Emodin-Conjugated PEGylation of Fe₃O₄ Nanoparticles for FI/MRI Dual-Modal Imaging and Therapy in Pancreatic Cancer

Figures & data

Figure 1 Schematic illustration of the synthesis of Fe₃O₄-PEG-Cy7-EMO (A) and the possible mechanism of Fe₃O₄-PEG-Cy7-EMO enabled FI/MRI dual-modal imaging and targeted therapy in pancreatic tumor xenografted mice based on the EPR effect (B).

Abbreviations: EMO, emodin; GFLG, Gly-Phe-Leu-Gly; PEG, polyethylene glycol; EDC, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; NHS, N-hydroxysuccinimide; EPR, enhanced permeation and retention.

Figure 2 Characterization of NPs.

Notes: (A) TEM image of Fe₃O₄-PEG, which has a core size of 9.1 ± 1.7 nm. (B) TEM image of Fe₃O₄-PEG-Cy7-EMO, which has a core size of 9.9 ± 1.2 nm. (C) The hysteresis curve shows that Fe₃O₄-PEG-Cy7-EMO has the characteristics of superparamagnetic property at room temperature. (D) FT-IR of Fe₃O₄-PEG (1101.16 cm⁻¹ C-O-C group). (E) The hydrodynamic size of Fe₃O₄-PEG (26.12 ± 5.13 nm) and Fe₃O₄-PEG-Cy7-EMO (27.16 ± 5.74 nm) was characterized by dynamic light scattering (DLS). (F) Hydrodynamic size distributions of Fe₃O₄-PEG-Cy7-EMO in different pH buffer solutions. (G) The zeta potential of Fe₃O₄-PEG (−40.2 ± 6.83 mV). (H) The zeta potential of Fe₃O₄-PEG-Cy7-EMO (−38.7 ± 6.32 mV). (I) Zeta potential distributions of Fe₃O₄-PEG-Cy7-EMO in different pH buffer solutions. (J and K) The stability of Fe₃O₄-PEG-Cy7-EMO in sodium chloride (0 to 1.0 mM, Figure 2J) solution (containing 5% BSA) and in different pH (3 to 11, Figure 2K) solution was observed for 7 days. No deposition is observed in Fe₃O₄-PEG-Cy7-EMO solutions that were stored at 4°C for 7 days.

Figure 3 Fe₃O₄-PEG-Cy7-EMO enabled FI/MRI dual-modal imaging in vitro.

Notes: (A) T2-weighted MR imaging of Fe₃O₄-PEG and Fe₃O₄-PEG-Cy7-EMO in vitro. The T2 signal intensities decrease with the increase of the concentration of Fe. (B) Fluorescence imaging of Fe₃O₄-PEG-Cy7-EMO in vitro. The fluorescence intensities increase with the increase of the concentration of Fe₃O₄-PEG-Cy7-EMO.

Figure 4 (A) The distribution of Fe₃O₄-PEG-Cy7-EMO in BxPC-3 and hTERT-HPNE cells examined by bio-TEM under 2 µm and 200 nm scale bars. (B) The distribution of Fe₃O₄-PEG-Cy7-EMO in BxPC-3 and hTERT-HPNE cells examined by Prussian Blue staining analysis.

Figure 5 (A) The MTT results of BxPC-3 and hTERT-HPNE cells incubated with Fe₃O₄-PEG, Fe₃O₄-PEG-Cy7-EMO, and EMO. (B) Apoptosis assays for BxPC-3 and hTERT-HPNE cells after different treatment. *P < 0.05 for Fe₃O₄-PEG vs Fe₃O₄-PEG-Cy7-EMO.

Table 1 The Apoptotic Rates of BxPC-3 and hTERT-HPNE Cells Incubated with Different Concentrations of NPs

Cell Lines	Control	Fe3O4-PEG (40μg/mL)	Fe3O4-PEG (80μg/mL)	Fe3O4-PEG-Cy7-EMO (40μg/mL)	Fe3O4-PEG-Cy7-EMO (80μg/mL)
BxPC-3	3.82%	11.38%	21.88%	33.85%	62.23%
hTERT-HPNE	1.20%	4.97%	7.19%	5.89%	7.62%

Note: Apoptotic rates = pre-apoptotic rate + post-apoptotic rate.

Figure 6 (A) The hemolysis experiments in vitro. Different treatments of tubes 1–5 are clarified in (B). (C) In vivo MRI images and (D) time-dependent intensity curves of Fe₃O₄-PEG and Fe₃O₄-PEG-Cy7-EMO groups. (E) In vivo fluorescence imaging and (F) ex vivo fluorescence imaging of hearts, livers, spleens, lungs, kidneys, and tumors.

Figure 7 (A) The H&E staining of different organs including hearts, livers, spleens, lungs, and kidneys. No obvious pathological changes were observed. (B) Prussian Blue staining analyses of ex vivo.

Figure 8 In vivo anti-tumor efficiency.

Notes: (A) Body weight changes of BxPC-3 tumor-bearing nude mice in different treatment groups. (B) Tumor volume changes of BxPC-3 tumor-bearing nude mice in different treatment groups. (C) Photos of tumors after treatment using 0.9% saline, Fe₃O₄-PEG, EMO, and Fe₃O₄-PEG-Cy7-EMO after two weeks.