Orthogonal self-assembly of an organoplatinum(II) metallacycle and cucurbit[8]uril that delivers curcumin to cancer cells

Significance

Despite decades of research, the development of efficient strategies that can effectively deliver poorly water-soluble anticancer drugs remains a challenge. Hierarchical self-assembly strategy allows combining multiple therapeutic agents to produce a synergistic effect, thus enhancing the therapeutic efficacy. Herein we describe a hierarchical approach to solubilize a hydrophobic anticancer 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 hexagonal Pt(II) metallacycle, cucurbit[8]uril, and curcumin exhibited enhanced anticancer activity against melanoma and breast cancer cells compared with the corresponding precursors. This paper provides a platform for efficient delivery of hydrophobic anticancer drugs to cancer cells by the judicious implementation of multiple orthogonal interactions in a single process.

Abstract

Curcumin (Cur) is a naturally occurring anticancer drug isolated from the Curcuma longa plant. It is known to exhibit anticancer properties via inhibiting the STAT3 phosphorylation process. However, its poor water solubility and low bioavailability impede its clinical application. Herein, we used organoplatinum(II) ← pyridyl coordination-driven self-assembly and a cucurbit[8]uril (CB[8])-mediated heteroternary host–guest complex formation in concert to produce an effective delivery system that transports Cur into the cancer cells. Specifically, a hexagon 1, containing hydrophilic methyl viologen (MV) units and 3,4,5-Tris[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]benzoyl groups alternatively at the vertices, has been synthesized 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, via a 1:1:1 heteroternary complex formation, resulting in a water-soluble host–guest complex 5. The host–guest complex 5 exhibited ca. 100-fold improved IC₅₀ values relative to free Cur against human melanoma (C32), melanoma of rodents (B16F10), and hormone-responsive (MCF-7) and triple-negative (MDA-MB231) breast cancer cells. Moreover, strong synergisms of Cur with 1 and 4 with combinatorial indexes of <1 across all of the cell lines were observed. An induced apoptosis with fragmented DNA pattern and inhibited expression of phosphor-STAT3 supported the improved therapeutic potential of Cur in heteroternary complex 5.

Coordination-driven self-assembly via metal–ligand interactions is an efficient strategy for preparing discrete supramolecular coordination complexes (SCCs) with predesigned shapes and sizes (1–6). The well-defined core structures of SCCs further facilitate the introduction of functional groups on the interior and/or exterior vertices of these frameworks, leading to the formation 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 coordination with other noncovalent interactions, such as hydrogen bonding, π–π stacking, van der Waals forces, and host–guest complexations, allows the facile construction of SCC-cored supramolecular polymer networks (SPNs) with self-healing properties and stimuli responsiveness (15). However, the majority of the known SPNs have been prepared in organic medium, due to the intrinsic hydrophobicity 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 repeating glycoluril units (16). A variety of neutral or positively charged guests can be encapsulated inside their cavities with high equilibrium association constants. The host–guest complexations in water are driven by a combination of ion-dipole, hydrophobic, and hydrogen-bonding interactions between the ureidyl C = O groups 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 sophisticated materials (17–21). For example, Scherman and coworkers (22) have combined microfluidic techniques with cucurbit[8]uril-mediated interfacial host–guest chemistry and prepared monodisperse supramolecular microcapsules that are useful in sensing and drug delivery.

Despite the recent advances in cancer research, how to improve the water solubility of hydrophobic drugs such as paclitaxel, curcumin (Cur), camptothecin, tamoxifen, and others is still a formidable challenge (23–27). Various nanocarriers including nanostructures (28–31), conjugates (32–34), hydrogels (35), carbon nanomaterials (36), and so on have been developed to overcome this problem. Likewise, the solubility, stability, and bioavailability of anticancer drugs have been significantly improved in physiological environments via host–guest complexations (37–40). Lippard and coworkers (41) reported a hexanuclear Pt(II) cage as a drug 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 anticancer drug, doxorubicin, to human cervical cancer (HeLa) cells (40). Herein, we utilized the bis-phosphine organoplatinum(II) ← pyridyl coordination-driven self-assembly and CB[8]-MV-mediated host–guest complexation in a single process to construct a delivery system that can solubilize Cur in water and transport it to the cancer cells.

