On the encapsulation and assembly of anticancer drugs in a cooperative fashion

Abstract

In this study, we report the remarkable recognition and assembly characteristics of D_3h symmetric basket 1⁶⁻ containing two adjoining and nonpolar cavities with six biocompatible GABA residues at their northern and southern termini. From the results of experimental (¹H NMR, fluorescence and UV-Vis spectroscopies) and computational (MM-MC/OPLS3e) investigations, we deduced that hexaanionic 1⁶⁻ captured two molecules of anticancer drug doxorubicin 2⁺ in water and accommodated them in its two deep cavities. The formation of stable 1⁶⁻⊂2₂²⁺ (K_a = 3 × 10¹² M⁻²) was accompanied by the exceptional homotopic cooperativity (α = 4K₂/K₁ = 112) in which K₁ = 3.2 ± 0.8 × 10⁵ M⁻¹ and K₂ = 9 ± 1 × 10⁶ M⁻¹. Furthermore, bolaamphiphilic 1⁶⁻⊂2₂²⁺ assembled into spherical nanoparticles (DLS, cryo-TEM and TEM) possessing 41% drug loading. The preorganization of abiotic receptor 1⁶⁻ and its complementarity to 2⁺ have been proposed to play a part in the positive cooperativity in which ten favorable noncovalent contacts (i.e. hydrogen bonds, salt bridges, C–H⋯π and π–π contacts) are formed between doxorubicin and the dual-cavity host. In the case of topotecan 3⁺, however, the absence of multiple and favorable basket⊂drug interactions resulted in the predominant formation of a binary 1⁶⁻ ⊂ 3⁺ complex (K₁ = 2.12 ± 0.01 × 10⁴ M⁻¹) and the negative homotopic allostery (α ≪ 1). To summarize, our study lays out a roadmap for creating a family of novel, accessible and multivalent hosts capable of complexing anticancer agents in a cooperative manner. As basket⊂drug complexes organize into highly loaded nanoparticles, the reported soft material is amenable to the bottom-up construction of stimuli-responsive nanomedicine capable of effective scavenging and/or delivery of drugs.

---

Introduction

Nanosystems carrying anticancer drugs, i.e. nanomedicine,¹ hold great promise in the area of targeted chemotherapy for selective detection and extermination of cancer cells.^1a,2 In particular, drug-delivery nanoparticles³ extravasate from abnormal blood vessels into the tumor microenvironment (EPR effect),⁴ which with their surface modification could improve the therapy.^3,5,6 Besides, stimuli-responsive and biocompatible⁷ cyclodextrins,⁸ calixarenes⁹ and cucurbiturils¹⁰ are also capable of transporting/releasing anticancer drugs.¹¹ As a result of the inclusion complexation,¹² abiotic hosts are expected to improve the therapy by allowing spatiotemporal control¹³ of drug release and also assisting with (a) loading capacity,¹⁴ (b) solubility,¹⁵ (c) bioavailability¹⁶ and (d) stability of drugs.¹⁷ In addition to such virtues, allosteric and chelate cooperativity¹⁸ operating in the host–guest complexation of pharmaceuticals is expected to give rise to an amplified response so that small changes in the external signal (i.e. stimulus) trigger a large non-linear outcome.¹⁹ The on/off mode²⁰ of delivery is, however, challenging to attain²¹ yet a sought-after strategy to achieve minimal release of the payload before reaching its targeted site followed by a burst in drug release.²²

Recently, we reported about dual-cavity baskets comprising six²³ (S) alanine residues at their periphery (Fig. 1) and acting in an allosteric manner²⁴ by trapping small molecules akin to nerve agents.²⁵ As these complexes assembled into vesicles,^25b we became increasingly curious about examining the capacity of deeper dual-cavity 1⁶⁻ (Fig. 1A) for including anticancer drugs doxorubicin 2⁺ (DOX) and topotecan 3⁺ in its two adjoining cavities. At first, the inner space of basket 1⁶⁻ seemed complementary²⁶ to 2⁺ and 3⁺ in size, shape and polarity (Fig. 1A). Moreover, structurally unique 1⁶⁻ possesses six biocompatible GABA (γ-aminobutyric acid) residues at its southern and northern termini for acting as “sticky” carboxylate fingers capable of grabbing functional drug molecules and holding them in two aromatic pockets.²⁷ The results of our experimental and computational studies have revealed a fascinating way by which dual-cavity 1⁶⁻ encapsulates two molecules of doxorubicin 2⁺ with exceptional homotopic cooperativity of α = 4K₂/K₁ = 112!^18,28 Moreover, stable ternary 1⁶⁻ ⊂ 2₂²⁺ complexes (K_a = 10¹² M⁻²) assembled into spherical nanoparticles possessing a high (41%) drug loading.²⁹

