Engineering a customized nanodrug delivery system at the cellular level for targeted cancer therapy

Abstract

Drug administration customized to individual cells could intrinsically address cancer heterogeneity and provide a safe and effective method for delivering personalized treatment. To accomplish this, we developed a smart nanodrug delivery system characterized by cancer cell-targeted drug delivery and intracellular biomarker-responsive drug activation. This system was composed of a long-nicked DNA duplex formed by tandem hybridization of two extended antisense oligonucleotides whose ends were separately blocked with a cancer cell-specific aptamer, AS1411, and a replaceable anti-biomarker probe (ABP). We demonstrated that this DNA nanodrug was directed to cancer cells with the guidance power of AS1411 and then activated by the presence of a given intracellular biomarker. By using such a belt-and-braces strategy, this DNA nanodrug system could safely and efficiently accelerate apoptosis of target cancer cells. Moreover, since the expression level of biomarkers tends to indicate the specific physiological state of individual cells, biomarker-responsive activation of the nanodrug is expected to enable customized drug administration at the cellular level.

Keywords: antisense oligonucleotides; aptamer; drug delivery; personalized medicine.

Figure 1

(a) Formation of nanodrugs characterized by atomic force microscope (AFM); (b) biomarker-responsive dissociation of the Apt-ND-ABP confirmed with 1.5% agarose gel electrophoresis. From Lanes 1 to 5: Apt-ND-ABP; Apt-ND-ABP+TA+TB; Apt-ND-ABP+TA+TB+BM; Apt-ND-rd; Apt-ND-rd+TA+TB+BM. Each sample was incubated at 37 °C for 1 h and then analyzed by agarose gel electrophoresis. The concentrations of A, B, TA, TB and BM were all fixed at 1 μM (color online).

Figure 2

Cell-specific binding and internalization of Apt-ND-ABP. Flow cytometry confirms the specific binding of Apt-ND-ABP to target A549 cells (a), but not control HBE cells (b). (c) CLSM images of A549 cells incubated with Apt-ND-ABP (i) and Lib-ND-ABP (ii) at 37 °C for 2 h. (d) CLSM images of HBE cells incubated with Apt (i) and Apt-ND-ABP (ii) at 37 °C for 2 h. The excitation wavelength of the FITC fluorophore was 488 nm (color online).

Figure 3

Biomarker-responsive feasibility of Apt-ND-ABP in cells. CLSM images of A549 cells incubated with Apt-ND-ABP (a) and Apt-ND-rd (b) at 37 °C for 2 h. From left to right: fluorescence image of Cy3, FRET fluorescence image of Cy5, and bright field. The 543-nm laser was used for excitation. (c) FRET ratio calculated with ImageJ software accordingly. (d) FRET ratio calculated from the CLSM images of the A549 cells separately transfected with different amounts of BM (black line) and anti-BM (red line) prior to incubation with Apt-ND-ABP (color online).

Figure 4

Viability of A549 cells treated with Apt-ND-rd, Apt-ND-ABP, Lib-ND-ABP, Apt-control-ABP, and Apt-ND, respectively. Error bars represent the standard deviation of three independent experiments (color online).

Scheme 1

Schematic illustration of the biomarker-responsive nanodrug delivery system for targeted cancer therapy at the cellular level. Apt-ND-ABP is composed of the nanodrug with two ends separately blocked with aptamer AS1411 and Anti-biomarker probe ABP. Apt-ND-ABP passes through the cell membrane guided by AS1411. In the absence of biomarker/ABP interaction, the nanodrug is quiescent. However, upon sensing the biomarker as an indicator of the specific physiological state of individual cells, ABP is removed by formation of a biomarker/ABP complex, followed by exposure of the nanodrug toehold for access to therapeutic target miRNAs (miRNA-21 and miRNA-150) for cell apoptosis (color online).