A water-soluble organoplatinum(II) hexagon 1 containing MV units and tri(ethylene glycol) groups (PEG) alternating at the vertices has been synthesized (Fig. 1). The MV units of 1 can form a host–guest complex with three equivalents (equiv.) of CB[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 solubility to prevent precipitation of the resulting supramolecular polymer 5 in aqueous (aq.) solution. At the same time, PEG can interact with biological membranes, which in turn provides better permeability and increased transmembrane transports (42). Due to all these unique characteristics, 5 showed ca. 100-fold improved IC₅₀ values relative to free 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 active drug delivery.

Fig. 1.

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

Results and Discussion

Stirring 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). Multinuclear NMR (¹H and ³¹P{¹H}) analysis of the isolated product revealed the formation of a discrete, highly symmetric entity (Fig. 2 A–E). The ³¹P{¹H} NMR spectrum of 1 showed a single sharp singlet at ∼16.98 ppm with concomitant ¹⁹⁵Pt satellites (J_Pt-P = 2314.5 Hz), which is in accord with the proposed structure (Fig. 2A). In the ¹H NMR spectrum of 1 (Fig. 2C), the protons of the pyridyl groups exhibited downfield shifts (Δδ[Hα] = 0.24 ppm; Δδ[Hβ] = 0.28 ppm) compared with those of the dipyridyl ligand 2′ (2·2NO₃⁻) (Fig. 2D). This is attributed to the loss of electron density that occurs upon pyridyl coordination with the Pt(II) metal center. Electrospray ionization (ESI) TOF MS supports the stoichiometry by showing a peak at m/z = 1,380.56 Da corresponding to [M – 5ONO₂]⁵⁺ species (Fig. 2F). This peak was isotopically well-resolved and in good accordance with its calculated theoretical distribution.

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⁺.

The reported equilibrium association constants of MV·CB[8] complexes are in the range of K_a1 ∼ 10⁵ M⁻¹, while a second equilibrium association of guaiacol or catechol into that complex is characterized by K_a2 ∼ 10⁴ M⁻¹, suggesting that the overall association constant for a 1:1:1 heteroternary complex is ca. β₁₂ = K_a1 × K_a2 = 10⁹–10¹⁰ M⁻² (44, 45). Based on this high association constant, we prepared complex 4 (Fig. 1) via the host–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 it via a heteroternary host–guest complex formation. Interestingly, only 1.5 equiv. of Cur (with respect to 1) could be solubilized by an aq. solution of 4 resulting in the formation of 5 (Fig. 1). Indeed, a further addition of Cur to 5 led to a yellow precipitation of free Cur. The host–guest complexation was characterized by ¹H NMR experiments (SI Appendix, Fig. S14). The ¹H NMR signal at 9.10 ppm of 1 was assigned to the H₁ and H₄ protons of the MV units (SI Appendix, Fig. S14A). They shifted upfield due to the host–guest complexation and appeared as a broad peak at 8.87 ppm in 4 and at 8.70 ppm in 5 (SI Appendix, Fig. S14 B and C). These changes in the chemical shifts are diagnostic for the heteroternary complexation (40, 43). Moreover, UV-visible spectra were recorded in water to understand the interactions present in the complexes (SI Appendix, Fig. S15A). The intensity of the absorption band near 280 nm associated with the MV units of 1 decreased in 4 (40, 46). This band was further decreased in 5 accompanied by the appearance of a new band around 400–500 nm, indicating the solubilization of Cur in water via 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 complexation of 1 with three equiv. of CB[8] in aq. medium led to the formation of aggregated nanospheres (75–150 nm) (SI Appendix, Fig. S16A). Cur-embedded heteroternary complex 5 formed micrometer-sized, honeycomb-like networks at a concentration of 0.28 mM (Fig. 3B) which subsequently transformed to tapes with widths of about 40–80 nm and lengths of several micrometers 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 aggregates ranged from 30 to 100 nm with an average of ∼75 nm. A similar type of concentration-dependent morphological variation was recently reported for a metallacycle-cored covalent polymer by Zhang et al. (47). Likewise, Huang and coworkers (48) also observed a morphological transition of a supramolecular polymer from honeycomb-like structure to microsphere upon dilution. Additionally, scanning TEM energy-dispersive spectroscopy spectra of the above samples further confirmed the presence of carbon, nitrogen, oxygen, phosphorus, and platinum in the aggregates (SI Appendix, Fig. S17).