Fig. 1 (A) van der Waals surfaces of energy-minimized basket 1⁶⁻, doxorubicin 2⁺ and topotecan 3⁺ (OPLS3e, Maestro). (B) Basket 1⁶⁻ was prepared from anthracene, vinyl acetate and GABA (Scheme S1†). (C) Top (left) and side (right) views of the aligned conformers of 1⁶⁻ (<1.5 kcal mol⁻¹) obtained from the Monte-Carlo conformational search (OPLS3e, Maestro) in implicit water.

Results and discussion

Dual-cavity 1 (Fig. 1B) was prepared from inexpensive anthracene, vinyl acetate and GABA by following a recently developed procedure (Scheme S1†).³⁰ After basket 1 was dissolved in 10 mM phosphate buffer (PBS) at pH = 7.0, its ¹H NMR spectrum showed a set of five, somewhat broad, resonances corresponding to, on average, D_3h symmetric 1⁶⁻ (Fig. 2A); note that for six remote carboxylates, we assumed pK_a < 5.³¹ In line with ¹H NMR results, the Monte-Carlo conformational sampling of 1⁶⁻, in implicit water solvent (OPLS3e, Maestro), revealed numerous conformers (Fig. 1C) populating the equilibrium (<1.5 kcal mol⁻¹). In solution, these conformers were likely to exchange at a high rate to give the observed ¹H NMR spectrum. Furthermore, six carboxylates cluster at the outer side of 1⁶⁻ to make the binding pockets deep, nonpolar and suitable for accommodating nonpolar segments of doxorubicin 2⁺ and topotecan 3⁺ (Fig. 1A). The phthalimide side arms are “bouncing” back and forth to alter the cavity size,³² which is central to the induced-fit mode of complexation of the first molecule of drug, as discussed in the text below.³³

Fig. 2 (A) ¹H NMR spectra (600 MHz, 298 K) of 0.04 mM basket 1⁶⁻ in 10 mM PBS at pH = 7.0 (bottom, green) before and after incremental addition of doxorubicin 2⁺ (top, blue-red). (B) UV-Vis absorbance (300 nm) of differently concentrated solutions of 1⁶⁻ in 10 mM PBS at pH = 7.0 as a function of its concentration. Each set of data (blue and red) was fit to a linear equation (R² > 0.99, SigmaPlot) using the least-square regression analysis. (C) The Tyndall effect can be seen for 0.4 mM (right) but not for a 0.05 mM (left) solution of 1⁶⁻. The size distribution of particles in a 0.76 mM solution of 1⁶⁻ (10 mM PBS at pH = 7.0) obtained from DLS measurement at 298.0 K. (D) Cryo-TEM image (left) of 0.6 mM solution of basket 1⁶⁻ in 10 mM PBS at pH = 7.0 and TEM image (right) of 0.2 mM 1⁶⁻ in 10 mM PBS at pH = 7.0 deposited on a copper grid and stained with uranyl acetate.

Variously concentrated solutions of 1⁶⁻ exhibited a sharp change in the UV-Vis extinction coefficient (ε₃₀₀, i.e. slopes in Fig. 2B) of this chromophore at ∼0.12 mM.²¹ The “segmental adherence to the Lambert–Beer law suggested that the basket transitioned from the monomeric to the aggregated state at 0.12 mM thereby depicting its critical aggregation concentration (CAC).^25b,34 Indeed, the results of dynamic light scattering measurements of 0.76 mM 1⁶⁻ (DLS, Fig. 2C) were in line with the formation of nanosized particles, while the Tyndall effect was apparent for 0.4 mM but absent for the 0.05 mM solution of 1⁶⁻ (Fig. 2C). At last, cryo-TEM and TEM imaging (Fig. 2D) revealed the formation of circa 40–50 nm spherical objects possessing uniform coloration with no distinguishable features (i.e. double layer). Accordingly, bolaamphiphilic 1⁶⁻ is likely populating the interior of the spherical aggregates with a packing mode that still needs to be elucidated.^34a

