Orthogonal self-assembly of an organoplatinum(II)metallacycle and cucurbit[8]uril that deliverscurcumin to cancer cellsSougata Dattaa, Santosh K. Misrab,c,d,e, Manik Lal Sahaa, Nabajit Lahiria, Janis Louiea,1, Dipanjan Panb,c,d,e,1,and Peter J. Stanga,1

aDepartment of Chemistry, University of Utah, Salt Lake City, UT 84112; bDepartment of Bioengineering, University of Illinois at Urbana–Champaign,Urbana, IL 61801; cMills Breast Cancer Institute, Carle Foundation Hospital, Urbana, IL 61801; dDepartment of Materials Science and Engineering, Universityof Illinois at Urbana–Champaign, Urbana, IL 61801; and eBeckman Institute, University of Illinois at Urbana–Champaign, Urbana, IL 61801

Contributed by Peter J. Stang, June 15, 2018 (sent for review March 5, 2018; reviewed by Jacqueline K. Barton and Alanna Schepartz)

Curcumin (Cur) is a naturally occurring anticancer drug isolatedfrom the Curcuma longa plant. It is known to exhibit anticancerproperties via inhibiting the STAT3 phosphorylation process. How-ever, its poor water solubility and low bioavailability impede itsclinical application. Herein, we used organoplatinum(II) ← pyridylcoordination-driven self-assembly and a cucurbit[8]uril (CB[8])-mediated heteroternary host–guest complex formation in concert toproduce an effective delivery system that transports Cur into thecancer cells. Specifically, a hexagon 1, containing hydrophilic methylviologen (MV) units and 3,4,5-Tris[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]benzoyl groups alternatively at the vertices, has been syn-thesized and characterized by several spectroscopic techniques.The MV units of 1 underwent noncovalent complexation with CB[8]to yield a host–guest complex 4. Cur can be encapsulated in 4, viaa 1:1:1 heteroternary complex formation, resulting in a water-soluble host–guest complex 5. The host–guest complex 5 exhibitedca. 100-fold improved IC50 values relative to free Cur against humanmelanoma (C32), melanoma of rodents (B16F10), and hormone-responsive (MCF-7) and triple-negative (MDA-MB231) breast cancercells. Moreover, strong synergisms of Cur with 1 and 4 with combina-torial indexes of <1 across all of the cell lines were observed. An in-duced apoptosis with fragmented DNA pattern and inhibitedexpression of phosphor-STAT3 supported the improved therapeu-tic potential of Cur in heteroternary complex 5.

supramolecular coordination complex | orthogonal self-assembly |metallacycle | cancer | drug delivery

Coordination-driven self-assembly via metal–ligand interac-tions is an efficient strategy for preparing discrete supra-

molecular coordination complexes (SCCs) with predesignedshapes and sizes (1–6). The well-defined core structures of SCCsfurther facilitate the introduction of functional groups on the in-terior and/or exterior vertices of these frameworks, leading to theformation of functional systems useful in selective encapsulation(7), sensing (8), optical and electronic materials (9), drug delivery(10), and so on (11–14). The orthogonality of metal–ligand coor-dination with other noncovalent interactions, such as hydrogenbonding, π–π stacking, van der Waals forces, and host–guest com-plexations, allows the facile construction of SCC-cored supramo-lecular polymer networks (SPNs) with self-healing properties andstimuli responsiveness (15). However, the majority of the knownSPNs have been prepared in organic medium, due to the intrinsichydrophobicity of SCCs, limiting their biomedical applications (15).Cucurbit[n]urils (CB[n]) (n = 5–8, 10, and 14) are a family of

barrel-shaped macrocyclic molecular hosts composed of re-peating glycoluril units (16). A variety of neutral or positivelycharged guests can be encapsulated inside their cavities with highequilibrium association constants. The host–guest complexationsin water are driven by a combination of ion-dipole, hydrophobic,and hydrogen-bonding interactions between the ureidyl C=Ogroups of CB[n] and the guest molecules. Among the CB[n]

homologs, CB[8] has a unique capability to accommodate two hetero/homo guests in its cavity, leading to the formation of sophisticatedmaterials (17–21). For example, Scherman and coworkers (22) havecombined microfluidic techniques with cucurbit[8]uril-mediated in-terfacial host–guest chemistry and prepared monodisperse supra-molecular microcapsules that are useful in sensing and drug delivery.Despite the recent advances in cancer research, how to im-

