 1 over a period of 2 weeks. Single crystal XRD analysis unambiguously established the formation of an open tetrafacial molecular barrel as shown in Figure 3. The molecular

Figure 3. Single crystal XRD structure of 1. (a) View along the crystallographic c-axis. (b) View along the crystallographic a-axis. Color codes: green = C, blue = N, red = Pd. Hydrogen atoms, counteranions, and solvent molecules have been omitted for clarity.

barrel crystallized in the tetragonal I4/m space group with the asymmetric unit containing only one molecule of a cage (Supporting Information, Figure S15). The crystal structure consists of four ligands (L) connected together by eight Pd(II) acceptors forming a square barrel as depicted in Figure 3. The panel type ligands (L) with five aromatic rings (4 pyridyl rings and 1 phenyl ring) form the four walls, connected to each other with eight 90° [cis-(en)Pd(NO3)2] acceptors at the corners, forming a highly symmetrical structure closely resembling a square barrel (Figure 3, Supporting Information, Figures S14). The dimensions of the cavity inside the barrel are 12.2 × 12.2 × 15.3 Å3. In the crystal structure, the opposite ligand panels of the centrosymmetric molecular barrel are identical with the central phenyl ring of each ligand (L) tilted toward the cavity with an angle of 60°. As a result, the singlet peak at 7.48 ppm for four phenyl protons of ligand L splits into two singlet peaks at 7.54 and 6.84 ppm, indicating the different chemical environment and hindered rotation of the central phenyl rings. Interestingly, the distance between the opposite walls of the cavity is 12.2 Å, which is good enough to accommodate a guest molecule without strain. The interior cavity of the molecular barrel is hydrophobic due to the skeletal carbon atoms of the phenyl and pyridine groups of the ligand, while

Figure 4. (a) Partial 1H NMR spectrum; and (b) DOSY NMR of 1⊂curcumin in D2O.

single band at log D = −9.5 in the diffusion-ordered NMR spectrum of 1⊂curcumin in D2O indicates the formation of a single host−guest complex (Supporting Information, Figures S7 and S8). Further confirmation for the encapsulation of curcumin comes from UV−vis spectroscopy. A suspension of curcumin in water at room temperature does not show any absorption band in the UV−vis spectrum; however, upon addition of 1 to the suspension, a new band appears at 430 nm, revealing the encapsulation of curcumin inside the barrel. Increase in the concentration of 1 shows a sharp increase in the intensity of the 5355

DOI: 10.1021/acs.inorgchem.7b00449 Inorg. Chem. 2017, 56, 5352−5360

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Figure 5. UV−visible spectra of (a) 1 in water and (b) 1⊂curcumin in aqueous medium with a gradual increase in the concentration of 1.

of the extracted curcumin displayed the characteristic molecular ion peak [M + H]+ at 369.1224 (Figure 7).

absorption band at 430 nm, which further endorses the encapsulation of curcumin (Figures 5 and S6). Stability of Curcumin in the Barrel. Curcumin is known to be photosensitive in solution phase upon exposure to sunlight or UV-radiation. This limits the storage of curcumin solution in the presence of light for practical use. In order to address the issues related to photostability, we made a comprehensive study of the stability of curcumin inside the barrel by UV−vis, 1H NMR spectroscopy, and ESI-MS spectrometry. A solution of 1⊂curcumin in D2O was exposed to daylight for 48 h followed by the extraction of curcumin from the host−guest complex by addition of CDCl3. The 1H NMR spectrum of the extracted curcumin showed no degradation even after 2 days inside the barrel (Figure S10). To see whether the encapsulated curcumin is stable under exposure of UV radiation, the aqueous solution of 1⊂curcumin was exposed under a 400 W UV lamp for 2 h. The UV−visible spectrum of the extracted curcumin showed the characteristic band at 430 nm, confirming the stability of 1⊂curcumin (Figure 6), which was further confirmed by 1H NMR analysis of 1⊂curcumin (Figure S11). Furthermore, the mass spectrum

Figure 7. ESI-MS of (a) 1⊂curcumin after 2 h of UV exposure, and of (b) free curcumin.

