Iodinated Echogenic Glycol Chitosan Nanoparticles for X-ray CT/US Dual Imaging of Tumor

Abstract

Development of biopolymer-based imaging agents which can access rapidly and provide detailed information about the diseases has received much attention as an alternative to conventional imaging agents. However, development of biopolymer-based nanomaterials for tumor imaging still remains challenging due to their low sensitivity and image resolution. To surmount of these limitations, multimodal imaging agents have been developed, and they were widely utilized for theranostic applications. Herein, iodine containing echogenic glycol chitosan nanoparticles are developed for x-ray computed tomography (CT) and ultrasound (US) imaging of tumor diagnosis. X-ray CT/US dual-modal imaging probe was prepared by following below two steps. First, iodine-contained diatrizoic acid (DTA) was chemically conjugated to the glycol chitosan (GC) for the CT imaging. DTA conjugated GC (GC-DTA NPs) formed stable nanoparticles with an average diameter of 315 nm. Second, perfluoropentane (PFP), a US imaging agent, was physically encapsulated into GC-DTA NPs by O/W emulsion method yielding GC-DTA-PFP nanoparticles (GC-DTA-PFP NPs). The GC-DTA-PFP NPs formed nanoparticles in physiological condition, and they presented the strong x-ray CT, and US signals in phantom test in vitro. Importantly, GC-DTA-PFP NPs were effectively accumulated on the tumor site by enhanced permeation and retention (EPR) effects. Moreover, GC-DTA-PFP NPs showed x-ray CT, and US signals in tumor tissues after intratumoral and intravenous injection, respectively. Therefore, GC-DTA-PFP NPs indicated that x-ray CT/US dual-modal imaging using iodinated echogenic nanoparticles could be provided more comprehensive and accurate diagnostic information to diagnosis of tumor.

Keywords: Computed tomography; Dual-modal imaging; Glycol chitosan nanoparticles; Tumor imaging; Ultrasound imaging.

Scheme 1

Schematic illustration of x-ray CT/US dual-modal imaging using GC-DTA-PFP NPs. GC-DTA-PFP NPs can discrimination of tumor site by x-ray CT and real-time monitoring using US imaging.

Figure 1

Synthetic scheme of (a) diatirizoic acid (DTA) and (b) 3,5-diacetamidobenzoic acid (DBA) conjugated glycol chitosan nanoparticles. (c) Schematic illustration of perfluoropentane (PFP) encapsulated DTA conjugated glycol chitosan nanoparticles (GC-DTA NPs) and DBA conjugated glycol chitosan nanoparticles (GC-DBA NPs) in aqueous conditions. (d) Schematic diagram of x-ray CT/US dual-modal imaging using GC-DTA-PFP NPs. GC-DTA-PFP NPs can discrimination of tumor site by x-ray CT and real-time monitoring using US imaging.

Figure 2

In vitro characterizations of GC-DTA NPs and GC-DBA NPs. (a) Structural analysis using ¹H-NMR. (b) Size distribution of GC-DTA NPs, GC-DBA NPs, GC-DTA-PFP NPs, and GC-DBA-PFP NPs by dynamic laser scattering (DLS) measurement. (c) TEM images of GC-DTA NPs, GC-DBA NPs, GC-DTA-PFP NPs, and GC-DBA-PFP NPs. Scale bar indicates 200 nm and 400 nm.

Figure 3

In vitro x-ray CT and US phantom images. (a) X-ray CT images of GC-DTA-PFP NPs and GC-DBA-PFP NPs (2.5 - 30 mg/ml). (b) Relative X-ray CT signal intensities of GC-DTA-PFP NPs and GC-DBA-PFP NPs. The error bars represent the standard deviation (n = 5). (c) Time-dependent US images of GC-DTA-PFP NPs and GC-DBA-PFP NPs at 37 ^oC. (d) Normalized US intensities of GC-DTA-PFP NPs and GC-DBA-PFP NPs. The error bars represent the standard deviation (n = 5). *Indicates difference at the p < 0.001 significance level.

Figure 4

In vitro cytotoxicity of (a) SCC7 cells and (b) NIH3T3 cells which were incubated with GC-DTA NPs, GC-DBA NPs, GC-DTA-PFP NPs, or GC-DBA-PFP NPs for 24 h at 37 ^oC. The error bars represent the standard deviation (n = 5).

Figure 5

In vivo biodistribution of Cy5.5 labeled GC-DTA-PFP NPs in SCC7 tumor bearing mice (n = 3). (a) Whole-body near-infrared fluorescence (NIRF) images after intravenous injection of GC-DTA-PFP NPs (10 mg/kg). White circle indicates tumor site. (b) Ex vivo NIRF image of organs and tumor at 48 h post-injection of Cy5.5 labeled GC-DTA-PFP NPs. (c) NIRF intensities in the organs and tumor in (b). The error bar represents the standard deviation (n = 3).

Figure 6

In vivo x-ray CT/US imaging in SCC7 tumor-bearing mice (n = 3). (a) X-ray CT images of 120 mg/kg of GC-DTA-PFP NPs (7 mg/kg of iodine) and GC-DBA-PFP NPs-treated SCC7 tumor-bearing mice, respectively. The white arrows indicate the direct-injection site in tumor tissue. (b) Relative x-ray CT signal intensities of GC-DTA-PFP NPs and GC-DBA-PFP NPs. The relative x-ray CT signal intensities were calculated by intensities ratio of normal tissues to tumor tissue. The error bars represent the standard deviation (n = 3). (c) Time-dependent US images of tumor tissue after intravenous injection of GC-DTA-PFP NPs (240 mg/kg, 0.3 mg/kg of PFP) into SCC7 tumor-bearing mice (n =3). (d) Time-dependent US signal intensities at tumor site. The error bars represent the standard deviation (n = 3). *Indicates difference at the p < 0.005 significance level.

Figure 7

Biosafety of GC-DTA-PFP NPs and GC-DBA-PFP NPs was evaluated by histopathological changes in the major organs (liver, lung, spleen, kidney, heart) based on H&E staining.