After an incremental addition of doxorubicin 2⁺ to basket 1⁶⁻ (Fig. 2A), there followed a broadening of ¹H NMR resonances from the host while signals corresponding to the drug's nuclei became ill defined. In particular, H_A–H_D signals from the doxorubicin's methoxybenzene (shown in red, Fig. 2A) were difficult to resolve, as they were likely becoming magnetically shielded and broadening into the baseline. Alternatively, H_E–H_N signals from the doxorubicin's non-aromatic side (shown in blue, Fig. 2A) were easier to identify by comparing the spectra. From the NMR results, we hypothesized that doxorubicin 2⁺ formed a stable host–guest complex with 1⁶⁻ to result in the complexation dynamics occurring at an intermediate rate and/or rapid T₂ relaxation of H_A–H_D protons 2⁺ inside the cavity(ies) of 1⁶⁻.³⁵ As the magnetic environment and dynamics of H_E–H_N were perturbed to a lesser degree than H_A–H_D protons, however, these nuclei stayed away from the concave aromatic pockets of 1⁶⁻. To further quantify the inclusion complexation, we noted that incremental addition of basket 1⁶⁻ to 2⁺ (Fig. 3A) quenched the emission from doxorubicin;³⁶ the quenching was static,³⁷ since in the absence of the complex formation the emission from the drug remained steady (Fig. S7†). The binding isotherm (Fig. 3B; see also Fig. S8† and www.supramolecular.org) fits well to the 1 : 2 complexation mode²⁸ with a random distribution of residuals³⁸ and K₁ = 3.2 ± 0.8 × 10⁵ M⁻¹/K₂ = 9 ± 1 × 10⁶ M⁻¹. The method of continuous variation (UV-Vis, Fig. 3C) was also in line with the formation of ternary 1⁶⁻⊂2₂²⁺ with the parabolic function peaking at [2⁺]/([1⁶⁻] + [2⁺]) ∼ 0.67.³⁸ The positive cooperativity of α = 4K₂/K₁ = 112 is large and quite unusual for an artificial host.^21,39 We therefore suspected that the formation of ternary 1⁶⁻⊂2₂²⁺ could be accompanied by an aggregation of these bolaamphiphilic complexes so that the observed cooperativity is in part arising from the chelate (multivalent) cooperativity.¹⁸ In support of such logic, the observed broadening of ¹H NMR signals of both 1⁶⁻ and 2⁺ in Fig. 2A could have, in part, resulted from their aggregation. First, DLS measurements of 1⁶⁻ ⊂ 2₂²⁺ showed the presence of circa 60–240 nm particles (Fig. 3D) having electrokinetic (zeta) potential of ζ = −33.6 (Fig. S9†) and therefore moderate stability.^25b Likewise, TEM (Fig. 3E) and cryo-TEM imaging (Fig. 3F) revealed the formation of nanoparticles whose size (circa 50 nm), shape and, perhaps, assembly mode were akin to those forming from 1⁶⁻. As nanoparticles are composed of dual-cavity hosts (M_w = 1536) with each holding two molecules of doxorubicin (M_w = 2 × 544 = 1088), it follows that the material's loading is circa 41%, which is highly desirable¹³ for nanomedicine.^1a

Fig. 3 (A) An incremental addition of basket 1⁶⁻ to a 1.0 μM solution of doxorubicin 2⁺ (10 mM PBS at pH = 7.0) was monitored with fluorescence spectroscopy (λ_ex = 500 nm; 298 K). (B) A change in the emission intensity of 2⁺ as a function of the increasing concentrations of 1⁶⁻ was subjected to global (550–590 nm) nonlinear regression analysis using a 2 : 1 stoichiometric model (note that doxorubicin 2⁺ is in this experimental setup virtually acting as a host with the stoichiometry depicted as 2 : 1); the reported K₁ = 3.2 ± 0.8 × 10⁵ M⁻¹ and K₂ = 9 ± 1 × 10⁶ M⁻¹ represent the arithmetic mean and standard deviation from two independent measurements (Fig. S8†); a random distribution of residuals is shown in the inset. (C) UV-Vis spectra of 0.04 mM doxorubicin 2⁺ (10 mM PBS at pH = 7.0) in the presence of different proportions of 1⁶⁻ with the Job plot (inset) obtained using the absorbance at 500 nm; for the y axis: (A_obs − A₀)[2⁺]₀. (D) The size distribution of particles in the solution of 1⁶⁻ (0.05 mM) and 2⁺ (0.1 mM) (10 mM PBS at pH = 7.0) was obtained from DLS measurement at 298.0 K. (E) TEM image of a 10 mM PBS solution of 1⁶⁻ (0.1 mM) and 2⁺ (0.2 mM) at pH = 7.0 deposited on a copper grid and stained with uranyl acetate. (F) Cryo-TEM image of a 0.1 mM solution of basket 1⁶⁻ containing 2⁺ (0.2 mM) in 10 mM PBS at pH = 7.0.

