Crystalline Nanoparticles of Water-Soluble Covalent Basket Cages (CBCs) for Encapsulation of Anticancer Drugs

Abstract

We herein describe the preparation, assembly, recognition characteristics, and biocompatibility of novel covalent basket cage CBC-11, composed of four molecular baskets linked to four trivalent aromatic amines through amide groups. The cage is tetrahedral in shape and similar in size to small proteins (M_w =8637 g/mol) with a spacious nonpolar interior for accommodating multiple guests. While 24 carboxylates at the outer surface of CBC-11 render it soluble in aqueous phosphate buffer (PBS) at pH=7.0, the amphiphilic nature prompts its assembly into nanoparticles (d=250 nm, DLS). Cryo-TEM examination of nanoparticles revealed their crystalline nature with wafer-like shapes and hexagonally arranged cages. Nanoparticulate CBC-11 traps anticancer drugs irinotecan and doxorubicin, with each cage binding up to four drug molecules in a non-cooperative manner. The inclusion complexation resulted in nanoparticles growing in size and precipitating. In media containing mammalian cells (HCT 116, human colon carcinoma), the IC₅₀ value of CBC-11 was above 100 μM. While this work presents the first example of a large covalent organic cage operating in water at the physiological pH and forming crystalline nanoparticles, it also demonstrates its biocompatibility and potential to act as a polyvalent binder of drugs for their sequestration or delivery.

Keywords: Anticancer Drugs; Covalent Organic Cages; Drug Delivery; Host-Guest Chemistry; Sequestration.

Figure 1.

(A) One-pot synthesis of covalent basket cage CBC-1 (OPLS4) occurs via reversible condensation of four tris-aldehyde baskets (green) and four tris-amines (blue). Covalent basket cage CBC-1 has twelve imines joining four tris-amine panels (blue) with four molecular baskets (green) in addition to twelve hexoxide groups (pink) for improving solubility in nonpolar media; for clarity, only three solubilizing groups (pink spheres) are shown. (B) Graphical, energy-minimized (OPLS4) representations of CBC-2. In this work we aimed to convert labile imines from CBC-1 like cage into robust amides (the Pinnick oxidation) and then install charged groups (i.e., ammoniums or carboxylates) at the outer surface for improving stability and solubility of such CBC-2 in aqueous media; for clarity, only three solubilizing groups (pink spheres) are shown. Four drug molecules (gray) were expected to occupy the cavity of CBC-2.

Figure 2.

(A) ¹H NMR spectra (850 MHz, 298 K) of CBC-11 in CD₃OD:CD₂Cl₂ = 1:3, CBC-10 in CD₂Cl₂), and CBC-11 in 30 mM PBS at pH = 7.0. (B) ESI-MS of CBC-11 showing the formation of its variously charged anions. (C) Top views of CPK models of energy-minimized CBC-11 (OPLS-4) showing left- and right-handed orientations of three thio-ether chains (magenta).

Figure 3.

(A) Dynamic light scattering (DLS) plot of 66 μM solution of CBC-11 in 30 mM PBS at pH = 7.0. (Right) A plot showing the total number of scattered photons (i.e., derived count rate) from 2-66 μM solutions of CBC-11; each point presents a mean of three measurements with one standard deviation as error bars. The data fits well to a linear function (SigmaPlot). (B) Cryo-TEM images of 200 μM CBC-11 in 30 mM PBS at pH = 7.0 showing a round nanocrystalline wafer (top left). Fourier transformation of the electron diffraction data confirms the nanomaterial’s crystallinity (top middle and right). An enlarged view of a nanocrystalline CBC-11 showing the proposed hexagonal packing.

Figure 4.

(A) Two representations of energy-minimized [12₄⊂CBC-11] (OPLS4) with a plot showing perturbation of chemical shifts (Δδ) of ¹H NMR signals corresponding to nuclei from CBC-11 (40.0 μM) in the presence of 3.0 molar equivalent of irinotecan 12⁺ in 30 mM PBS at pH = 7.0 (Figures S46-S48). (B) Emission spectra of 2.0 μM irinotecan 12⁺ in 30 mM PBS at pH = 7.0 (λ_ex = 380 nm) obtained after an incremental addition of CBC-11. The binding isotherm corresponding to fluorescence intensity of irinotecan 12⁺ as a function of increasing proportion of CBC-11 was fit to 1:1 stoichiometric model to give K_a = 7.7 ± 0.9 · 10⁴ M⁻¹; a random distribution of residuals is shown on top.

Figure 5.

(A) A pictorial representation of van der Waals surface of energy-minimized [13₄⊂CBC-11] (OPLS4). (Middle) Emission spectra of 1.0 μM doxorubicin 13⁺ in 30 mM PBS at pH = 7.0 (λ_ex = 500 nm) obtained after incremental addition of CBC-11. (Right) The binding isotherm corresponding to fluorescence intensity of doxorubicin 13⁺ as a function of increasing proportion of CBC-11 was fit to 1:1 stoichiometric model (supramolecular.org) to give K_a = 1.8 ± 0.1 · 10⁵ M⁻¹; the distribution of residuals is shown on top (see also Figure S56). (B) Dynamic light scattering data from 12 μM solution of CBC-11 in 30 mM PBS at pH = 7.0 in the presence of 0, 12, 48, 72 and 96 μM of 13⁺. (C) Luminescent intensity of HCT 116 cell line in a medium containing resazurin dye (i.e., CellTiter-Blue cell viability assay) as a function of CBC-11 concentration after 48 hours of incubation. The result of a control experiment without cells (death control) is shown in red; for other controls see SI. Each point represents a mean of four measurements with standard deviation as an error.

Scheme 1.

A scheme showing the synthesis of covalent basket cages (CBC); for clarity, only a representative part of chemical structures is shown. (Left, bottom) Energy-minimized structure (OPLS-4) of CBEC-11 holding three thio-ether chains. (Right, bottom) Energy-minimized structure (OPLS4; Monte Carlo) of the most stable conformer of the model compound holding a thio-ether chain.