The UV-stability of the curcumin inside barrel 1 is due to the shielding of the high UV absorption cross-section area of the surrounding aromatic walls of barrel 1. In an aqueous solution of 1⊂curcumin, a high proportion of the incident light is absorbed by the surrounding aromatic walls of barrel 1, which reduces the fraction of incident photons absorbed by curcumin to a great extent and thus shields the encapsulated curcumin from photodegradation. A good carrier for drug delivery should be stable under physiological conditions inside the cells. Thus, the stability of the barrel 1 has been investigated under these conditions. No degradation of the cage was noticed in the cell culture media and in the sodium phosphate buffer of pH 6, 7, and 8 even after 24 h, demonstrating the in vitro stability of the molecular barrel (Figures S12 and S16). Curcumin Loading in the Barrel and Cytotoxicity Analysis of 1⊂curcumin. Though different methods have been used previously to load and increase the solubility of curcumin in water, it is important to determine the exact amount of curcumin loaded in different carriers. For this, as in most studies, methanol or DMSO was used as a solvent of choice to solubilize the curcumin completely and was used as a reference. As the focus of the present study is to increase the solubility of curcumin in aqueous medium, the study was limited to an approximation based analysis. For this, the amount of curcumin encapsulated in different concentrations of the barrel 1 was estimated using a standard curcumin amount (Figure S13). For the estimation of the amount of curcumin loaded in the barrel, a series of solutions of (1) was prepared with varying concentrations. Experiments showed a negligible amount of curcumin encapsulation in 1.6 μM, 5.0 μM, and 8.3

Figure 6. UV−visible spectra of curcumin and 1⊂curcumin after 2 h UV exposure. 5356

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Inorganic Chemistry μM solutions of 1. However, the curcumin concentrations in 11.6 μM and 15.0 μM solutions of 1 were significant and found to be 9.7 μM and 16.3 μM, respectively (Figure S13). Once the solubility and stability of curcumin were increased in the presence of the aqueous barrel, it was imperative to analyze whether the encapsulated curcumin retains its intrinsic antiproliferative activity toward cancer cells. To analyze the efficacy of 1⊂curcumin in inhibiting cell proliferation, a cytotoxicity analysis was performed in HeLa cells using an MTT assay. HeLa cells seeded overnight were incubated with host−guest systems 1 (11.6 μM) and 1 (15.0 μM) for 48 h. The host−guest systems with 1 (5.0 μM) and 1 (8.3 μM) were used as negative control in the analysis, as no significant amount of curcumin was entrapped. The incubated HeLa cells were then processed further in an MTT assay, and the absorbance at 540 nm was recorded. A significant reduction in cell growth was observed in cells incubated with host−guest systems 1 (11.6 μM) and 1 (15.0 μM), which is in good agreement with the high quantity of curcumin encapsulated in these concentrations of 1⊂curcumin (Figure 8). Overall, the data confirmed that the encapsulated curcumin retains its intrinsic cytotoxic property in the cancer cells.

Figure 9. Fluorescence based analysis in support of cellular uptake of 1⊂curcumin. Representative images for HeLa cells incubated with each (a) 1 (15 μM)⊂curcumin, (b) curcumin in water, (c) 1 (15 μM) only, and (d) cells loaded with curcumin in DMSO as a positive control.

of the complex. This was tested by evaluating its minimum inhibitory concentration in comparison to the curcumin solubilized in DMSO (where it dissolves completely). An IC50 value of 10 μM was determined from a dose response analysis in HeLa cells incubated with various doses of curcumin−DMSO solution as shown in Figure 10.

Figure 8. Effect of the 1⊂curcumin system on cell proliferation. The bioactivity of 1⊂curcumin complexes was analyzed using a standard MTT based assay. (n ≥ 3; p values; ns, ≥0.05; **, ≤0.01; calculated w.r.t. curcumin solution in water, wherein n represents the number of independent biological replicates, p represents the probability value used in statistics to measure the significance of an experiment as marked, “ns” represents the nonsignificant data, and ** represents data of high significance.