prove the water solubility of hydrophobic drugs such as pacli-taxel, curcumin (Cur), camptothecin, tamoxifen, and others isstill a formidable challenge (23–27). Various nanocarriers in-cluding nanostructures (28–31), conjugates (32–34), hydrogels(35), carbon nanomaterials (36), and so on have been developedto overcome this problem. Likewise, the solubility, stability, andbioavailability of anticancer drugs have been significantly improvedin physiological environments via host–guest complexations (37–40).Lippard and coworkers (41) reported a hexanuclear Pt(II) cage as adrug delivery vehicle to deliver a Pt(II) prodrug to cancer cells.Likewise, a Fujita-type Pd(II)-organic polyhedron capped with CB[8] units, via the host–guest complexation with its methyl viologens(MV) functionalities, has been used to deliver a water-soluble an-ticancer drug, doxorubicin, to human cervical cancer (HeLa) cells

Significance

Despite decades of research, the development of efficient strate-gies that can effectively deliver poorly water-soluble anticancerdrugs remains a challenge. Hierarchical self-assembly strategy al-lows combining multiple therapeutic agents to produce a syner-gistic effect, thus enhancing the therapeutic efficacy. Herein wedescribe a hierarchical approach to solubilize a hydrophobic anti-cancer drug, curcumin in water via a combination of coordination-driven self-assembly and host–guest interactions. The water-soluble orthogonal self-assembly constructed by a hexagonalPt(II) metallacycle, cucurbit[8]uril, and curcumin exhibited en-hanced anticancer activity against melanoma and breast cancercells compared with the corresponding precursors. This paperprovides a platform for efficient delivery of hydrophobic anti-cancer drugs to cancer cells by the judicious implementation ofmultiple orthogonal interactions in a single process.

Author contributions: S.D., S.K.M., D.P., and P.J.S. designed research; S.D., S.K.M., M.L.S.,and N.L. performed research; S.D. contributed new reagents/analytic tools; S.D., S.K.M.,M.L.S., D.P., and P.J.S. analyzed data; and S.D., S.K.M., M.L.S., N.L., J.L., D.P., and P.J.S.wrote the paper.

Reviewers: J.K.B., California Institute of Technology; and A.S., Yale University.

The authors declare no conflict of interest.

Published under the PNAS license.1To whom correspondence may be addressed. Email: louie@chem.utah.edu, dipanjan@illinois.edu, or stang@chem.utah.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1803800115/-/DCSupplemental.

Published online July 23, 2018.

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(40). Herein, we utilized the bis-phosphine organoplatinum(II) ←pyridyl coordination-driven self-assembly and CB[8]-MV-mediatedhost–guest complexation in a single process to construct a deliverysystem that can solubilize Cur in water and transport it to thecancer cells.A water-soluble organoplatinum(II) hexagon 1 containing MV

units and tri(ethylene glycol) groups (PEG) alternating at thevertices has been synthesized (Fig. 1). The MV units of 1 canform a host–guest complex with three equivalents (equiv.) ofCB[8], resulting in 4, which further encapsulated 1.5 equiv. of Cur(with respect to 1) via a heteroternary host–guest complex formation.The hydrophilic PEG groups of 1 further induce additional solubilityto prevent precipitation of the resulting supramolecular polymer 5 inaqueous (aq.) solution. At the same time, PEG can interact withbiological membranes, which in turn provides better permeability andincreased transmembrane transports (42). Due to all these uniquecharacteristics, 5 showed ca. 100-fold improved IC50 values relative tofree Cur against human melanoma (C32), melanoma of rodents(B16F10), and hormone-responsive (MCF-7) and also triple-negative, hard-to-treat (MDA-MB231) breast cancer cells (43),providing a nanoformulation pathway to the field of activedrug delivery.