Encouraged by the results with DOX 2⁺, we turned to examine the entrapment of another therapeutic anticancer drug, topotecan 3⁺ (Fig. 4). This flat molecule is in size and shape similar to doxorubicin (Fig. 1A) therefore making a good candidate for occupying the binding pockets of 1⁶⁻. Upon an incremental addition of a standard solution of 3⁺ to 1⁶⁻, there were small but steady magnetic perturbations of ¹H NMR resonances from the basket (green in Fig. 4A). In particular, a greater proportion of 3⁺ resulted in the H_L singlet from D_3h symmetric 1⁶⁻ turning into a doublet. The observation was taken as a sign depicting the predominant formation of pseudo C_3v symmetric 1⁶⁻⊂3⁺ in which a single cavity of the host is holding the drug. Furthermore, H_A/B/C and H_H signals from topotecan 3⁺ (red in Fig. 4A) experienced a greater magnetic shielding (Δδ ∼ 0.3–0.4 ppm, red in Fig. 4A) than its other nuclei to denote the lactone but also dimethyl ammonium portions of the drug residing in the host's aromatic cavity. The method of continuous variation (Fig. 4B) indicated the formation of a binary 1⁶⁻⊂3⁺ complex, while the fluorescence binding isotherm (Fig. 4C; see also Fig. S10†) fits well to the 1 : 1 stoichiometric model with a random distribution of residuals (K₁ = 2.12 ± 0.01 × 10⁴ M⁻¹);³⁸ note that the fluorescence of topotecan 3⁺ was, as in the case of doxorubicin, quenched by the basket. At last, the results from DLS (Fig. S11†), TEM (Fig. S12†) and cryo-TEM (Fig. S13†) measurements were consistent with 1⁶⁻ ⊂ 3⁺ assembling into circa 35 nm nanoparticles with the morphology akin to 1⁶⁻ and 1⁶⁻⊂2₂²⁺ nanoparticles.

Fig. 4 (A) ¹H NMR spectra (850 MHz, 298 K) depicting an incremental addition of topotecan 3⁺ (red, top) to 0.1 mM basket 1⁶⁻ (green, bottom) in 10 mM PBS at pH = 7.0. (B) UV-Vis spectra of 0.03 mM topotecan 3⁺ (10 mM PBS at pH = 7.0) in the presence of different proportions of 1⁶⁻ with the Job plot (inset) obtained using the absorbance at 382 nm; for the y axis: (A_obs − A₀)[3⁺]₀. (C) A change in the emission intensity of 3⁺ (2 μM in 10 mM PBS at pH = 7.0) as a function of the increasing concentrations of 1⁶⁻ was subjected to global (520–540 nm) nonlinear regression analysis using a 1 : 1 stoichiometric model to give K₁ = 2.12 ± 0.01 × 10⁴ M⁻¹, representing the arithmetic mean and standard deviation from two independent measurements (Fig. S10†); a random distribution of residuals is shown in the inset. (Right) Fluorescence spectra of 2 μM 3⁺ in the presence of increasing concentrations of 1⁶⁻ (λ_ex = 420 nm; 298 K) in 10 mM PBS at pH = 7.0.

