A Unique Core-Shell Structured, Glycol Chitosan-Based Nanoparticle Achieves Cancer-Selective Gene Delivery with Reduced Off-Target Effects

Abstract

The inherent instability of nucleic acids within serum and the tumor microenvironment necessitates a suitable vehicle for non-viral gene delivery to malignant lesions. A specificity-conferring mechanism is also often needed to mitigate off-target toxicity. In the present study, we report a stable and efficient redox-sensitive nanoparticle system with a unique core-shell structure as a DNA carrier for cancer theranostics. Thiolated polyethylenimine (PEI-SH) is complexed with DNA through electrostatic interactions to form the core, and glycol chitosan-modified with succinimidyl 3-(2-pyridyldithio)propionate (GCS-PDP) is grafted on the surface through a thiolate-disulfide interchange reaction to form the shell. The resulting nanoparticles, GCS-PDP/PEI-SH/DNA nanoparticles (GNPs), exhibit high colloid stability in a simulated physiological environment and redox-responsive DNA release. GNPs not only show a high and redox-responsive cellular uptake, high transfection efficiency, and low cytotoxicity in vitro, but also exhibit selective tumor targeting, with minimal toxicity, in vivo, upon systemic administration. Such a performance positions GNPs as viable candidates for molecular-genetic imaging and theranostic applications.

Keywords: gene delivery; molecular-genetic imaging; reporter–probe pair; systemic delivery; toxicity.

Scheme 1

Schematic illustration showing the preparation (A) and cellular trafficking (B) of GNPs.

Figure 1

GCS-PDP/PEI-SH/DNA NP maintains stability under different conditions. (A) Hydrodynamic size changes of GNPs in different media; (B) hydrodynamic size change of PEI/DNA and GNPs in 10 mM TBS buffer at 37 ℃ over 30 days) (* indicates PEI/DNA complex aggregation, with polydispersity index = 0.806); (C) agarose gel electrophoresis assay of GNPs in the presence of heparin and 10% FBS; and (D) agarose gel electrophoresis assay of GNPs in the presence of DNase I. Each bar represents mean ± standard deviation (n = 3).

Figure 2

GNPs release DNA selectively in a high reducing environment. (A) The hydrodynamic size changes of GNPs in media of high redox potential. The ordinate represents the weight ratio of GCS-PDP to PEI-SH to DNA. Each bar represents mean ± standard deviation (n = 3); (B) agarose gel electrophoresis assay of GNPs and GCS-ss-PEI/DNA NPs in the presence of DTT and heparin; and (C) typical TEM images of GNPs before (a) and after (b) co-incubation with DTT and heparin (scale bar =100 nm).

Figure 3

Uptake of GNP (1/1/1) in PC3 cells. (A) Confocal microscopy indicates the cellular distribution of GNPs and PEI/DNA NPs in PC3 cells after 4 h of incubation. GCS-PDP was labeled with Cy5.5 and DNA was labeled with YOYO-1; (B) FACS indicates the uptake of GNPs in PC3 cells at different time points; and (C) confocal images of PC3 cells after co-incubation with GNPs for 1 h. Small white arrows point to plasmids that are not in the lysotracker-positive compartment (lysosomes), and (D) the effect of intracellular GSH level on the transfection efficacy of redox-sensitive nanoparticles (data are analyzed by unpaired Student’s t test, * p < 0.05).

Figure 4

Optimization of GNPs to achieve high transfection efficiency and low cytotoxicity in PC3 cells. (A) In vitro transfection efficiency of different formulations measured by bioluminescence; (B) cytotoxicity of different formulations as determined by MTT assay; and (C,D) the percentage of early apoptotic and necrotic cells after treatment with GNPs or PEI/DNA NPs was quantified by flow cytometry. Each bar represents mean ± standard deviation. All data are analyzed by one-way ANOVA and Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Figure 5

Transgene expression of different formulations in an experimental model of metastatic prostate cancer. (A) In vivo BLI of PC3-ML tumor-bearing mice after treatment with GNP and in vivo jetPEI/DNA NPs for 72 h; (B) ex vivo BLI images of in vivo jetPEI, 1/1/1, 1/1.5/1 and 1/2/1 GNPs treated mice; and (C–E) summary of luciferase expression level in the lung (C), liver (D), and kidney (E) at 72 h (n = 3–5 mice per group).

Figure 6

Complete blood analyses and clinical chemistry parameters of BALB/c (A) and CD-1 (B) mice treated with in vivo JetPEI or NPs (1/1.5/1 and 1/2/1). White blood cell (WBC), red blood cell (RBC), platelet (PLT), blood urea nitrogen (BUN), glucose (GLU), alkaline phosphatase (ALP), total protein (T-Pro), alanine aminotransferase (ALT), and creatinine (Cre) were measured (n = 5–7 mice per group; and data were analyzed by one-way ANOVA; ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Figure 7

Histopathology of various NPs in BALB/c and CD1 mice. (A) H&E staining of liver from Balb/c mice treated with in vivo jetPEI/DNA NPs and GNPs (1/1.5/1, 1/2/1) under 10× (A) and 1.25× magnification (B); (C) H&E staining of liver from CD1 mice treated with in vivo jetPEI/DNA NPs and GNPs (1/1.5/1, 1/2/1) NPs under 20× magnification. Scale bar: 200 µm.