Results and DiscussionStirring a 1:1 mixture of MV-functionalized 120° dipyridyl donor,2·2PF6

−, and 120° organoplatinum(II) acceptor 3 in an H2O:acetone (1:2 vol/vol) mixture at room temperature for 12 h,followed by anion exchange using tetrabutylammonium nitrate,resulted in a self-assembled [3 + 3] hexagon 1 (Fig. 1). Multi-nuclear NMR (1H and 31P{1H}) analysis of the isolated productrevealed the formation of a discrete, highly symmetric entity

(Fig. 2 A–E). The 31P{1H} NMR spectrum of 1 showed a singlesharp singlet at ∼16.98 ppm with concomitant 195Pt satellites(JPt-P = 2314.5 Hz), which is in accord with the proposedstructure (Fig. 2A). In the 1H NMR spectrum of 1 (Fig. 2C), theprotons of the pyridyl groups exhibited downfield shifts (Δδ[Hα] =0.24 ppm; Δδ[Hβ] = 0.28 ppm) compared with those of thedipyridyl ligand 2′ (2·2NO3

−) (Fig. 2D). This is attributed to the lossof electron density that occurs upon pyridyl coordination with the Pt(II) metal center. Electrospray ionization (ESI) TOF MS supportsthe stoichiometry by showing a peak at m/z = 1,380.56 Da corre-sponding to [M – 5ONO2]

5+ species (Fig. 2F). This peak was iso-topically well-resolved and in good accordance with its calculatedtheoretical distribution.The reported equilibrium association constants of MV·CB[8]

complexes are in the range of Ka1 ∼ 105 M−1, while a secondequilibrium association of guaiacol or catechol into that complexis characterized by Ka2 ∼ 104 M−1, suggesting that the overallassociation constant for a 1:1:1 heteroternary complex is ca.β12 = Ka1 × Ka2 = 109–1010 M−2 (44, 45). Based on this highassociation constant, we prepared complex 4 (Fig. 1) via thehost–guest complexation of the MV units of 1 and three equiv.of CB[8] in water. Since Cur contains two 4-hydroxy-3-methoxyphenyl groups, we envisioned that 4 can encapsulate itvia a heteroternary host–guest complex formation. Interestingly,only 1.5 equiv. of Cur (with respect to 1) could be solubilized byan aq. solution of 4 resulting in the formation of 5 (Fig. 1). In-deed, a further addition of Cur to 5 led to a yellow precipitationof free Cur. The host–guest complexation was characterized by1H NMR experiments (SI Appendix, Fig. S14). The 1H NMRsignal at 9.10 ppm of 1 was assigned to the H1 and H4 protons ofthe MV units (SI Appendix, Fig. S14A). They shifted upfield due

Fig. 1. (A) Synthesis of MV-functionalized discrete metallacycle 1 and cartoon representation of the formation of host–guest complex 5 from the hierarchicalself-assembly of 1, CB[8], and Cur. (B) Host–guest complexation of 2′ with CB[8] and Cur.

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to the host–guest complexation and appeared as a broad peak at8.87 ppm in 4 and at 8.70 ppm in 5 (SI Appendix, Fig. S14 B andC). These changes in the chemical shifts are diagnostic for theheteroternary complexation (40, 43). Moreover, UV-visiblespectra were recorded in water to understand the interactionspresent in the complexes (SI Appendix, Fig. S15A). The intensityof the absorption band near 280 nm associated with the MVunits of 1 decreased in 4 (40, 46). This band was further de-creased in 5 accompanied by the appearance of a new bandaround 400–500 nm, indicating the solubilization of Cur in watervia a heteroternary host–guest complexation.Transmission electron microscopy (TEM) was employed to

examine the morphology of 1, 4, and 5 in water. As shown in Fig.3A, 1 self-assembled to form nanoparticles with a diameter of ca.7–8 nm. Subsequently, a noncovalent host–guest complexationof 1 with three equiv. of CB[8] in aq. medium led to the for-mation of aggregated nanospheres (75–150 nm) (SI Appendix,Fig. S16A). Cur-embedded heteroternary complex 5 formedmicrometer-sized, honeycomb-like networks at a concentrationof 0.28 mM (Fig. 3B) which subsequently transformed to tapeswith widths of about 40–80 nm and lengths of several micro-meters when the concentration reached 0.14 mM (Fig. 3C).These tapes further converted into vesicle-like aggregates (Fig.3D) upon dilution (0.014 mM). The diameters of these aggre-gates ranged from 30 to 100 nm with an average of ∼75 nm. Asimilar type of concentration-dependent morphological variationwas recently reported for a metallacycle-cored covalent polymerby Zhang et al. (47). Likewise, Huang and coworkers (48) alsoobserved a morphological transition of a supramolecular poly-mer from honeycomb-like structure to microsphere upon di-lution. Additionally, scanning TEM energy-dispersive spectroscopyspectra of the above samples further confirmed the presence ofcarbon, nitrogen, oxygen, phosphorus, and platinum in the aggregates(SI Appendix, Fig. S17).Dynamic light scattering (DLS) experiments were used to