Why is the encapsulation of doxorubicin characterized with positive cooperativity (α = 112) while topotecan prefers to occupy a single pocket of dual-cavity 1⁶⁻ (i.e. negative cooperativity α ≪ 1)?^21,24 In order to elucidate the origin of the allostery, we first subjected 1⁶⁻⊂2⁺ and 1⁶⁻⊂2₂²⁺ complexes to the Monte-Carlo conformational search using the OPLS3e force field⁴⁰ in implicit water solvent. The global minimum was, in all cases, assessed by running multiple calculations, each starting with the drug assuming a different pose in the basket's cavity (see the ESI†). The conformational sampling of 1⁶⁻⊂2⁺ returned numerous yet similar conformers populating the equilibrium (<1.5 kcal mol⁻¹, Fig. 5A): all structures encompass drug 2⁺ docked within the host's top cavity and the methoxybenzene ring poised to form C–H⋯π (D, Fig. 5A) and π–π stacking (E, Fig. 5A) interactions with the surrounding aromatic box. Moreover, three GABA carboxylates from basket 1⁶⁻ form two hydrogen bonds A/B and salt bridge C with the drug. While the occupied cavity snugs the guest by reducing the average imide d_N–N distance to 9.19–9.30 Å (arithmetic mean of three d_N–N distances in Fig. 5A), the unoccupied (bottom) cavity responds by flexing^25a,41 its phthalimide arms to d_N–N = 9.92–10.01 Å (Fig. 5D; for empty 1⁶⁻, d_N–N = 9.71–9.77 Å). For the doubly populated 1⁶⁻ ⊂ 2₂²⁺, the drug molecule docked in the top cavity preserves all five noncovalent contacts A–E with the cavity 1⁶⁻⊂2₂²⁺ (Fig. 5B). Doxorubicin residing in the bottom cavity, though, assumes an equivalent position to that in the top one with three GABA residues forming hydrogen bonds A and B and the salt bridge C to hold it in its place. The transition of singly 1⁶⁻⊂2⁺ into doubly populated 1⁶⁻⊂2₂²⁺ (Fig. 5C) is accompanied by the top cavity retaining its size d_N–N = 9.20–9.30 Å and the bottom one shrinking to d_N–N = 9.07–9.27 Å. The second binding event thus necessitates a reduction in the size of the bottom cavity while concurrently maintaining the shape of the top one (Fig. 5D). We reason that such increase in the steric strain (ΔH° > 0) following the second complexation is likely overcompensated by favourable noncovalent A–E contacts (ΔH° ≪ 0).

Fig. 5 (A) A side view of 155 conformers of 1⁶⁻⊂2⁺ (<1.5 kcal mol⁻¹) obtained by the Monte-Carlo conformational search (OPLS3e, Schrodinger) in implicit water. (Middle) Chemical structure of doxorubicin 2⁺ forming favourable A–E noncovalent contacts with basket 1⁶⁻. (Right) Top view of the most stable conformer of 1⁶⁻⊂2⁺. (B) A side view of 33 conformers of 1⁶⁻⊂2₂²⁺ (<1.5 kcal mol⁻¹) obtained by the Monte-Carlo conformational search (OPLS-3, Schrodinger) in implicit water. (Right) The most stable conformer of 1⁶⁻⊂2⁺ showing DOX 2⁺ (red) forming favourable A–E contacts with the basket. (C) The most stable conformers of 1⁶⁻⊂2⁺ (blue) and 1⁶⁻⊂2₂²⁺ are aligned to demonstrate the induced-fit mode of binding in the bottom cavity. (D) A schematic representation of the induced-fit model of binding of doxorubicin 2⁺ with dual-cavity 1⁶⁻.

The formation of binary 1⁶⁻⊂2⁺ is a favourable process (K₁ = 3.2 ± 0.8 × 10⁵ M⁻¹) supported with the notion that the basket's top grips onto the drug by using all three of its phthalimide arms and engaging in five favourable non-covalent interactions (Fig. 5A): the top cavity shrinks to hold the drug, while the bottom one expands; the hydrophobic effect is expected to contribute to this complexation as well.⁴² With all three of the top cavity's sides being “tied”, the bottom three phthalimides become more rigid^25a and thus more preorganized to, via favourable entropy, increase the affinity for the second drug molecule (K₂ = 9 ± 1 × 10⁶ M⁻¹). That is to say, the first complexation preorganizes the host for the second one to occur with a minimal loss in entropy, and gain in enthalpy and the overall positive homotopic cooperativity. The role of the assembly of 1⁶⁻ ⊂ 2₂²⁺ has not been taken into consideration although it is more than likely that the process has an effect on the cooperativity and the mechanism by which the recognition takes place.

A validation for our computational findings comes from the results of ¹H NMR spectroscopic experiments (Fig. 2A). The methoxybenzene nuclei from doxorubicin 2⁺ sustained the greatest degree of magnetic perturbation to implicate it occupying the host's cavities as the theory suggests (Fig. 5).