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.

Dynamic light scattering (DLS) experiments were used to determine the size-distribution profiles of these supramolecular aggregates. Compound 1 showed a narrow size distribution with an average hydrodynamic diameter (D_h) of 7 nm (Fig. 4A), which is 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. of CB[8], D_h increased to 100 nm with a relatively broader size distribution, presumably because of the aggregated morphological features of 4 (SI Appendix, Fig. S19A). The heteroternary complex 5 showed a much larger size distribution (about 1–2 μm) at a higher concentration (0.14 mM), indicating the formation larger aggregates (SI Appendix, Fig. S19B). As the concentration of 5 was decreased to 0.014 mM, D_h was found to be 80 nm (Fig. 4B), which is consistent with the vesicular morphology observed under TEM (Fig. 3D).

Fig. 4.

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

We investigated the release of Cur at different time intervals from the host–guest complex 5 by a dialysis method. The released amount was quantified by measuring the absorbance (A_{425 nm}) of the medium outside the dialysis membrane. Interestingly, controlled release of Cur from host–guest complex 5 was observed (SI Appendix, Fig. S20), and a higher release rate was obtained at lower pHs. All these results indicate the applicability of 5 as a drug delivery system that could minimize its exposure to the healthy tissues having higher pH and enhance the accumulation of the drug in tumor 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. The variability in cell toxicity of 5 was studied by comparing its anticancer activity in C32 and B16F10 cells of the same skin origin from humans and rats, respectively. MCF-7 and MDA-MB231 were also used as cancer cells of the same human breast origin with two different properties of ER(+) and TNBC. Furthermore, a comparison of C32 with MCF-7 and MDA-MB231 has provided information about the difference in anticancer activity of 5 across different human organs. Thus, the use of these four cell lines could establish the broader impact of anticancer activity for 5 with reasonable variations. The anticancer activity of Cur-loaded 5 was 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 growth inhibition of all used cancer cell lines. Moreover, the growth inhibition efficiency was significantly improved in these cells, as supported by the decrease of IC₅₀ values to a biostatistically significant level of P < 0.0001 (SI Appendix, Table S1). The anticancer efficacy of 5 was further compared (Fig. 5 E and F) with the activities of free Cur 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 used cells. This is likely due to its low water solubility, which eventually impedes its bioavailability. Likewise, both CB[8]-capped complexes, that is, 4 and 4′, are less effective relative to their free precursors 1 and 2′, respectively. The activity of these CB[8]-capped complexes was, however, drastically improved upon incorporation of Cur, as supported by the fact that 5 and 5′ exhibited higher growth inhibition efficiencies compared with all other formulations. Notably, 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 corroborated well with their efficacy as calculated from an MTT assay (SI Appendix, Figs. S22–S25).

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 for 48 h before performing the MTT assay. Cells were treated at concentrations of 50, 5, 0.5, and 0.05 μM aq. formulations of free Cur, 1, 4, 5, 2′, 4′, and 5′ (concentrations of 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, and MDA-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 test comparing cell viability from Cur treatment with other samples. Here *, **, ***, and **** represent P values < 0.05, 0.01, 0.001, and 0.0001, respectively.

