Desmin- and vimentin-mediated hepatic stellate cell-targeting radiotracer 99mTc-GlcNAc-PEI for liver fibrosis imaging with SPECT

Abstract

Extracellular matrix (ECM) accumulation in liver fibrosis is caused by the activation of hepatic stellate cells (HSCs). The goal of this study was to develop a ^99mTc-labeled N-acetylglucosamine (GlcNAc) that specifically interacts with desmin and vimentin expressed on activated HSCs to monitor the progression and prognosis of liver fibrosis using single-photon emission computed tomography (SPECT) imaging. Methods: GlcNAc-conjugated polyethylenimine (PEI) was first prepared and radiolabeled with ^99mTc. Noninvasive SPECT imaging with ^99mTc-GlcNAc-PEI was used to assess liver fibrosis in a carbon tetrachloride (CCl₄) mouse model. The liver uptake value (LUV) of ^99mTc-GlcNAc-PEI was measured by drawing the region of interest (ROI) of the whole liver as previously suggested. The LUV of the CCl₄ groups was compared with that of the olive oil group. Next, we estimated the correlation between the results of SPECT imaging and physiological indexes. After treatment with clodronate liposome, the LUV of ^99mTc-GlcNAc-PEI in fibrotic mice was compared with that in control mice. Results:^99mTc-GlcNAc-PEI is a hydrophilic compound with high radiochemical purity (>98%) and good stability. It could specifically target desmin and vimentin on the surface of activated HSCs with high affinity (the K_d values were 53.75 ± 9.50 nM and 20.98 ± 3.56 nM, respectively). The LUV of ^99mTc-GlcNAc-PEI was significantly different between the CCl₄ and control groups as early as 4 weeks of CCl₄ administration (3.30 ± 0.160 vs 2.34 ± 0.114%/cc; P ˂ 0.05). There was a strong correlation between the LUV and Sirius Red quantification (R = 0.92, P ˂ 0.001). Compared with control, clodronate liposome treatment reduced the LUV of ^99mTc-GlcNAc-PEI (4.62 ± 0.352 vs 2.133 ± 0.414%/cc; P ˂ 0.05). Conclusion:^99mTc-GlcNAc-PEI SPECT/CT was useful in assessing liver fibrosis and monitoring the treatment response.

Keywords: 99mTc-GlcNAc-PEI; SPECT imaging; desmin; hepatic fibrosis; hepatic stellate cells; vimentin.

Fig 1

Synthesis and radiolabeling of GlcNAc-PEI with ^99mTc.

Fig 2

Characterization of a CCl₄-induced mouse model of liver fibrosis. (A) Representative images of Sirius Red staining after CCl₄ administration for 4 or 8 weeks. Scale bar: 200 μm. (B) Red area of Sirius Red staining was quantified using Image pro-pus software. (C) Hydroxyproline level in the liver of CCl₄-induced mice was calculated by HPLC analysis. (D) Ishak score of the liver tissue in CCl₄-induced fibrotic mice. (E) Correlation between total collagen (hydroxyproline) and red area of Sirius Red staining. (F) Correlation between the total Ishak score and red area of Sirius Red staining. (G) Correlation between the Ishak score and total collagen (hydroxyproline). ^*P <0.05 and ^**P <0.01.

Fig 3

MicroSPECT/CT imaging with ^99mTc-GlcNAc-PEI to assess liver fibrosis. (A) SPECT/CT imaging of CCl₄-induced fibrotic mice with ^99mTc-GlcNAc-PEI. Both images were rendered at the same scale. (B) Hepatic uptake of control and fibrotic mice by drawing the ROI of the whole liver. (C) Biodistribution of ^99mTc-GlcNAc-PEI in CCl₄-induced fibrotic mice (n = 3). (D) Correlation between hepatic uptake and Sirius Red quantification. (E) Correlation between hepatic uptake and total collagen (hydroxyproline). (F) Immunofluorescence co-localization analysis of desmin and GlcNAc-PEI-Cy5.5 in the liver tissue of CCl₄-induced fibrotic mice. Scale bar: 200 μm. ^*P <0.05 and ^**P <0.01.

Fig 4

Blocking studies of ^99mTc-GlcNAc-PEI in fibrotic mice. (A) SPECT/CT imaging of fibrotic mice at 30 min after the intravenous injection of ^99mTc-GlcNAc-PEI (18.5 MBq) with or without GlcNAc-PEI for blocking. Images were adjusted using the same scale for all animals. (B) Cell uptake of GlcNAc-PEI-Cy5.5 in the fibrotic liver tissue. Blue: DAPI; Red: GlcNAc-PEI-Cy5.5. Scale bar: 50 μm. (C) Hepatic uptake of ^99mTc-GlcNAc-PEI derived from SPECT imaging by drawing the ROI of the whole liver. (D) Competitive binding assay of ^99mTc-GlcNAc-PEI with or without the presence of excess GlcNAc-PEI in HSCs separated from the liver of fibrotic mice. (E) Saturation binding assay of ^99mTc-GlcNAc-PEI with desmin protein.

Fig 5

^99mTc-GlcNAc-PEI SPECT/CT imaging of liver fibrosis mice with clodronate liposome treatment. (A) ^99mTc-GlcNAc-PEI SPECT/CT imaging of fibrotic mice with clodronate liposome treatment. (B) Hepatic uptake of ^99mTc-GlcNAc-PEI by drawing the ROI of the whole liver. (C) Representative images of Sirius Red staining after clodronate liposome treatment. Scale bar: 200 μm. (D) Quantization of the red area of Sirius Red staining after treatment with clodronate liposome. (E) Hydroxyproline level in the liver of CCl₄-induced mice after treatment with clodronate liposomes. (F) Immunofluorescence staining of fibrotic liver tissue after treatment with clodronate liposome. Blue: DAPI; green: anti-CD₆₈ staining FITC; Red: GlcNAc-PEI-Cy5.5. Scale bar: 200 μm. ^*P <0.05 and ^**P <0.01.