The Monte-Carlo conformational sampling of 1⁶⁻⊂3⁺ (Fig. 6) revealed topotecan docking in the basket's cavity by anchoring its lactone ring (Fig. 6A) or dimethyl ammonium group in the aromatic box (Fig. 6B). The apparent absence of additional noncovalent contacts between 3⁺ and 1⁶⁻ (except one hydrogen bond A in Fig. 6B) makes this host–guest pair less complementary than the prior one. The basket is thus loosely held by topotecan to make the 1⁶⁻⊂3⁺ complex less preorganized for binding another molecule of the drug. Moreover, a weaker complexation could also be alleged to provide insufficient driving force for overcoming the strain required in the second binding event. In support of topotecan 3⁺ being less complementary to dual-cavity 1⁶⁻ than doxorubicin 2⁺, we note that binary 1⁶⁻⊂3⁺ is an order of magnitude less stable (K₁ = 2.12 ± 0.01 × 10⁴ M⁻¹) than 1⁶⁻⊂2⁺ (K₁ = 3.2 ± 0.8 × 10⁵ M⁻¹). On another note, the results of the NMR titration (Fig. 4A) showed that protons at both termini of topotecan became magnetically shielded to, perhaps, indicate the concurrent existence of both computed poses shown in Fig. 6.

Fig. 6 Side views of 467 (A) and 271 (B) conformers of 1⁶⁻⊂3⁺ (<1.5 kcal mol⁻¹) obtained by the Monte-Carlo conformational search (OPLS3e, Maestro) in implicit water. Note that topotecan 3⁺ inserts (A) its lactone ring or (B) dimethyl ammonium group in the cavity of basket 1⁶⁻. ΔE represents the computed difference in steric energies of two most stable conformers in (A) and (B).

Conclusions

We discovered an abiotic host capable of encapsulating two molecules of anticancer drug doxorubicin in a positive cooperative fashion with ternary drug–host complexes assembling into nanoparticles. The origin of the cooperativity can, in part, be traced back to extraordinary complementarity of the drug to the abiotic receptor in addition to its preorganization. That is to say, at least ten favorable noncovalent contacts are established between doxorubicin and the dual-cavity basket, resembling those found for receptors in the natural world. The basket acts as a “claw grabber” capturing drug molecules with its six “sticky carboxylate fingers” and snugging them within the adjoining aromatic cavities in an induced-fit manner. In the case of topotecan, however, we posited that the paucity of favorable noncovalent contacts between the basket and the drug, i.e. lack of complementarity, played the key role in the observed complexation stoichiometry. In general, our discovery sets the stage for (a) improving our fundamental understanding of cooperativity in artificial systems and (b) creating a family of novel hosts capable of complexing plane-shaped anticancer agents with high affinity and selectivity.

Holding a high drug payload (41%),¹³ nanoparticles release doxorubicin at a lower pH⁴³ (Fig. S14/S15†) or in the presence of spermine⁴⁴ (Fig. S16†), as signaled by the increase in fluorescence.^36b,45 Accordingly, our aims shift to the examination of this soft material for delivery⁴⁶ as well as scavenging⁴⁷ of anticancer drugs in biological and other competitive media.²² In fact, with dual-cavity baskets amenable to screening protocols,³⁰ one should be able to optimize their capacity for selective removal⁴⁸ as well as amplified delivery²² of targeted pharmaceuticals.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was financially supported with funds obtained from the Army Research Office (W911NF-17-1-0140). We would like to thank Prof. C. M. Hadad from the Ohio State University, Department of Chemistry and Biochemistry, for useful suggestions and inspiring discussions.

