Complementary Nucleobase Interactions Drive Co-Assembly of Drugs and Nanocarriers for Selective Cancer Chemotherapy

Abstract

A new concept in cooperative adenine-uracil (A-U) hydrogen bonding interactions between anticancer drugs and nanocarrier complexes was successfully demonstrated by invoking the co-assembly of water soluble, uracil end-capped polyethylene glycol polymer (BU-PEG) upon association with the hydrophobic drug adenine-modified rhodamine (A-R6G). This concept holds promise as a smart and versatile drug delivery system for the achievement of targeted, more efficient cancer chemotherapy. Due to A-U base pairing between BU-PEG and A-R6G, BU-PEG has high tendency to interact with A-R6G, which leads to the formation of self-assembled A-R6G/BU-PEG nanogels in aqueous solution. The resulting nanogels exhibit a number of unique physical properties, including extremely high A-R6G-loading capacity, well-controlled, pH-triggered A-R6G release behavior, and excellent structural stability in biological media. Importantly, a series of in vitro cellular experiments clearly demonstrated that A-R6G/BU-PEG nanogels improved the selective uptake of A-R6G by cancer cells via endocytosis and promoted the intracellular release of A-R6G to subsequently induce apoptotic cell death, while control rhodamine/BU-PEG nanogels did not exert selective toxicity in cancer or normal cell lines. Overall, these results indicate that cooperative A-U base pairing within nanogels is a critical factor that improves selective drug uptake and effectively promotes apoptotic programmed cell death in cancer cells.

Keywords: adenine–uracil base pair; complementary hydrogen bonded drug carrier system; controlled drug delivery; selective cytotoxicity; supramolecular nanogels.

Scheme 1

Schematic illustration of the co-assembly process, fluorescent properties, and cancer cell-selective cytotoxic behavior of hydrogen-bonded A-R6G/BU-PEG complexes. The upper-right green and red arrows represent the association and dissociation of the complementary A–U base pairs under various pH conditions.

Figure 1

(a) Determination of the CMC values of PEG and BU-PEG in water in the presence of pyrene. (b) Hydrodynamic particle size of the BU-PEG polymer in water as determined by DLS at 25 °C. Surface morphologies of spin-coated BU-PEG polymers obtained by (c) AFM and (d) SEM at 25 °C.

Figure 2

(a) AMF images of A-R6G-loaded BU-PEG nanogels measured at 25 °C. (b) PL spectra of A-R6G-loaded and R6G-loaded BU-PEG nanogels in water at 25 °C. Inset: photographs of A-R6G-loaded and R6G-loaded BU-PEG nanogels in water exposed to (1) natural lighting and (2) broadband UV lighting conditions. (c) Time-dependent DLS analysis of the structural stability of A-R6G-loaded and R6G-loaded BU-PEG nanogels in media containing 10% FBS at pH 7.4 for 24 h. (d) In vitro hemolytic assay of different concentrations of A-R6G-loaded and R6G-loaded BU-PEG nanogels on SRBCs. Inset: photographs showing SRBCs incubated with varying concentrations (1–100 μg/mL) of A-R6G-loaded or R6G-loaded BU-PEG nanogels at 37 °C in 5% CO₂ for 4 h.

Figure 3

In vitro drug release profiles of (a) A-R6G-loaded and (b) R6G-loaded BU-PEG nanogels. In vitro cytotoxicity of A-R6G-loaded and R6G-loaded BU-PEG nanogels against (c) NIH/3T3 cells and (d) HeLa cells after 24 h incubation.

Figure 4

CLSM images of (a) NIH/3T3 cells and (b) HeLa cells cultured with A-R6G-loaded BU-PEG nanogels at normal physiological conditions (pH 7.4 and 37 °C) for 3, 12, or 24 h. The scale bars in all CLSM images are 20 µm.

Figure 5

Flow cytometry histograms of (a) NIH/3T3 cells and (b) HeLa cells cultured with A-R6G-loaded or R6G-loaded BU-PEG nanogels at normal physiological conditions (pH 7.4 and 37 °C) for 1, 3, 6, 12, or 24 h.

Figure 6

Flow cytometric dot plot quadrant charts of NIH/3T3 and HeLa cells cultured with A-R6G-loaded and R6G-loaded BU-PEG nanogels at normal physiological conditions (pH 7.4 and 37 °C) for 1, 3, 6, or 12 h before staining with BV421 Annexin V and GDR780. Figures (a)–(p) represent the results of flow cytometry for different time points during the co-culture period. The graph quadrants from the lower left to the upper left (turning anti-clockwise) represent viable cells (BV421 Annexin V^-, GDR780⁻), early apoptotic cells (BV421 Annexin V⁺, GDR780⁻), late apoptotic cells (BV421 Annexin V⁺, GDR780⁺), and necrotic cells (BV421 Annexin V⁻, GDR780⁺). The numbers inside each quadrant refer to the proportions of cells.