Design and construction of a magnetic targeting pro-coagulant protein for embolic therapy of solid tumors

Figures & data

Figure 1. The schematic diagram of the mechanism of the magnetic targeting pro-coagulant protein.

Figure 2. The process of synthesis of magnetic targeting pro-coagulant protein.

Table 1. Treatment strategy and grouping of experiments in mice.

	Live imaging	Tumour growth inhibition test	HCC model treatment
Models	subcutaneous	subcutaneous	orthotopic
Grouping	4 groups of 3 each	4 groups of 10 each	4 groups of 3 each
Treatment	Group a: 100 μL of saline Group b: 100 μL of OCMC/Fe3O4 & magnetic field Group c: 100 μL of tTF-EG3287(0.5 mg/mL) Group d: 100 μL of tTF-EG3287@OCMC/Fe3O4 (0.5 mg/mL) & magnetic field

Figure 3. Identification of the fusion protein tTF-EG3287. (A) Analysis of different induction time (0–6 h) of the fusion protein tTF-EG3287 by Coomassie-stained SDS-PAGE. (B) Purification of the fusion protein. The proteins were purified from E. coli by use of nickel column chromatography. Shown is a crude lysate (Lane1), column flow-through (Lane2), wash (Lane3,4), and elution(Lane5) for the fusion protein tTF-EG3287. (C) Western blot analysis of the purified fusion protein samples, using an anti-His-Tag antibody. (D) The standard curve of the BCA assay.

Figure 4. Characterisation of Fe₃O₄, OCMC/Fe₃O₄ and tTF-EG3287@OCMC/Fe₃O₄. (A) Transmission electron microscope image of Fe₃O₄(a), OCMC/Fe₃O₄(b) and tTF-EG3287@OCMC/Fe₃O₄(c), scale bar = 50nm; (B)The particle size analysis of Fe₃O₄(a), OCMC/Fe₃O₄ (b) and tTF-EG3287@OCMC/Fe₃O₄ (c) calculated by Malvern laser diffraction analyser; (C) X-ray diffraction (XRD) patterns of Fe₃O₄(a), OCMC/Fe₃O₄(b) and tTF-EG3287@OCMC/Fe₃O₄(c); (D) Fourier transform infra-red (FTIR) spectra of Fe₃O₄(a), OCMC(b) and OCMC/Fe₃O₄(c); (E) Magnetisation curve for the uncoated at Fe₃O₄(a), OCMC/Fe₃O₄(b) and tTF-EG3287@OCMC/Fe₃O₄(c) at 150K. (F) Stability analysis of Fe₃O₄(a), OCMC/Fe₃O₄(b) and tTF-EG3287@OCMC/Fe₃O₄(c) after standing for 24 h.

Figure 5. Biological functions of the magnetic targeting pro-coagulant protein. (A) Spectrozyme FXa Assay of the tTF, tTF-EG3287 and tTF-EG3287@OCMC/Fe₃O₄. PBS and OCMC/Fe₃O₄ were used as negative control. Data represent the means ± SE of five independent experiments. Confocal microscopy (B) and flow cytometry (C) were used to evaluate the NRP-1 binding ability of the magnetic procoagulant protein on HUVEC cell. (D) Cytotoxicity on HepG2 and HUVEC cells after treated with tTF-EG3287@OCMC/Fe₃O₄ for 24 h. The values presented are the means ± SE of three independent experiments.

Figure 6. Magnetic targeting ability of the magnetic targeting pro-coagulant protein detected by in vivo imaging system. (A) Delegate in vivo fluorescence images at various times after tail vein infusion with saline(a), OCMC/Fe₃O₄(b) and Cy5.5-tTF-EG3287(c) and Cy5.5-tTF-EG3287@OCMC/Fe₃O₄(d). As the reference scale on the right, different colours indicates different fluorescence intensities. Blue indicates strong fluorescence signal and red colour indicates a weak signal. (B) Cy5.5 fluorescence intensity in tumour tissues collected at 72 h following injection. Results were presented as means ± SE for a group of 3 mice.

Figure 7. Effect of the magnetic procoagulant protein on growth of tumour in mice models. (A) Tumour volume of HepG2 tumour-bearing mice of each groups. Data are presented as means ± SE of two independent experiments. Statistical comparisons were done for each time point. Asterisks, statistical significance between tTF-EG3287@OCMC/Fe₃O₄ and controls (saline, OCMC/Fe₃O₄ and tTF-EG3287). Arrows, time points of injection. (B) Average body weight of mice in each group, data represent the means ± SE of two independent experiments. (C) Average tumour weights of HepG2 tumour-bearing mice of each groups after treatment, data are presented as means ± SE of two independent experiments. (D) Representative pictures of the HepG2 tumour bearing mice at the end of treatment (Day 7) by saline (a), OCMC/Fe₃O₄ (b), tTF-EG3287 (c) and tTF-EG3287@OCMC/Fe₃O₄ (d).

Table 2. Antitumor activity of the magnetic procoagulant protein in mice.

Treatment	Tumour volume (mm^3)		Tumour growth index*	p value
	Day 0	Day 7		vs/ saline	vs tTF-EG3287
saline	98.9 ± 11.7	807.8 ± 127.7	8.1	–	<.05
OCMC/Fe3O4	82.6 ± 12.0	678.8 ± 97.5	8.2	NS	NS
tTF-EG3287	87.2 ± 9.6	632.7 ± 106.5	7.3	<.05	–
tTF-EG3287@OCMC/Fe3O4	86.5 ± 8.9	88.9 ± 16.2	1.0	<.001	<.001

Figure 8. Histology studies of the treatment effect of the magnetic procoagulant protein in subcutaneous transplantation tumour models. (A ∼ C) Representative picture of the cancer tissue after treatment of controls groups (saline (A), OCMC/Fe₃O₄ (B) and tTF-EG3287 (C)) stained with haematoxylin and eosin. Obvious thrombus (D ∼ F) and necrotic area (G ∼ I) were found in the blood vessels of hepatic carcinoma after treated by the MTPCP tTF-EG3287@OCMC/Fe₃O₄ for 4 consecutive days.

Figure 9. Thrombotic risk assessment in the normal organs of the treated mice. Histological analysis of various normal organs of tumour-bearing mice treated with the MTPCP. Sections of heart (A), liver (B), spleen (C), lung (D), kidney (E) and brain (F) were stained with haematoxylin and eosin (H&E).

Figure 10. Schematic diagrams of TACE/TAE (A) and the magnetic targeting pro-coagulant protein (B).