Novel aptamer-nanoparticle bioconjugates enhances delivery of anticancer drug to MUC1-positive cancer cells in vitro

Abstract

MUC1 protein is an attractive target for anticancer drug delivery owing to its overexpression in most adenocarcinomas. In this study, a reported MUC1 protein aptamer is exploited as the targeting agent of a nanoparticle-based drug delivery system. Paclitaxel (PTX) loaded poly (lactic-co-glycolic-acid) (PLGA) nanoparticles were formulated by an emulsion/evaporation method, and MUC1 aptamers (Apt) were conjugated to the particle surface through a DNA spacer. The aptamer conjugated nanoparticles (Apt-NPs) are about 225.3 nm in size with a stable in vitro drug release profile. Using MCF-7 breast cancer cell as a MUC1-overexpressing model, the MUC1 aptamer increased the uptake of nanoparticles into the target cells as measured by flow cytometry. Moreover, the PTX loaded Apt-NPs enhanced in vitro drug delivery and cytotoxicity to MUC1(+) cancer cells, as compared with non-targeted nanoparticles that lack the MUC1 aptamer (P<0.01). The behavior of this novel aptamer-nanoparticle bioconjugates suggests that MUC1 aptamers may have application potential in targeted drug delivery towards MUC1-overexpressing tumors.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Construction of the S2.2-spacer aptamer and PTX-Apt-NPs.

(A) Structure of MUC1 aptamer S2.2-spacer. The S2.2-spacer is made of the MUC1 aptamer S2.2 (as the targeting agent) and a designed sequence of single strand DNA (as the linking spacer). (B) Preparation procedure for PTX-Apt-NP using the emulsion/evaporation method.

Figure 2. FCM analysis of MCF-7 and HepG2 cells incubated with random DNA, MUC1 aptamer S2.2, or S2.2-spacer.

(A) Histograms of FCM analysis. FITC-labeled random DNA, S2.2, or S2.2-spacer was incubated separately with MCF-7 (left) and HepG2 (right) cells. (B) The mean fluorescent intensity of MCF-7 and HepG2 cells incubated with random DNA and different aptamers.

Figure 3. Analysis of aptamer conjugation to microparticles or nanoparticles.

The PLGA microparticles (MPs) or nanoparticles (NPs) reacted with aptamers in the absence (-EDC&NHS) or presence (+EDC&NHS) of the catalysts. (A) Histograms of FCM analysis of MP reacted with FITC-labeled aptamers in the absence (-EDC&NHS, left) or presence (+EDC&NHS, right) of the catalysts. Plain microparticles that had not reacted with aptamers were used as control. (B) UV absorption of DNA at 260 nm (n = 3) on the particles that underwent conjugation process with aptamers. The control group (NPs) indicates plain nanoparticles that have not reacted with DNA aptamers.

Figure 4. In vitro drug release profile of the PTX encapsulated Apt-NPs.

The experiment was conducted in phosphate buffer saline (PBS, pH 7.4) containing 0.5% (w/v) poloxamer using the membrane diffusion technique, (n = 3).

Figure 5. FCM analysis of cellular uptake of Apt-NPs and NPs by MCF-7 (left) and HepG2 cells (right).

The cells were incubated with FITC encapsulated Apt-NPs or NPs at 50 µg/ml for 2 hours before subject to analysis.

Figure 6. Confocal fluorescent scanning microscopy images detecting cellular uptake of Apt-NPs (top row) or NPs (bottom row) in MCF-7 cells.

Green fluorescent FITC was encapsulated in Apt-NPs and NPs. The nuclei were stained blue with DAPI. The right column showed the merged images of the FITC and the DAPI channels. MCF-7 cells were exposed to FITC-encapsulated Apt-NPs or NPs at 100 µg/ml for 2 hours.

Figure 7. Cytotoxicity assays of MCF-7 (Left) and HepG2 cells (Right) treated for 4 hours with plain nanoparticles (NP), free paclitaxel (PTX), PTX-loaded NPs (PTX-NP), PTX-loaded NPs conjugated to random DNA (PTX-R-NP), or PTX-loaded NPs conjugated to MUC1 aptamer (PTX-Apt-NP).

The cells were subsequently washed and incubated in culture media for a total of 48 hours, before cell viability in each group was assessed with a standard MTS assay (n = 6, mean ± SD).