determine the size-distribution profiles of these supramolecularaggregates. Compound 1 showed a narrow size distribution withan average hydrodynamic diameter (Dh) of 7 nm (Fig. 4A), whichis in accordance with the TEM results (Fig. 3A). Furthermore,no obvious size variation was detected at various pHs (7.4, 6.8,and 5) in buffer containing 10% FBS over 48 h (SI Appendix, Fig.S18) (10). Upon host–guest complexation of 1 with 3 equiv. ofCB[8], Dh increased to 100 nm with a relatively broader sizedistribution, presumably because of the aggregated morpholog-ical features of 4 (SI Appendix, Fig. S19A). The heteroternarycomplex 5 showed a much larger size distribution (about 1–2 μm)at a higher concentration (0.14 mM), indicating the formationlarger aggregates (SI Appendix, Fig. S19B). As the concentrationof 5 was decreased to 0.014 mM, Dh was found to be 80 nm (Fig.

4B), which is consistent with the vesicular morphology observedunder TEM (Fig. 3D).We investigated the release of Cur at different time intervals

from the host–guest complex 5 by a dialysis method. The releasedamount was quantified by measuring the absorbance (A425 nm) ofthe medium outside the dialysis membrane. Interestingly, controlledrelease of Cur from host–guest complex 5 was observed (SI Ap-pendix, Fig. S20), and a higher release rate was obtained at lowerpHs. All these results indicate the applicability of 5 as a drug de-livery system that could minimize its exposure to the healthy tissueshaving higher pH and enhance the accumulation of the drug intumor regions having lower pH.We chose four different cancer cell lines of different origins,

stages, and animal types in our study: human melanoma (C32),melanoma of rodents (B16F10), and hormone-responsive (MCF-7) and triple-negative (MDA-MB231) breast cancer cells. Thevariability in cell toxicity of 5 was studied by comparing its an-ticancer activity in C32 and B16F10 cells of the same skin originfrom humans and rats, respectively. MCF-7 and MDA-MB231were also used as cancer cells of the same human breast originwith two different properties of ER(+) and TNBC. Furthermore,a comparison of C32 with MCF-7 and MDA-MB231 has pro-vided information about the difference in anticancer activity of 5across different human organs. Thus, the use of these four celllines could establish the broader impact of anticancer activity for5 with reasonable variations. The anticancer activity of Cur-loaded 5was examined in vitro (Fig. 5 A–D). The results obtained from 3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyltetrazolium bromide (MTT)assay (49) suggested that 5 was effective in Cur-regulated cell growthinhibition of all used cancer cell lines. Moreover, the growth in-hibition efficiency was significantly improved in these cells, as sup-ported by the decrease of IC50 values to a biostatistically significantlevel of P < 0.0001 (SI Appendix, Table S1). The anticancer efficacy of5 was further compared (Fig. 5 E and F) with the activities of freeCur and other negative controls, including 2′, 4′, 5′, 1, and 4 (Fig. 1),suggesting that free Cur was the least effective against all of the usedcells. This is likely due to its low water solubility, which eventually

Fig. 2. (A and B) The 31P and partial (C–E) 1H NMR spectra (CD3OD, 25 °C) of (A and C) hexagonal metallacycle 1, (B and E) acceptor 3, and (D) donor 2’. (F)Experimental (red) and calculated (blue) ESI TOF MS spectra of discrete metallacycle [M − 5ONO2]

5+.

Fig. 3. TEM images of self-assembled nanostructures obtained from aq.solutions of (A) 1 at a concentration of 0.14 mM and 5 at concentrations of(B–D) 0.28, 0.14, and 0.014 mM, respectively.

