Evaluation of Chitosan Derivative Microparticles Encapsulating Superparamagnetic Iron Oxide and Doxorubicin as a pH-Sensitive Delivery Carrier in Hepatic Carcinoma Treatment: An in vitro Comparison Study

Abstract

We developed a novel, pH-sensitive drug delivery microparticle based on N-palmitoyl chitosan (NPCS) to transport the superparamagnetic iron oxide (SPIO) and anticancer drug doxorubicin (DOX). The characteristics of NPCS were characterized through nuclear magnetic resonance. Our results based on testing of volume swelling in multiple pH aqueous solutions revealed that the modified chitosan had a pH-sensitive property. The morphology and size of the DOX-SPIO/NPCS microparticles were investigated using transmission electron microscopy and scanning electron microscopy. The statistical result of microparticles had diameter of 185 ± 87 nm. Surface chemical moieties of DOX-SPIO/NPCS microparticles were confirmed using attenuated total reflection Fourier transform infrared spectroscopy and indicated the existence of mostly hydrophilic groups such as -OH, -C=O, and -C-O-C-. Transmission electron microscopy revealed the dark contrast of SPIO dots encapsulated in the NPCS matrix. Nuclear magnetic resonance T2-weighted magnetic resonance imaging confirmed that the produced DOX-SPIO/NPCS microparticles still exhibited T2 relaxation durations as short as 37.68 ± 8.69 ms (under administration of 2.5 μg/mL), which is comparable to the clinically required dosage. In the drug release profile, the DOX-SPIO/NPCS drug delivery microparticle was accelerated in an acidic environment (pH 6.5) compared with that in a basic environment. Microparticles in a cytotoxicity assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay) revealed that DOX-SPIO/NPCS microparticles had better antitumor ability than did free-form of DOX. Additionally, microparticles loaded with 0.5-5 μg/mL DOX in an acidic environment (pH 6.5) demonstrated higher efficacy against Hep G2 cell growth, possibly because of the swelling effect of NPCS, resulting in volume expansion and easy drug release. Accordingly, these large DOX-SPIO/NPCS microparticles showed potential for application as a pH-sensitive drug delivery system and as chemoembolization particles for hepatic carcinoma therapy.

Keywords: doxorubicin; electrospray; microparticle; nanotechnology; superparamagnetic iron oxide.

Scheme 1

Synthetic procedure of N-palmitoyl chitosan.

Figure 1

¹H NMR spectrum of (A) N-hydroxysuccinimide ester, and (B) N-palmitoyl chitosan. All of the samples were dissolved formic acid-d2 (95% w/w in D₂O).

Figure 2

(A) schematic illustration of the plausible swelling mechanism of the produced DOX-SPIO/NPCS microparticle, and (B) pH-dependent of statistical results of volume change of NPCS gel droplets measured at different time-point. The volume changing ration can be seen as an index for evaluating the swelling degree of NPCS. **p < 0.01, and ***p < 0.001, respectively.

Figure 3

Fourier-transform infrared (FT-IR) spectra of (A) Chitosan powder, (B) palmitic acid N-hydroxysuccinimide ester powder, (C) NPCS powder, (D) DOX powder, and (E) DOX-SPIO/NPCS microparticles.

Figure 4

SEM images and statistic bar charts of DOX-SPIO/NPCS microparticle prepared under (A,C,E) formic acid, (B,D,F) formic acid/acetone solvent system.

Figure 5

TEM images of (A,C) DOX/NPCS, (B,D) DOX-SPIO/NPCS microparticles prepared under parameters as follows: working distance 6.5 cm high, applied voltage ranged from 10 to 17 kV, and flow rate 2 μL/min.

Figure 6

Signals of T2-weighted FSE MRI images from a series samples: (A) a series of DOX solution with concentration ranging from 0.5 to 62.5 μg/mL, (B) a series of DOX-SPIO/NPCS microparticle suspension with concentration ranging from 0 to 0.5 μg/mL and (C) quantatative value of T2 relaxation time.

Figure 7

In vitro drug-release profiles of (•) DOX, and (▴) DOX-SPIO/NPCS microparticle soaked in PBS solution at pH 7.4 and 37°C for up to 72 h.

Figure 8

DOX-SPIO/NPCS microparticle suspension effectively suppressed hepatic cancer cell proliferation at concentrations of 0.5–50 μg/mL as compared to that of free form of DOX: (A) free form of DOX, and (B) DOX-SPIO/NPCS microparticle suspension. The same concentration of free DOX was also tested simultaneously for comparison. All cell viabilities shown in this figure are normalized to the negative control (NC) group treated with equivalent amounts of serum-free DMEM medium. n = 3 was tested for each group of trial. **p < 0.01 and ***p < 0.001, respectively.