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 and negative controls, respectively. A triple-negative breast cancer cell line, MDA-MB231, was used as a model cell line for the study due to its low cytotoxicity response against all of the used formulations. As shown in SI Appendix, Fig. S26, a noncytotoxic nature of CB[8] and a slight improvement in cell growth regression by Cur-CB[8] were observed. Cur-CB[8] reduced the cell growth to a level of 65 ± 5 compared with 75 ± 10% by free Cur 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 C32 is the most difficult cell line to effect cell growth, probably due to the 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 resistive natures for entry of foreign reagents (52). However, 5 showed promising anticancer activity in C32 cells, plausibly due to the presence of Cur in high local concentration that negates the effect of lower cellular entry of 5. Notably, 1, 4, and 5 affected overall cell growth and IC₅₀ more efficiently compared with 2′, 4′, and 5′ across all of the cell lines, probably due the presence of cell-growth-inhibiting moieties with a high local concentration. The results obtained from the treatment with 1, 4, and 5 were found to be similar for C32 and B16F10, probably due to the similar skin melanoma nature of the cells even though they originated from humans and rats, respectively. IC₅₀ values of 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 the growth inhibition properties of the different formulations in the different cell types not only depend on the combined effect of their chemistry, local concentration, and effective bioavailability of Cur but also on the origin of the cell itself. It also revealed the biostatistically significant improvement in cell growth inhibition of 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:

$$\mathrm{CI}=\left\{\left(\mathrm{I}{\mathrm{C}}_{50}\right)\mathrm{A}/\left(\mathrm{I}{\mathrm{C}}_{50}\right)\mathrm{A}+\mathrm{B}\right\}+\left\{\left(\mathrm{I}{\mathrm{C}}_{50}\right)\mathrm{B}/\left(\mathrm{I}{\mathrm{C}}_{50}\right)\mathrm{A}+\mathrm{B}\right\},$$ [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 > 1.3 indicates 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 various samples we concluded that in 5 Cur acts as a synergistic component with 1 as well as 4 with CI < 1 for all of the cell lines used in the study (SI Appendix, Table S2).

Propidium iodide (PI) is a known DNA intercalator that selectively enters into dead cells with an increase in its red fluorescence (53). A flow-assisted cell study was performed on C32 cells after separately treating them with Cur, 2′, 4′, 5′, 1, 4, and 5 for 48 h, followed by incubation with PI (1 μg/mL) for 20 min. A high-PI-loaded population was observed for cells treated with 2′ compared with 1, indicating 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 a higher cell-killing efficiency due to some nonobvious reasons. However, the addition of CB[8] lowers their cytotoxicity such that the corresponding complexes 4 and 4′ have comparable cytotoxicity (SI Appendix, Fig. S27B). The cytotoxicity was further enhanced and differed after the loading of Cur, resulting in a higher extent of PI accumulation in cells treated with 5 compared with 5′ (Fig. 6A). The results suggest that CB[8] is a noncytotoxic component in these systems and acts as a host to transport Cur to cancer cells.

Fig. 6.

(A) Histograms of C32 cells obtained after PI staining posttreatment with 5′ and 5. Cells without treatment and without PI staining were used as controls, while cells with PI staining and without treatment were secondary controls. Cur-treated cells were used as positive controls. Mechanistic studies on representative C32 cell lines. (B) DNA fragmentation assay performed on cells 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 polynucleotide length and molecular weight. (C) Protein bands captured from blots 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 cell protein (band I) and (D) comparison of percentage protein expression from different treatments. Cells were treated with free Cur, 5, and 5′ at a concentration of 10 μM (concentrations of 5 and 5′ are chosen such that they have the same molar equivalence of Cur).