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impedes its bioavailability. Likewise, both CB[8]-capped complexes,that is, 4 and 4′, are less effective relative to their free precursors 1and 2′, respectively. The activity of these CB[8]-capped complexeswas, however, drastically improved upon incorporation of Cur, assupported by the fact that 5 and 5′ exhibited higher growth in-hibition efficiencies compared with all other formulations. Nota-bly, as expected, almost all of the cells showed a high deterioration(50) in cell morphology and growth density when treated with 5(SI Appendix, Fig. S21). The effects of all formulations are cor-roborated well with their efficacy as calculated from anMTT assay(SI Appendix, Figs. S22–S25).To evaluate the role of CB[8] in the bioavailability of Cur,

Cur-CB[8] mixture (1:1) and CB[8] were used as positive andnegative controls, respectively. A triple-negative breast cancercell line, MDA-MB231, was used as a model cell line for thestudy due to its low cytotoxicity response against all of the usedformulations. As shown in SI Appendix, Fig. S26, a noncytotoxicnature of CB[8] and a slight improvement in cell growth re-gression by Cur-CB[8] were observed. Cur-CB[8] reduced thecell growth to a level of 65 ± 5 compared with 75 ± 10% by freeCur at a concentration of 50 μM.Interestingly, the IC50 values for different formulations were

also dependent on the type of the cell line. It is reported that C32is the most difficult cell line to effect cell growth, probably due tothe nature of the cell line (51). C32 is the human melanoma cell

line of skin origin, which is known to have one of the most re-sistive natures for entry of foreign reagents (52). However, 5showed promising anticancer activity in C32 cells, plausibly dueto the presence of Cur in high local concentration that negatesthe effect of lower cellular entry of 5. Notably, 1, 4, and 5 af-fected overall cell growth and IC50 more efficiently comparedwith 2′, 4′, and 5′ across all of the cell lines, probably due thepresence of cell-growth-inhibiting moieties with a high localconcentration. The results obtained from the treatment with 1, 4,and 5 were found to be similar for C32 and B16F10, probablydue to the similar skin melanoma nature of the cells even thoughthey originated from humans and rats, respectively. IC50 valuesof 1 and 4 were also similar in MCF-7 and MDA-MB231,probably due to the same origin of cells from human breast.Interestingly, the IC50 of 5 was significantly higher in MDA-MB231 compared with MCF-7, probably due to the triple-negative nature of MDA-MB231. All of the differences in thegrowth inhibition properties of the different formulations in thedifferent cell types not only depend on the combined effect oftheir chemistry, local concentration, and effective bioavailabilityof Cur but also on the origin of the cell itself. It also revealed thebiostatistically significant improvement in cell growth inhibitionof Cur across all of the used cell lines irrespective of their origin.To evaluate synergistic effect of Cur with 1 and 4 in the form

of 5 the combinatorial index (CI) was calculated:

CI= fðIC50ÞA=ðIC50ÞA+Bg+ fðIC50ÞB=ðIC50ÞA+Bg, [1]

where (IC50)A is IC50 of sample A, (IC50)B is IC50 of sample B,and (IC50)A + B is IC50 of combined samples A + B. CI > 1.3indicates antagonism, CI = 1.1–1.3 moderate antagonism, CI =0.9–1.1 additive effect, CI = 0.8–0.9 slight synergism, CI = 0.6–0.8 moderate synergism, CI = 0.4–0.6 synergism, and CI = 0.2–0.4 strong synergism. After performing a CI analysis on varioussamples we concluded that in 5 Cur acts as a synergistic compo-nent with 1 as well as 4 with CI < 1 for all of the cell lines used inthe study (SI Appendix, Table S2).Propidium iodide (PI) is a knownDNA intercalator that selectively

enters into dead cells with an increase in its red fluorescence (53). A

Fig. 5. MTT assay results showing cell viability in (A) C32, (B) B16F10, (C) MCF-7, and (D) MDA-MB231 cells; 10,000 cells were plated in 96-well plates and treated for48 h before performing theMTT assay. Cells were treated at concentrations of 50, 5, 0.5, and 0.05 μMaq. formulations of free Cur, 1, 4, 5, 2′, 4′, and 5′ (concentrationsof 5 and 5′ are chosen such that they have the same molar equivalence of Cur). Comparison of IC50 values obtained from treatment of C32, B16F10, MCF-7, andMDA-MB231 cells using samples (E) 2′, 4′, and 5′ and (F) 1, 4, and 5. Biostatistical analysis was performed using two-way ANOVA with Dunnett’s multiple comparisons testcomparing cell viability from Cur treatment with other samples. Here *, **, ***, and **** represent P values < 0.05, 0.01, 0.001, and 0.0001, respectively.

