Poly(2-(dimethylamino)ethyl methacrylate)-Grafted Amphiphilic Block Copolymer Micelles Co-Loaded with Quercetin and DNA

Abstract

The synergistic effect of drug and gene delivery is expected to significantly improve cancer therapy. However, it is still challenging to design suitable nanocarriers that are able to load simultaneously anticancer drugs and nucleic acids due to their different physico-chemical properties. In the present work, an amphiphilic block copolymer comprising a biocompatible poly(ethylene glycol) (PEG) block and a multi-alkyne-functional biodegradable polycarbonate (PC) block was modified with a number of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) side chains applying the highly efficient azide-alkyne "click" chemistry reaction. The resulting cationic amphiphilic copolymer with block and graft architecture (MPEG-b-(PC-g-PDMAEMA)) self-associated in aqueous media into nanosized micelles which were loaded with the antioxidant, anti-inflammatory, and anticancer drug quercetin. The drug-loaded nanoparticles were further used to form micelleplexes in aqueous media through electrostatic interactions with DNA. The obtained nanoaggregates-empty and drug-loaded micelles as well as the micelleplexes intended for simultaneous DNA and drug codelivery-were physico-chemically characterized. Additionally, initial in vitro evaluations were performed, indicating the potential application of the novel polymer nanocarriers as drug delivery systems.

Keywords: DNA; click; codelivery; graft copolymer; metabolic activity; micelleplex; multifunctional; quercetin.

Scheme 1

Synthetic path towards (a) azido-end functional PDMAEMA; (b) cationic amphiphilic graft copolymer MPEG-b-(PC-g-PDMAEMA).

Figure 1

¹H NMR spectra in CDCl₃ of (a) azide end-functional polymer (PDMAEMA-N₃); (b) alkyne-multifunctional amphiphilic block copolymer backbone (MPEG-b-PC); and (c) cationic amphiphilic graft copolymer MPEG-b-(PC-g-PDMAEMA).

Figure 2

CMC determination for MPEG-b-(PC-g-PDMAEMA) graft copolymer: I₁/I₃ ratios from pyrene excitation spectra vs. copolymer concentration.

Figure 3

Size distributions (a) and zeta potentials (b) obtained from dynamic light scattering measurements of aqueous dispersions of empty micelles (M: d = 157.91 ± 2.42 nm, PdI: 0.221, ζ = 20.08 ± 3.73 mV), quercetin-loaded micelles (M/Que: d = 126.88 ± 2.26 nm, PdI: 0.312, ζ = 21.42 ± 4.10 mV), and quercetin-loaded micelleplexes prepared at N/P = 10:1 (MP 10:1: d = 91.80 ± 1.49 nm, PdI: 0.173, ζ = 21.64 ± 1.65 mV).

Figure 4

TEM micrographs of (a) graft copolymer micelles (M). The inset shows the quercetin-loaded micelle (M/Que) and (b) quercetin-loaded micelleplexes obtained at N/P = 10:1 (MP 10:1).

Figure 5

In vitro release profiles in phosphate buffer (PBS, pH 7.4) containing 10% (v/v) ethanol of (a) free quercetin (Que) and (b) quercetin from the cationic graft copolymer micelles (M/Que). The data are expressed as mean value ± SD, n = 3.

Figure 6

Ethidium bromide displacement assay for miclelleplexes prepared at different N/P ratios. The data are expressed as mean value ± SD, n = 3.

Figure 7

Stability of quercetin-loaded micelleplexes (MP 10:1) in aqueous media assessed using DLS measurements as a function of incubation time.

Figure 8

Cell metabolic activity defined via the MTT test after 24 h of incubation of HepG2 cells treated with different concentrations of (a) empty cationic graft copolymer micelles (M); (b) free (Que) and micellar (M/Que) quercetin; and (c) quercetin-loaded micelleplex obtained at N/P = 10:1 (MP 10:1). The data are presented as percentages of untreated controls and are expressed as mean value ± SE, n = 3.