Folic acid-nanoscale gadolinium-porphyrin metal-organic frameworks: fluorescence and magnetic resonance dual-modality imaging and photodynamic therapy in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and severely threatens human health. Since the prognosis of advanced HCC remains poor, there is an urgent need to develop new therapeutic approaches. Porphyrin metal-organic frameworks are a class of porous organic-inorganic hybrid functional materials with good biocompatibility.

Methods: Gadolinium-porphyrin metal-organic frameworks were used as a skeleton for folic acid (FA) conjugation to synthesize a novel type of nanoparticle, denoted as folic acid-nanoscale gadolinium-porphyrin metal-organic frameworks (FA-NPMOFs). The FA-NPMOFs were characterized using transmission electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric-differential thermal analysis. The biotoxicity and imaging capability of the FA-NPMOFs were determined using HepG2 cells and embryonic and larval zebrafish. The delivery and photodynamic therapeutic effect of FA-NPMOFs were explored in transgenic zebrafish with doxycycline-induced HCC.

Results: FA-NPMOFs were spherical in structure with good dispersion and water solubility. They showed low biotoxicity, emitted bright red fluorescence, and exhibited an excellent magnetic resonance imaging capability, both in vitro and in vivo. Meanwhile, the FA-NPMOFs exhibited a strong affinity for folate receptor (FR)-expressing cells and were delivered to the tumor site in a targeted manner. Moreover, HCC tumor cells were eliminated following laser irradiation.

Conclusion: FA-NPMOFs can be used for dual-modality imaging and photodynamic therapy in HCC and show promise for use as a carrier in new therapies for HCC and other FR-positive tumors.

Keywords: dual-modality imaging; folic acid; hepatocellular carcinoma; nanoscale gadolinium-porphyrin metal-organic frameworks; photodynamic therapy.

Figure 1

Synthesis and characterization of FA-NPMOFs. Notes: (A) Schematic illustration of FA-NPMOF synthesis. (B) Transmission electron microscopy images. (C) NPMOF (purple) and FA-NPMOF (black) composition determined by Fourier transform infrared spectroscopy. (D) TGA of NPMOFs and FA-NPMOFs. (E) FA-NPMOF composition determined by TGA. (F) UV-vis absorbance (black) and normalized fluorescence (blue, λex =530 nm). Scale bar in (B) 200 nm and 50 nm (inset). Abbreviations: DMF, N,N-dimethylformamide; FA-NPMOF, folic acid-nanoscale gadolinium-porphyrin metal-organic framework; PEI, polyetherimide; TCPP, 5,10,15,20-tetrakis(4-carboxyl)-21H,23H-porphine; CTAB, cetyltrimethylammonium bromide.

Figure 2

In vitro cytotoxicity of FA-NPMOFs. Notes: (A) Fluorescence images of HepG2 cells; red signals are from FA-NPMOFs, blue signals are from DAPI staining. Note that the FA-NPMOFs are localized at the membrane and in the cytoplasm. (B) Statistical analysis of fluorescence intensity in the control and FA-NPMOF-exposed groups. (C) Statistical analysis of viability of L02 cells treated with FA-NPMOFs (white), HepG2 cells treated with FA-NPMOFs (gray), and HepG2 cells treated with FA-NPMOFs and photodynamic therapy (black). Scale bar in (A): 20 µm. *P<0.05, ***P<0.001. Abbreviation: FA-NPMOF, folic acid-nanoscale gadolinium-porphyrin metal-organic framework; PDT, photodynamic therapy; NS, not significant.

Figure 3

Fluorescence imaging and MRI of FA-NPMOFs in zebrafish embryos. Notes: (A, B) Bright-field and fluorescence images of FA-NPMOF-exposed zebrafish embryos. Note that the red fluorescence was emitted by FA-NPMOFs. (C, D) MRI scans on the (C) sagittal and (D) horizontal planes. Scale bar in (A, B): 100 µm. Abbreviations: FA-NPMOF, folic acid-nanoscale gadolinium-porphyrin metal-organic framework; MRI, magnetic resonance imaging.

Figure 4

Time-lapse distribution of FA-NPMOFs in zebrafish larvae. Notes: (A, B) Fluorescence and bright-field images of FA-NPMOF-exposed larvae at 5 days post fertilization. Note that the red fluorescence was emitted by FA-NPMOFs. (C) Merged images from (A and B). Scale bar: 100 µm. Abbreviation: FA-NPMOF, folic acid-nanoscale gadolinium-porphyrin metal-organic framework; hpe, hours post exposure.

Figure 5

HCC in kras^G12V zebrafish. Notes: (A) Bright-field, GFP fluorescence and H&E staining images in wild-type (control, the upper line) and kras^G12V zebrafish (the lower line) at 15 dpi. (B) FR immunostaining in the liver from kras^G12V zebrafish at 15 dpi. Scale bar in (A): 500 µm; 10 µm; (B): 20 µm. Abbreviations: dpi, days post induction; FR, folate receptor; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; WT, wild type.

Figure 6

Time-lapse retention of FA-NPMOFs in the liver of HCC-bearing kras^G12V zebrafish. Notes: (A) Retention of FA-NPMOFs in the liver of HCC-bearing zebrafish at 0 (control), 24, 48, 72, and 96 hpt. (B) Images of GFP fluorescence and DAPI staining in the same liver from (A). (D) Merged images of (A–C). (E) Magnetic resonance imaging scans and thermal images of FA-NPMOF-treated HCC-bearing zebrafish at 48 hpt. Scale bar: 20 µm. Abbreviations: FA-NPMOF, folic acid-nanoscale gadolinium-porphyrin metal-organic framework; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; hpt, hours post treatment; MRI, magnetic resonance imaging.

Figure 7

Tumor inhibition after PDT in HCC-bearing zebrafish. Notes: (A–C) Bright-field, fluorescence, thermal, and H&E staining images of HCC-bearing zebrafish before (FA-NPMOF; left column) and after (FA-NPMOF + PDT; right column) PDT. (D) Images of tumors in the FA-NPMOF and NPMOF + PDT groups. (E) Statistical analysis of tumor volume between the two groups. Scale bar in (A): 500 µm, (C): 20 µm. ***P<0.001. Abbreviations: FA-NPMOF, folic acid-nanoscale gadolinium-porphyrin metal-organic framework; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; PDT, photodynamic therapy.