Fig. 4. DLS plots of 1 and 5 at concentrations of (A) 0.14 and (B) 0.014 mM.

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flow-assisted cell study was performed on C32 cells after separatelytreating them with Cur, 2′, 4′, 5′, 1, 4, and 5 for 48 h, followed byincubation with PI (1 μg/mL) for 20 min. A high-PI-loaded pop-ulation was observed for cells treated with 2′ compared with 1, in-dicating that the free ligand is more cytotoxic than the metallacycle,but the MTT assay showed a reverse trend (SI Appendix, Fig. S27A).Probably, 1 has a high cell-growth-inhibition property, while 2′ has ahigher cell-killing efficiency due to some nonobvious reasons. How-ever, the addition of CB[8] lowers their cytotoxicity such that thecorresponding complexes 4 and 4′ have comparable cytotoxicity (SIAppendix, Fig. S27B). The cytotoxicity was further enhanced anddiffered after the loading of Cur, resulting in a higher extent of PIaccumulation in cells treated with 5 compared with 5′ (Fig. 6A). Theresults suggest that CB[8] is a noncytotoxic component in these sys-tems and acts as a host to transport Cur to cancer cells.Cellular entry of the host–guest complexes 4 and 5 was verified

by inductively coupled plasma detection of Pt. Since Pt is not apart of any component of the cells, detection of Pt can be at-tributed to the cellular internalization of 4 and 5. The level of Ptwas detected in cancer cells treated with 4 and 5 at a concen-tration of 5 μM. Triple-negative breast cancer cell line MDA-MB231 was used as a model cell line for the study due to its lowcytotoxicity response against all of the used agents, while MCF-7is the best of the responding cells. Cancer cells incubated with 5showed a Pt level of ∼3 and 4 ppm in MCF-7 and MDA-MB231cells, respectively, while ∼1 ppm Pt level was observed in each ofthe aforesaid cell lines treated with 4 (SI Appendix, Fig. S28A).As shown in SI Appendix, Fig. S28B, ∼200 and 400% higher Ptdelivery was observed with 5 compared with 4 in MCF-7 and MDA-MB231 cells, respectively. The higher Pt content in cancer cells treatedwith 5 compared with 4 is likely due to higher cellular internalizationof 5 compared with 4. Cells treated with Cur and Cur-CB[8] were

used as negative controls along with cells alone, where no significantlevel of Pt was detected in any of the used cell lines.We further investigated the apoptotic potential of Cur loaded

in 5 via a DNA fragmentation assay (54). C32 cells were chosenas a representative cell line for the DNA fragmentation assaydue to either comparable or lower cell growth inhibition effi-ciency of all of the reagents in it. A DNA fragmentation study inthis cell line would reflect the clear possibilities of DNA frag-mentation in other cell lines at the same concentration. GenomicDNA was first collected from C32 cells treated with either freeCur, 5, or 5′ for 48 h as well as from untreated cells and thensubjected to gel analysis. The results are shown in Fig. 6B. Thetreatment of 5 produced bands of fragmented DNA (representedby white arrows in lane II), while no significant fragmentationswere observed for untreated cells and cells treated with Cur(lane I) or 5′ (lane V), indicating an apoptosis-induced frag-mentation of genomic DNA enhanced by 5.The apoptosis-induction ability of Cur is likely because of its

STAT3 down-regulation ability (55). To further evaluate thismechanism, we performed a protein expression analysis. C32cells were treated with free Cur, 5, and 5′ for 48 h and the totalprotein was extracted. The collected and quantified cell proteinwas run on an SDS gel for 1.5 h. Protein bands were transferredto blots which were further exposed against the primary anti-bodies of phosphor-STAT3 (pSTAT3) and background proteinβ-actin followed by secondary antibodies. The β-actin proteinbands were used for normalizing the protein expression intreated cells. Protein bands from both the blots were imagedusing a chemilumeniscence agent (Fig. 6C). The treatment of 5(band IV) reduced the expression of pSTAT3 to more than 50%compared with protein expression in untreated cells (band I),while only ca. 30% and 15% reduction were observed for 5′(band III) and Cur (band II), respectively (Fig. 6D). The datasupport that 5 can efficiently deliver Cur to C32 cells, causing anapoptosis via pSTAT3 down-regulation pathways.

