Theranostic nanoparticles based on bioreducible polyethylenimine-coated iron oxide for reduction-responsive gene delivery and magnetic resonance imaging

Abstract

Theranostic nanoparticles based on superparamagnetic iron oxide (SPIO) have a great promise for tumor diagnosis and gene therapy. However, the availability of theranostic nanoparticles with efficient gene transfection and minimal toxicity remains a big challenge. In this study, we construct an intelligent SPIO-based nanoparticle comprising a SPIO inner core and a disulfide-containing polyethylenimine (SSPEI) outer layer, which is referred to as a SSPEI-SPIO nanoparticle, for redox-triggered gene release in response to an intracellular reducing environment. We reveal that SSPEI-SPIO nanoparticles are capable of binding genes to form nano-complexes and mediating a facilitated gene release in the presence of dithiothreitol (5-20 mM), thereby leading to high transfection efficiency against different cancer cells. The SSPEI-SPIO nanoparticles are also able to deliver small interfering RNA (siRNA) for the silencing of human telomerase reverse transcriptase genes in HepG2 cells, causing their apoptosis and growth inhibition. Further, the nanoparticles are applicable as T2-negative contrast agents for magnetic resonance (MR) imaging of a tumor xenografted in a nude mouse. Importantly, SSPEI-SPIO nanoparticles have relatively low cytotoxicity in vitro at a high concentration of 100 μg/mL. The results of this study demonstrate the utility of a disulfide-containing cationic polymer-decorated SPIO nanoparticle as highly potent and low-toxic theranostic nano-system for specific nucleic acid delivery inside cancer cells.

Keywords: MR imaging; RNA interference; SSPEI; disulfide; hTERT; nanoparticles; tumor.

Figure 1

Schematic illustration of SSPEI-SPIO nanoparticles, which efficiently bind genes and mediate subsequent gene release triggered by an intracellular reducing environment. Abbreviations: SPIO, superparamagnetic iron oxide; SSPEI, disulfide-containing polyethylenimine; siRNA, small interfering RNA.

Figure 2

(A) Illustration of the structure and component of SSPEI-SPIO nanoparticles; (B) atom force microscopy images of SSPEI60-SPIO nanoparticles; (C) size distribution of SSPEI60-SPIO nanoparticles in deionized water; (D) Fourier transform infrared spectra of SSPEI, SPIO and SSPEI60-SPIO nanoparticles; (E) size distribution of SSPEI60-SPIO/DNA complexes at a mass ratio of 10:1 in HEPES buffer (20 mM, pH 7.4). Abbreviations: SPIO, superparamagnetic iron oxide; SSPEI, disulfide-containing polyethylenimine; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane sulfonic acid; PAA, poly(acrylic acid); PDI, polydispersity index.

Figure 3

(A) Agarose gel retardation analysis of the complexes of SSPEI12-SPIO, SSPEI20-SPIO, and SSPEI60-SPIO at different mass ratios; (B) agarose gel retardation analysis of the complexes of SSPEI60-SPIO in 5 or 20 mM DTT; (C) EB accessibility assay of complexes of SSPEI60-SPIO/DNA at different mass ratios in the absence (w/o) and presence (w/) of 20 mM DTT. Abbreviations: SSPEI, disulfide-containing polyethylenimine; SPIO, super para-magnetic iron oxide; DTT, dithiothreitol; EB, ethidium bromide; w/o, without; w/, with.

Figure 4

(A) Cytotoxicity evaluation of SSPEI-SPIO nanoparticles, SSPEI, and branched PEI against MCF-7 cells at varying concentrations. The percentage of relative cell viability was determined relative to control cells (untreated), taken as 100% cell viability (***P<0.001, SSPEI-SPIO, SSPEI versus PEI); (B,C) Gene expression levels and cell viability afforded by SSPEI60–SPIO/DNA in MCF-7 cells in vitro as a function of the mass ratios. The polyplexes of branched PEI at a mass ratio of 1:1 was used as a control. ***P<0.001 and *P<0.01: PEI versus blank; (D) Typical GFP-expressing MCF-7 cells observed under fluorescence microscopy after transfection with SSPEI60-SPIO nanoparticles and branched PEI; (E) GFP-expressing PC-3, HepG2 and SKOV-3 cells after transfection with SSPEI60-SPIO nanoparticles. Note: scale bar: 50 μm. Abbreviations: FI, fluorescence intensity; AU, arbitrary units; SSPEI, disulfide-containing polyethylenimine; SPIO, superparamagnetic iron oxide; PEI, polyethylenimine; GFP, green fluorescent protein.

Figure 5

(A) RT-PCR for hTERT mRNA expression in HepG2 cells at 48 hours after a 4 hour exposure in the complexes of SSPEI60-SPIO/hTERT-siRNA (siRNA-NC) or untreated cells (blank) at the mass ratio of 10:1. The expression of the housekeeping gene β-actin was used as an internal control; (B) Western blot assay showing the expression of hTERT protein at 48 hours after a 4 hour exposure in SSPEI60/hTERT-siRNA or siRNA-NC (control) at the mass ratio of 10:1 as well as untreated cells (blank), respectively. The expression of the housekeeping gene β-action was used as a reference. The semi-quantification of Western blot band intensity (mean × pixel) is shown as hTERT/β-actin ratio and results are relative to the blank (taken as 1.00); (C) effect of hTERT gene silencing induced by SSPEI60-SPIO/hTERT-siRNA complexes on HepG2 cell growth. **P<0.01: siRNA3 versus blank; (D) effect of SSPEI60-SPIO/siRNA3-induced hTERT gene silencing on apoptosis in HepG2 cells. A formulation of Lipofectamine 2000/siRNA 1–4 was used at an optimal ratio, SSPEI60-SPIO and Lipofectamine 2000 were used as controls. Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; siRNA, small interfering RNA; M, marker; hTERT, human telomerase reverse transcriptase; SSPEI, disulfide-containing polyethylenimine; SPIO, superparamagnetic iron oxide; Lipo2000, Lipofectamine 2000.

Figure 6

(A) In vivo near infrared imaging of Cy5.5-labeled SSPEI60-SPIO nanoparticles in HepG2 tumor bearing in nude mice at different time intervals; (B) ex vivo imaging of Cy5.5-labeled SSPEI60-SPIO nanoparticles in tumor and organs after 60 minutes post-injection; (C) accumulation kinetics of Cy5.5-SSPEI60-SPIO nanoparticles in tumor after post-injection at different times; (D) body biodistribution of Cy5.5-labeled SSPEI-SPIO nanoparticles or Cy5.5 24 hours post-injection. Note: **P<0.05. Abbreviations: ID %, percentage of injection dose; SSPEI, disulfide-containing polyethylenimine; SPIO, superparamagnetic iron oxide; min, minutes.

Figure 7

(A) Magnetization curves of SSPEI60-SPIO nanoparticles; (B) phantom images and quantified MR signal intensity of SSPEI60-SPIO nanoparticles encapsulated in 0.7% agarose gel at different Fe concentrations; (C) in vivo MR images of tumor-xenografted nude mice of SSPEI60-SPIO nanoparticles before (pre) and at 1, 2, and 3 hours post-IV injection. The arrows indicate tumor location; (D) Prussian blue staining of tissue sections 4 hours after IV injection of SSPEI60-SPIO nanoparticles. Abbreviations: H, magnetic intensity; T₂, relaxation time; MR, magnetic resonance; SSPEI, disulfide-containing polyethylenimine; SPIO, superparamagnetic iron oxide; IV, intravenous.