Doxorubicin and indocyanine green loaded superparamagnetic iron oxide nanoparticles with PEGylated phospholipid coating for magnetic resonance with fluorescence imaging and chemotherapy of glioma

Abstract

Background: Glioma represents the most common malignant brain tumor. Outcomes of surgical resection are often unsatisfactory due to low sensitivity or resolution of imaging methods. Moreover, the use of traditional chemotherapeutics, such as doxorubicin (DOX), is limited due to their low blood-brain barrier (BBB) permeability. Recently, the development of nanotechnology could overcome these obstacles.

Materials and methods: Hydrophobic superparamagnetic iron oxide nanoparticles (SPIO NPs) were prepared with the use of thermal decomposition method. They were coated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000) and DOX using a thin-film hydration method followed by loading of indocyanine green (ICG) into the phospholipid layers. Details regarding the characteristics of NPs were determined. The in vitro biocompatibility and antitumor efficacy were established with the use of MTT assay. In vivo fluorescence and magnetic resonance (MR) imaging were used to evaluate BBB penetration and accumulation of NPs at the tumor site. Antitumor efficacy was evaluated using measures of tumor size, median survival times, body weights, and H&E staining.

Results: The multifunctional NPs generated had an average diameter of 22.9 nm, a zeta potential of -38.19 mV, and were capable of providing a sustained release of DOX. In vitro experiments demonstrated that the SPIO@DSPE-PEG/DOX/ICG NPs effectively enhanced cellular uptake of DOX as compared with that of free DOX. In vivo fluorescence and MR imaging revealed that the NPs not only effectively crossed the BBB but selectively accumulated at the tumor site. Meanwhile, among all groups studied, C6 glioma-bearing rats treated with the NPs exhibited the maximal degree of therapeutic efficacy, including smallest tumor volume, lowest body weight loss, and longest survival times, with no obvious side effects.

Conclusion: These results suggest that the SPIO@DSPE-PEG/DOX/ICG NPs can not only function as a nanoprobe for MR and fluorescence bimodal imaging, but also as a vehicle to deliver chemotherapeutic drugs to the tumor site, to achieve the theranostic treatment of glioma.

Keywords: BBB; MR imaging; SPIO NPs; chemotherapy; fluorescence imaging.

Figure 1

Schematic illustration. SPIO@DSPE-PEG/DOX/ICG NPs preparation procedure and MR/NIR fluorescence dual-modal imaging and chemotherapy of glioma through intravenous injection. Abbreviations: BBB, blood–brain barrier; DOX, doxorubicin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]; ICG, indocyanine green; MR, magnetic resonance; SPIO NPs, superparamagnetic iron oxide nanoparticles; NIR, near-infrared.

Figure 2

Characterization of nanoparticles. Notes: TEM images of hydrophobic SPIO NPs (A) and SPIO@DSPE-PEG/DOX/ICG NPs (C). The bar represents 40 nm. Particle size distribution of hydrophobic SPIO NPs (B) and SPIO@DSPE-PEG/DOX/ICG NPs (D) by DLS measurements. Fluorescence spectra of nanoparticles with an excitation wavelength of 480 nm (E) and 740 nm (F). In vitro release profiles of DOX in PBS at 37°C (G). Data are presented as mean ± SD, n=3. Abbreviations: DLS, dynamic light scattering; DOX, doxorubicin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]; ICG, indocyanine green; SPIO NPs, superparamagnetic iron oxide nanoparticles; TEM, transmission electron microscopy.

Figure 3

Cellular uptake of nanoparticles. Note: CLSM images of U251 cells incubated with free DOX and SPIO@DSPE-PEG/DOX/ICG NPs at a DOX concentration of 6 µg/mL for 1 hour. Abbreviations: CLSM, confocal laser scan microscopy; DOX, doxorubicin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]; ICG, indocyanine green; SPIO NPs, superparamagnetic iron oxide nanoparticles.

Figure 4

Biocompatibility and cytotoxicity of nanoparticles in vitro. Notes: Viability of HUVEC cells after incubation with blank SPIO@DSPE-PEG NPs at varying concentrations (equivalent to Fe concentrations) for 24, 48, and 72 hours (A). In vitro antitumor efficiency of SPIO@DSPE-PEG/DOX/ICG NPs or free DOX (control) against U251 cells at different concentrations for 24 hours (B), 48 hours (C), and 72 hours (D). Data are mean ± SD, n=3, ^*P<0.05 vs free DOX group. Abbreviations: DOX, doxorubicin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]; HUVEC, human umbilical vein endothelial cells; ICG, indocyanine green; SPIO NPs, superparamagnetic iron oxide nanoparticles.

Figure 5

In vivo fluorescence imaging efficacy. Notes: Fluorescence images of glioma-bearing nude mice at different times after tail vein injection of free ICG and SPIO@DSPE-PEG/DOX/ICG NPs (A); arrow indicates direction of the glioma. Ex vivo fluorescence images (B) and quantitative analysis (C) of main organs from glioma-bearing nude mice after 72 hours of tail vein injection. Data are mean ± SD, n=3, ^*P<0.05, ^**P<0.01 vs flurensence intensity in tumor. Abbreviations: DOX, doxorubicin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]; ICG, indocyanine green; SPIO NPs, superparamagnetic iron oxide nanoparticles.

Figure 6

In vivo MRI efficacy. Notes: T₂-weighted MR images of glioma-bearing Wistar rats at different times after tail vein injection of SPIO@DSPE-PEG/DOX/ICG NPs (A); arrow indicates direction of the glioma. The quantitative analyses of the T₂ value in brain (B) and tumor (C). Data are mean ± SD, n=3, ^*P<0.05 and ^**P<0.01 vs 0 hours. Abbreviations: DOX, doxorubicin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]; ICG, indocyanine green; MRI, magnetic resonance imaging; SPIO NPs, superparamagnetic iron oxide nanoparticles.

Figure 7

Antitumor efficacy in vivo. Notes: T₁-weighted brain MR images of glioma-bearing Wistar rats treated with saline, SPIO@DSPE-PEG NPs, free DOX, and SPIO@DSPE-PEG/DOX/ICG NPs on days 7, 14, 21, and 28 after tumor implantation (A); arrow indicates direction of the glioma. Tumor volumes monitored by MRI (B). Kaplane–Meier survival curves (C) of rats. Data are shown as mean ± SD, n=6. ^***P<0.001 vs control group, ^#P<0.001 vs free DOX group. Abbreviations: DOX, doxorubicin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]; ICG, indocyanine green; MR, magnetic resonance; SPIO NPs, superparamagnetic iron oxide nanoparticles.

Figure 8

Histological sections of brain and tumor stained with H&E. Abbreviations: DOX, doxorubicin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]; ICG, indocyanine green; SPIO NPs, superparamagnetic iron oxide nanoparticles.