ConclusionIn summary, we report a host–guest complex, composed of awater-soluble organoplatinum(II) metallacycle 1 and CB[8], thatacts as an aq. carrier of Cur and delivers it to cancer cells. TheMV motifs and tri(ethylene glycol) groups of 1 overcome thehydrophobic barrier attributed to the hexagonal aromatic coreand triethylphosphine groups, bestowing it with water solubility.The MV units of 1 allow it to undergo host–guest complexationwith CB[8] in water. The resulting host–guest complex 4 can befurther used to encapsulate Cur via a heteroternary complexa-tion to form 5. The host–guest complexes were characterized by 1HNMR and UV-visible spectroscopies. The host–guest complexation-mediated morphological transformation processes were investigatedby TEM and DLS experiments. Under TEM, Cur-embedded het-eroternary complex 5 exhibits various morphological features, such ashoneycomb-like networks, fibers, and vesicles, depending upon theconcentration.The anticancer activity of 5 was established in vitro using 2D

cultures of different cancer cell lines. Primarily therapeutic ef-ficacy was determined by performing cell viability assays followedby studying the changes in cell growth patterns and morphol-ogies. Cur showed significant synergism with 1 and 4, havingCI < 1 irrespective of the cell lines used for the study. Further,flow-assisted assay, DNA fragmentation, and protein expressionstudies were corroborated with submicromolar IC50 for opti-mized sample 5 via down-regulation of pSTAT3. Given these results,this work shows how a judicious combination of coordination-drivenself-assembly and host–guest interactions can be utilized for hydro-phobic drug delivery with improved efficacy.

Fig. 6. (A) Histograms of C32 cells obtained after PI staining posttreatmentwith 5′ and 5. Cells without treatment and without PI staining were used ascontrols, while cells with PI staining and without treatment were secondarycontrols. Cur-treated cells were used as positive controls. Mechanistic studieson representative C32 cell lines. (B) DNA fragmentation assay performed oncells untreated (lane III) or treated with 5 (lane II), Cur (lane I), and 5′ (lane V);lane IV represents the DNA ladder with fragments of different poly-nucleotide length and molecular weight. (C) Protein bands captured fromblots with pSTAT3 and β-actin expression after treatment of cells with Cur(band II), 5′ (band III), and 5 (band IV) and compared with untreated cellprotein (band I) and (D) comparison of percentage protein expression fromdifferent treatments. Cells were treated with free Cur, 5, and 5′ at a con-centration of 10 μM (concentrations of 5 and 5′ are chosen such that theyhave the same molar equivalence of Cur).

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Materials and MethodsAll reagents were commercially available and used as supplied withoutfurther purification. Deuterated solvents were purchased from CambridgeIsotope Laboratory. 1H NMR and 13C NMR spectra were recorded on a Var-ianInova 400 MHz spectrometer. The 31P{1H} NMR spectra were recorded ona Varian Unity 300-MHz spectrometer. The 1H and 13C NMR chemical shiftsare reported relative to residual solvent signals, and 31P{1H} NMR chemicalshifts are referenced to an external unlocked sample of 85% H3PO4 (δ 0.0).Mass spectra were recorded on a Micromass Quattro II triple-quadrupolemass spectrometer using ESI with a MassLynx operating system. The UV-visible experiments were conducted on a Hitachi U-4100 absorption

spectrophotometer. DLS experiments were carried out on a DynaProNanoStar instrument. TEM images were acquired at an accelerating voltageof 80 keV on a JEOL JEM-2800 instrument and the images were processedusing Gatan DigitalMicrograph software. The details of the synthesis, char-acterization, and biological experiments are given in SI Appendix.

ACKNOWLEDGMENTS. We thank Indrajit Srivastava for helping with theflow cytometry experiment. This work was supported by NIH Grant R01-CA215157 (to P.J.S.), a Science and Engineering Research Board Indo-USPostdoctoral Fellowship (to S.D.), and University of Illinois at Urbana–Champaign (D.P.).

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