Cellular entry of the host–guest complexes 4 and 5 was verified by inductively coupled plasma detection of Pt. Since Pt is not a part of any component of the cells, detection of Pt can be attributed to the cellular internalization of 4 and 5. The level of Pt was detected in cancer cells treated with 4 and 5 at a concentration of 5 µM. Triple-negative breast cancer cell line MDA-MB231 was used as a model cell line for the study due to its low cytotoxicity response against all of the used agents, while MCF-7 is the best of the responding cells. Cancer cells incubated with 5 showed a Pt level of ∼3 and 4 ppm in MCF-7 and MDA-MB231 cells, respectively, while ∼1 ppm Pt level was observed in each of the aforesaid cell lines treated with 4 (SI Appendix, Fig. S28A). As shown in SI Appendix, Fig. S28B, ∼200 and 400% higher Pt delivery was observed with 5 compared with 4 in MCF-7 and MDA-MB231 cells, respectively. The higher Pt content in cancer cells treated with 5 compared with 4 is likely due to higher cellular internalization of 5 compared with 4. Cells treated with Cur and Cur-CB[8] were used as negative controls along with cells alone, where no significant level 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 chosen as a representative cell line for the DNA fragmentation assay due to either comparable or lower cell growth inhibition efficiency of all of the reagents in it. A DNA fragmentation study in this cell line would reflect the clear possibilities of DNA fragmentation in other cell lines at the same concentration. Genomic DNA was first collected from C32 cells treated with either free Cur, 5, or 5′ for 48 h as well as from untreated cells and then subjected to gel analysis. The results are shown in Fig. 6B. The treatment of 5 produced bands of fragmented DNA (represented by white arrows in lane II), while no significant fragmentations were observed for untreated cells and cells treated with Cur (lane I) or 5′ (lane V), indicating an apoptosis-induced fragmentation 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 this mechanism, we performed a protein expression analysis. C32 cells were treated with free Cur, 5, and 5′ for 48 h and the total protein was extracted. The collected and quantified cell protein was run on an SDS gel for 1.5 h. Protein bands were transferred to blots which were further exposed against the primary antibodies of phosphor-STAT3 (pSTAT3) and background protein β-actin followed by secondary antibodies. The β-actin protein bands were used for normalizing the protein expression in treated cells. Protein bands from both the blots were imaged using 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 data support that 5 can efficiently deliver Cur to C32 cells, causing an apoptosis via pSTAT3 down-regulation pathways.

Conclusion

In summary, we report a host–guest complex, composed of a water-soluble organoplatinum(II) metallacycle 1 and CB[8], that acts as an aq. carrier of Cur and delivers it to cancer cells. The MV motifs and tri(ethylene glycol) groups of 1 overcome the hydrophobic barrier attributed to the hexagonal aromatic core and triethylphosphine groups, bestowing it with water solubility. The MV units of 1 allow it to undergo host–guest complexation with CB[8] in water. The resulting host–guest complex 4 can be further used to encapsulate Cur via a heteroternary complexation to form 5. The host–guest complexes were characterized by ¹H NMR and UV-visible spectroscopies. The host–guest complexation-mediated morphological transformation processes were investigated by TEM and DLS experiments. Under TEM, Cur-embedded heteroternary complex 5 exhibits various morphological features, such as honeycomb-like networks, fibers, and vesicles, depending upon the concentration.

The anticancer activity of 5 was established in vitro using 2D cultures of different cancer cell lines. Primarily therapeutic efficacy was determined by performing cell viability assays followed by studying the changes in cell growth patterns and morphologies. Cur showed significant synergism with 1 and 4, having CI < 1 irrespective of the cell lines used for the study. Further, flow-assisted assay, DNA fragmentation, and protein expression studies were corroborated with submicromolar IC₅₀ for optimized sample 5 via down-regulation of pSTAT3. Given these results, this work shows how a judicious combination of coordination-driven self-assembly and host–guest interactions can be utilized for hydrophobic drug delivery with improved efficacy.

Materials and Methods

All reagents were commercially available and used as supplied without further purification. Deuterated solvents were purchased from Cambridge Isotope Laboratory. ¹H NMR and ¹³C NMR spectra were recorded on a VarianInova 400 MHz spectrometer. The ³¹P{¹H} NMR spectra were recorded on a Varian Unity 300-MHz spectrometer. The ¹H and ¹³C NMR chemical shifts are reported relative to residual solvent signals, and ³¹P{¹H} NMR chemical shifts are referenced to an external unlocked sample of 85% H₃PO₄ (δ 0.0). Mass spectra were recorded on a Micromass Quattro II triple-quadrupole mass 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 DynaPro NanoStar instrument. TEM images were acquired at an accelerating voltage of 80 keV on a JEOL JEM-2800 instrument and the images were processed using Gatan DigitalMicrograph software. The details of the synthesis, characterization, and biological experiments are given in SI Appendix.