Targeting Cancer Cells Overexpressing Folate Receptors with New Terpolymer-Based Nanocapsules: Toward a Novel Targeted DNA Delivery System for Cancer Therapy

Abstract

Chemotherapeutics represent the standard treatment for a wide range of cancers. However, these agents also affect healthy cells, thus leading to severe off-target effects. Given the non-selectivity of the commonly used drugs, any increase in the selective tumor tissue uptake would represent a significant improvement in cancer therapy. Recently, the use of gene therapy to completely remove the lesion and avoid the toxicity of chemotherapeutics has become a tendency in oncotherapy. Ideally, the genetic material must be safely transferred from the site of administration to the target cells, without involving healthy tissues. This can be achieved by encapsulating genes into non-viral carriers and modifying their surface with ligands with high selectivity and affinity for a relevant receptor on the target cells. Hence, in this work we evaluate the use of terpolymer-based nanocapsules for the targeted delivery of DNA toward cancer cells. The surface of the nanocapsules is decorated with folic acid to actively target the folate receptors overexpressed on a variety of cancer cells. The nanocapsules demonstrate a good ability of encapsulating and releasing DNA. Moreover, the presence of the targeting moieties on the surface of the nanocapsules favors cell uptake, opening up the possibility of more effective therapies.

Keywords: DNA delivery; active targeting; cancer; folic acid; gene therapy; nanocapsules; polymeric nanoparticles; terpolymer.

Figure 1

Schematic of the coupling reaction between folic acid (FA) and terpolymer-based nanoparticles (NPs). Activation of carboxylic groups of FA (chemical structure shown at the top right) by EDC/NHS; formation of covalent bonds among activated esters of FA and tertiary amine of DMAEMA monomer units of terpolymer-based nanoparticles.

Figure 2

(a) Percentage conversion for each monomeric unit; SEM representative micrographs of (b) PMMA sacrificial core, (c) NCs before and (d) after extraction of the core.

Figure 3

FT-IR analysis. (a) Spectrum of folic acid; (b) comparison among the spectra of pure terpolymer-based NCs, functionalized NCs, and NCs after adsorption of FA before extraction and (c) a magnification; (d) comparison between of pure terpolymer-based NCs, functionalized NCs, and NCs after adsorption of FA after removal procedure, and (e) a magnification.

Figure 4

In vitro cytotoxicity tests. (a) Cytotoxicity MTT assay of functionalized NCs after 24- and 72-h incubation with NIH3T3 cells. A comparison with negative control is shown; (b) Propidium Iodide Flow Cytometry assay of functionalized NCs after 24- and 72-h incubation with HeLa cells. A comparison with negative control is shown; (c) Propidium Iodide Flow Cytometry assay of functionalized NCs after 24- and 72-h incubation with NIH3T3 cells. A comparison with negative control is shown (* p < 0,05; t-test; mean ± SD; n = 3).

Figure 5

DNA adsorption and release assay. (a) DNA adsorption into non-functionalized and functionalized NCs; (b) percentage cumulative release of DNA from functionalized NCs. The inset represents a comparison of DNA released from functionalized NCs and non-functionalized NCs expressed as total mass released.

Figure 6

Cell uptake test. Fluorescence-activate cell sorting (FACS) analysis representing the dot plots of physical cell parameters and histogram plots of fluorescence emitting HeLa cells after (a) 6-h and (b) 24-h incubation with functionalized and non-functionalized NCs. Black histograms represent cells stained with 100 μg/mL⁻¹ NCs. Gray histograms represent unstained control cells; (c) confocal microscopy images of HeLa cells after 24 h of incubation with 100 μg mL⁻¹ functionalized and non-functionalized NCs. Scale bar 20 μm.