Though the encapsulated curcumin retained its cytotoxic property, yet the cellular uptake of curcumin has been shown to be limited.15a With this premise, the presence of curcumin loaded in cells as a host−guest system was analyzed using the intrinsic green fluorescence of the curcumin. A microscopic analysis of HeLa cells loaded with 1⊂curcumin showed a significantly higher fluorescence compared to 1 alone or curcumin in aqueous medium (Figure 9). It is interesting to note here that while the fluorescence of curcumin in DMSO solution can be seen both in the cytoplasm as well as in the nucleus of the cells, only the cytoplasmic localization was observed in the 1⊂curcumin system after 6 h of incubation. This possibly hints that the availability of free curcumin might be slower when trapped in the barrel compared to free curcumin in DMSO solution. The toxicity analysis of host−guest system 1 (15.0 μM) and its uptake inside the cells led us to further explore the efficiency

Figure 10. MTT based analysis performed on cells treated with varying concentration of curcumin solutions in DMSO. The graph represents a decrease in percentage cell survival as a function of increase in curcumin concentration for IC50 calculation.

Using the same assay, the IC50 value for 1⊂curcumin was determined by incubating HeLa cells with different amounts of the host−guest system, viz. 0, 2, 4, 6, 8, and 10 μL of 150 μM 1⊂curcumin. An inhibition of 50% growth was recorded when ∼8 μL of the host−guest complex was used (Figure 11). Given that 16.3 μM of curcumin is present in the volume of 10 μL of host−guest complex (150 μM) 1⊂curcumin (as calculated by using the standard plot shown in Figure S13), it corresponds 5357

DOI: 10.1021/acs.inorgchem.7b00449 Inorg. Chem. 2017, 56, 5352−5360

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Inorganic Chemistry

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Additional 1D, 2D NMR, UV−vis, IR spectra, and ESIMS spectrometry (PDF) CIF data for 1 (CIF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D. K Saini). *E-mail: [email protected] (P. S. Mukherjee). ORCID

Partha Sarathi Mukherjee: 0000-0001-6891-6697 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.S.M. is grateful to SERB-India for financial support. I.A.B. and M.M.S. are grateful to UGC (New Delhi) for an SRF and D. S. Kothari postdoctoral fellowship, respectively.

Figure 11. Efficiency analysis of 1⊂curcumin. HeLa cells treated with different concentrations of 1⊂curcumin for 48 h showed a linear decrease in cell growth. The graph plotted for percentage cell survival of 1⊂curcumin w.r.t. 1 is shown.

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closely to the IC50 of curcumin (10 μM) in an organic solvent such as DMSO (as shown in Figure 10). The data reported showed that ∼16.3 μM curcumin entrapped in 1 (15.0 μM) (IC50 ∼ 14 μM) is as efficient as curcumin in high-boiling toxic DMSO solvent for inhibiting the cancer cell proliferation and thus signifies the potential of the barrel for carrying curcumin in purely aqueous medium with significant cytotoxicity.

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CONCLUSION In conclusion, we report here the synthesis of a water-soluble tetragonal molecular nanobarrel (1) by facile coordination driven self-assembly of a symmetrical tetrapyridyl donor with the 90° ditopic acceptor cis-[(en)Pd(NO3)2]. The hydrophobic cavity of the water-soluble molecular barrel was found to encapsulate hydrophobic curcumin, as evident from UV−vis and NMR studies. Such encapsulation makes hydrophobic curcumin highly soluble in water at room temperature in the presence of the barrel. In addition to enhanced solubility of curcumin upon encapsulation, the panel-shaped aromatic walls of the barrel stabilize and protect highly photosensitive curcumin from photodegradation under sunlight/UV-irradiation. Moreover, the drastic increase in cell uptake of curcumin in the barrel in aqueous medium demonstrates the potential of (1) as a successful drug carrier. Detailed studies disclosed the potential of the tetragonal barrel as a potential carrier for the transportation of anticancer drug curcumin into HeLa cell lines. The determined IC50 value of ∼14 μM for 1⊂curcumin in a biocompatible aqueous medium reflects that this curcumin composite is as efficient as is free curcumin in toxic high-boiling organic solvent like dimethyl sulfoxide (DMSO). The multifold increased solubility of curcumin in combination with its enhanced cell uptake, high stability, and slow drug release upon encapsulation make the present barrel (1) a rare example of a discrete self-assembled coordination architecture as a safe aqueous carrier of curcumin.

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REFERENCES

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DOI: 10.1021/acs.inorgchem.7b00449 Inorg. Chem. 2017, 56, 5352−5360

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