Notes and references

1. (a) W. C. W. Chan, Acc. Chem. Res., 2017, 50, 627; (b) F. Caruso, T. Hyeon and V. M. Rotello, Chem. Soc. Rev., 2012, 41, 2537.
2. (a) S. Aftab, A. Shah, A. Nadhman, S. Kurbanoglu, S. Aysil Ozkan, D. D. Dionysiou, S. S. Shukla and T. M. Aminabhavi, Int. J. Pharm., 2018, 540, 132; (b) R. Awasthi, A. Roseblade, P. M. Hansbro, M. J. Rathbone, K. Dua and M. Bebawy, Curr. Drug Targets, 2018, 19, 1696; (c) T. J. Anchordoquy, Y. Barenholz, D. Boraschi, M. Chorny, P. Decuzzi, M. A. Dobrovolskaia, Z. S. Farhangrazi, D. Farrell, A. Gabizon, H. Ghandehari, B. Godin, N. M. La-Beck, J. Ljubimova, S. M. Moghimi, L. Pagliaro, J.-H. Park, D. Peer, E. Ruoslahti, N. J. Serkova and D. Simberg, ACS Nano, 2017, 11, 12; (d) L. Isaacs, Adv. Drug Delivery Rev., 2012, 64, 763; (e) C. M. Hartshorn, M. S. Bradbury, G. M. Lanza, A. E. Nel, J. Rao, A. Z. Wang, U. B. Wiesner, L. Yang and P. Grodzinski, ACS Nano, 2018, 12, 24.
3. S. Wilhelm, A. J. Tavares, Q. Dai, S. Ohta, J. Audet, H. F. Dvorak and W. C. W. Chan, Nat. Rev. Mater., 2016, 1, 16014.
4. C. Kinnear, T. L. Moore, L. Rodriguez-Lorenzo, B. Rothen-Rutishauser and A. Petri Fink, Chem. Rev., 2017, 117, 11476.
5. V. P. Torchilin, Handb. Exp. Pharmacol., 2010, 197, 3.
6. R. Mout, F. Moyano Daniel, S. Rana and M. Rotello Vincent, Chem. Soc. Rev., 2012, 41, 2539.
7. Y.-R. Zheng, K. Suntharalingam, T. C. Johnstone and S. J. Lippard, Chem. Sci., 2015, 6, 1189.
8. K. Uekama, F. Hirayama and T. Irie, Chem. Rev., 1998, 98, 2045.
9. M. Giuliani, I. Morbioli, F. Sansone and A. Casnati, Chem. Commun., 2015, 51, 14140.
10. L. Cao, G. Hettiarachchi, V. Briken and L. Isaacs, Angew. Chem., Int. Ed., 2013, 52, 12033.
11. X. Ma and Y. Zhao, Chem. Rev., 2015, 115, 7794.
12. L. Wang, L.-l. Li, Y.-s. Fan and H. Wang, Adv. Mater., 2013, 25, 3888.
13. K. Park, ACS Nano, 2013, 7, 7442.
14. C. Weeden, K. J. Hartlieb and L. Y. Lim, J. Pharm. Pharmacol., 2012, 64, 1403.
15. D. Ma, G. Hettiarachchi, D. Nguyen, B. Zhang, J. B. Wittenberg, P. Y. Zavalij, V. Briken and L. Isaacs, Nat. Chem., 2012, 4, 503.
16. W. Shao, X. Liu, G. Sun, X.-Y. Hu, J.-J. Zhu and L. Wang, Chem. Commun., 2018, 54, 9462.
17. A. Abdul Karim, Q. Dou, Z. Li and X. J. Loh, Chem.–Asian J., 2016, 11, 1300.
18. C. A. Hunter and H. L. Anderson, Angew. Chem., Int. Ed., 2009, 48, 7488.
19. Y. Li, T. Zhao, C. Wang, Z. Lin, G. Huang, J. Gao, C. Wang and D. Sumer Baran, Nat. Commun., 2016, 7, 13214.
20. J. A. Riddle, X. Jiang and D. Lee, Analyst, 2008, 133, 417.
21. S. Shinkai, M. Ikeda, A. Sugasaki and M. Takeuchi, Acc. Chem. Res., 2001, 34, 494.
22. Y. Li, Y. Wang, G. Huang and J. Gao, Chem. Rev., 2018, 118, 5359.
23. K. C. Tjandra and P. Thordarson, Bioconjugate Chem., 2019, 30, 503.
24. J. D. Badjic, A. Nelson, S. J. Cantrill, W. B. Turnbull and J. F. Stoddart, Acc. Chem. Res., 2005, 38, 723.
25. (a) S. Chen, M. Yamasaki, S. Polen, J. Gallucci, C. M. Hadad and J. D. Badjic, J. Am. Chem. Soc., 2015, 137, 12276; (b) S. Chen, L. Wang, S. M. Polen and J. D. Badjic, Chem. Mater., 2016, 28, 8128.
26. J. B. Wittenberg and L. Isaacs, Supramol. Chem.: Mol. Nanomater., 2012, 1, 25.
