Dually Responsive Nanoparticles for Drug Delivery Based on Quaternized Chitosan

Abstract

In this work, we report the fabrication and functional demonstration of a kind of dually responsive nanoparticles (NPs) as a potential drug delivery vector. The pH value, corresponding to the acidic microenvironment at the tumor site, and mannitol, to the extracellular trigger agent, were employed as the dually responsive factors. The function of dual responses was achieved by breaking the dynamic covalent bonds between phenylboronic acid (PBA) groups and diols at low pH value (pH 5.0) and/or under the administration of mannitol, which triggered the decomposition of the complex NPs and the concomitant release of anticancer drug of doxorubicin (DOX) loaded inside the NPs. The NPs were composed of modified chitosan (PQCS) with quaternary ammonium and PBA groups on the side chains, heparin (Hep), and poly(vinyl alcohol) (PVA), in which quaternary ammonium groups offer the positive charge for the cell-internalization of NPs, PBA groups serve for the formation of dynamic bonds in responding to pH change and mannitol addition, PVA furnishes the NPs with diol groups for the interaction with PBA groups and the formation of dynamic NPS, and Hep plays the roles of reducing the cytotoxicity of highly positively-charged chitosan and forming of complex NPs for DOX up-loading. A three-step fabrication process of drug-loaded NPs was described, and the characterization results were comprehensively demonstrated. The sustained drug release from the drug-loaded NPs displayed obvious pH and mannitol dependence. More specifically, the cumulative DOX release was increased more than 1.5-fold at pH 5.0 with 20 mg mL^-1 mannitol. Furthermore, the nanoparticles were manifested with effective antitumor efficient and apparently enhanced cytotoxicity in response to the acidic pH value and/or mannitol.

Keywords: chitosan; drug delivery; dual response; dynamic bond; nanoparticle.

Figure 1

A schematic illustration of the procedures for the preparation of DOX-loaded nanoparticles (NPs), the mechanism of the dually responsive drug release, and cellular uptake of NPs. QCS = quaternized chitosan; PQCS = PBA-modified QCS; PBA = phenylboronic acid; Hep = heparin; PVA = poly(vinyl alcohol); DOX = doxorubicin.

Figure 2

(a) ¹H NMR spectrum for PQCS in D₂O, the inset shows the chemical structure of PQCS. The solvent peak are marked with asterisk (*). (b) Fluorescence spectra of AR (0.034 mg mL⁻¹)/PQCS PBS solutions, excitation: 468 nm; (c) photograph of AR (0.034 mg mL⁻¹)/PQCS aqueous solutions.

Figure 3

Characterization of DOX-loaded Hep/PQCS-PVA NPs. (a) Z-average sizes and zeta potentials of Hep/PQCS aqueous solutions, (b) DOX-loaded Hep/PQCS-PVA aqueous solutions, and (c) DOX-loaded Hep/PQCS-PVA multilayer NPs in aqueous solutions measured by DLS; (d) The particle size of DOX-loaded Hep/PQCS-PVA NPs aqueous solutions measured by dynamic light scattering (DLS) for three times. The concentration of each solution: 0.1 mg mL⁻¹.

Figure 4

TEM images of the DOX-loaded Hep/PQCS-PVA NPs, the samples came from (a) deionized water and (b) DMEM. The images on the right are taken in high resolution mode.

Figure 5

Dually responsive DOX release in vitro. (a,b) TEM images of DOX-loaded Hep/PQCS-PVA NPs in DMEM solutions at pH 5.0 and in the presence of mannitol (20 mg mL⁻¹). (c,d) Cumulative release of DOX from the DOX-loaded Hep/PQCS-PVA NPs with or without treatment with 20 mg mL⁻¹ mannitol at pH 7.4 or pH 5.0. **: p < 0.01, ***: p < 0.001.

Figure 6

Cell viability measured by MTT assay. Cell viability of DOX-loaded Hep/PQCS-PVA NPs and free DOX toward Hep G2 cells at (a) 1st day and (b) 3rd day. (c) Cell viability of empty Hep/PQCS-PVA NPs toward Hep G2 cells, HeLa cells, and FIB cells. (d) Cell viability of DOX-loaded Hep/PQCS-PVA NPs toward Hep G2 cells with or without treatment with 20 mg mL⁻¹ mannitol at pH 7.4 or pH 5.0. *: p < 0.05, **: p < 0.01, ***: p < 0.001.