27. K. Hermann, M. Nakhla, J. Gallucci, E. Dalkilic, A. Dastan and J. D. Badjic, Angew. Chem., Int. Ed., 2013, 52, 11313.
28. P. Thordarson, Chem. Soc. Rev., 2011, 40, 1305.
29. S. Mitragotri and P. Stayton, MRS Bull., 2014, 39, 219.
30. T. Neal, W. Wang, L. Zhiquan, R. Peng, P. Soni, H. Xie and J. D. Badjic, Chem.–Eur. J., 2019, 25, 1115.
31. L. Zhiquan, S. Polen, C. M. Hadad, T. V. RajanBabu and J. D. Badjic, J. Am. Chem. Soc., 2016, 138, 8253.
32. Y. Ruan, H. A. Taha, R. J. Yoder, V. Maslak, C. M. Hadad and J. D. Badjic, J. Phys. Chem. B, 2013, 117, 3240.
33. L. Zhiquan, H. Xie, S. E. Border, J. Gallucci, R. Z. Pavlovic and J. D. Badjic, J. Am. Chem. Soc., 2018, 140, 11091.
34. (a) S. E. Border, R. Z. Pavlovic, L. Zhiquan, M. J. Gunther, H. Wang, H. Cui and J. D. Badjic, Chem. Commun., 2019 10.1039/c8cc08938h; (b) L. Wang, T. Neal, S. Chen and J. D. Badjic, Chem.–Eur. J., 2017, 23, 8829.
35. S. Chen, Y. Ruan, J. D. Brown, J. Gallucci, V. Maslak, C. M. Hadad and J. D. Badjic, J. Am. Chem. Soc., 2013, 135, 14964.
36. (a) S. Chen, S. M. Polen, L. Wang, M. Yamasaki, C. M. Hadad and J. D. Badjic, J. Am. Chem. Soc., 2016, 138, 11312; (b) W.-C. Geng, S. Jia, Z. Zheng, Z. Li, D. Ding and D.-S. Guo, Angew. Chem., Int. Ed., 2019 DOI:10.1002/anie.201813397.
37. D.-S. Guo, V. D. Uzunova, X. Su, Y. Liu and W. M. Nau, Chem. Sci., 2011, 2, 1722.
38. F. Ulatowski, K. Dabrowa, T. Balakier and J. Jurczak, J. Org. Chem., 2016, 81, 1746.
39. I. Saha, J. H. Lee, H. Hwang, T. S. Kim and C.-H. Lee, Chem. Commun., 2015, 51, 5679.
40. E. Harder, W. Damm, J. Maple, C. Wu, M. Reboul, J. Y. Xiang, L. Wang, D. Lupyan, M. K. Dahlgren, J. L. Knight, J. W. Kaus, D. S. Cerutti, G. Krilov, W. L. Jorgensen, R. Abel and R. A. Friesner, J. Chem. Theory Comput., 2016, 12, 281.
41. P. Thordarson, E. J. A. Bijsterveld, J. A. A. W. Elemans, P. Kasak, R. J. M. Nolte and A. E. Rowan, J. Am. Chem. Soc., 2003, 125, 1186.
42. L. Zhiquan, S. M. Polen, C. M. Hadad, T. V. RajanBabu and J. D. Badjic, Org. Lett., 2017, 19, 4932.
43. (a) J.-Z. Du, H.-J. Li and J. Wang, Acc. Chem. Res., 2018, 51, 2848; (b) B. Li, Z. Meng, Q. Li, X. Huang, Z. Kang, H. Dong, J. Chen, J. Sun, Y. Dong, J. Li, X. Jia, J. L. Sessler, Q. Meng and C. Li, Chem. Sci., 2017, 8, 4458.
44. Y. Chen, Z. Huang, H. Zhao, J.-F. Xu, Z. Sun and X. Zhang, ACS Appl. Mater. Interfaces, 2017, 9, 8602.
45. Y. Yuan and B. Liu, Chem. Sci., 2017, 8, 2537.
46. D. Bousmail, L. Amrein, J. J. Fakhoury, H. H. Fakih, J. C. C. Hsu, L. Panasci and H. F. Sleiman, Chem. Sci., 2017, 8, 6218.
47. H. J. Oh, M. S. Aboian, M. Y. J. Yi, J. A. Maslyn, W. S. Loo, X. Jiang, D. Y. Parkinson, M. W. Wilson, T. Moore, C. R. Yee, G. R. Robbins, F. M. Barth, J. M. DeSimone, S. W. Hetts and N. P. Balsara, ACS Cent. Sci., 2019 DOI:10.1021/acscentsci.8b00700.
48. J.-C. Leroux, Nat. Nanotechnol., 2007, 2, 679.

---

Footnote

† Electronic supplementary information (ESI) available: The preparation and characterization of 1 with additional experimental and computational details. See DOI: 10.1039/c9sc01380f

---