Figures & data
Table 1 Parameters of synthesized M-HFn nanoparticles with different core sizes
Figure 1 TEM analysis of M-HFn nanoparticles.
Notes: (A) TEM graphs of M-HFn1000, M-HFn3000, M-HFn5000, and M-HFn7000. Scale bar is 10 nm. (B) The high-resolution TEM images of these four M-HFn samples. Scale bar is 1 nm. (C) Corresponding SAED patterns of M-HFn nanoparticles. (D) Size histograms of M-HFn nanoparticles.
Abbreviations: M-HFn, ferrimagnetic H-ferritin; TEM, transmission electron microscope; SAED, selected area electron diffraction.
![Figure 1 TEM analysis of M-HFn nanoparticles.Notes: (A) TEM graphs of M-HFn1000, M-HFn3000, M-HFn5000, and M-HFn7000. Scale bar is 10 nm. (B) The high-resolution TEM images of these four M-HFn samples. Scale bar is 1 nm. (C) Corresponding SAED patterns of M-HFn nanoparticles. (D) Size histograms of M-HFn nanoparticles.Abbreviations: M-HFn, ferrimagnetic H-ferritin; TEM, transmission electron microscope; SAED, selected area electron diffraction.](/cms/asset/59d81627-04e7-4262-8fd5-c1c9cd20d62c/dijn_a_80025_f0001_b.jpg)
Figure 2 Native PAGE analysis of assembled HFn cage structure of M-HFn nanoparticles.
Notes: Gel was stained with (A) potassium ferrocyanide and (B) Coomassie Brilliant Blue R250. Lane 1, HFn cage; lane 2, M-HFn1000; lane 3, M-HFn3000; lane 4, M-HFn5000; and lane 5, M-HFn7000. (C) CD spectra determination of the secondary protein structural stability of M-HFn nanoparticles.
Abbreviations: HFn, H chain ferritin; M-HFn, ferrimagnetic H-ferritin; PAGE, polyacrylamide gel electrophoresis; CD, circular dichroism; PBS, phosphate-buffered saline.
![Figure 2 Native PAGE analysis of assembled HFn cage structure of M-HFn nanoparticles.Notes: Gel was stained with (A) potassium ferrocyanide and (B) Coomassie Brilliant Blue R250. Lane 1, HFn cage; lane 2, M-HFn1000; lane 3, M-HFn3000; lane 4, M-HFn5000; and lane 5, M-HFn7000. (C) CD spectra determination of the secondary protein structural stability of M-HFn nanoparticles.Abbreviations: HFn, H chain ferritin; M-HFn, ferrimagnetic H-ferritin; PAGE, polyacrylamide gel electrophoresis; CD, circular dichroism; PBS, phosphate-buffered saline.](/cms/asset/f57234eb-ea44-4ad6-852b-8cea804591c6/dijn_a_80025_f0002_c.jpg)
Figure 3 Size dependence of magnetic properties of M-HFn nanoparticles.
Notes: The relationship between the core size of M-HFn nanoparticles and their (A) R-value, (B) Ms, and (C) Hc.
Abbreviations: M-HFn, ferrimagnetic H-ferritin; Ms, saturation magnetization; Hc, coercivity; R, magnetostatic interaction parameter.
![Figure 3 Size dependence of magnetic properties of M-HFn nanoparticles.Notes: The relationship between the core size of M-HFn nanoparticles and their (A) R-value, (B) Ms, and (C) Hc.Abbreviations: M-HFn, ferrimagnetic H-ferritin; Ms, saturation magnetization; Hc, coercivity; R, magnetostatic interaction parameter.](/cms/asset/90499f6a-526b-4cc5-8888-f2e255fbcf15/dijn_a_80025_f0003_b.jpg)
Figure 4 Peroxidase-like activity assays of M-HFn nanoparticles.
Notes: M-HFn nanoparticles with different sizes of core catalyzed the oxidation of peroxidase substrates in the presence of H2O2. (A) TMB as the substrate to give a deep blue color product. (B) DAB as the substrate to give a deep brown color product. Color intensity changes with (a) HFn cage, (b) M-HFn1000, (c) M-HFn3000, (d) M-HFn5000, and (e) M-HFn7000. (C) The peroxidase-like activity of M-HFn nanoparticle is size dependent; larger M-HFn nanoparticles show higher peroxidase-like activity (TMB as the substrate).
Abbreviations: HFn, H chain ferritin; M-HFn, ferrimagnetic H-ferritin; TMB, 3,3′,5,5′-tetramethylbenzidine; DAB, 3,3′-diaminobenzidine tetrahydrochloride.
![Figure 4 Peroxidase-like activity assays of M-HFn nanoparticles.Notes: M-HFn nanoparticles with different sizes of core catalyzed the oxidation of peroxidase substrates in the presence of H2O2. (A) TMB as the substrate to give a deep blue color product. (B) DAB as the substrate to give a deep brown color product. Color intensity changes with (a) HFn cage, (b) M-HFn1000, (c) M-HFn3000, (d) M-HFn5000, and (e) M-HFn7000. (C) The peroxidase-like activity of M-HFn nanoparticle is size dependent; larger M-HFn nanoparticles show higher peroxidase-like activity (TMB as the substrate).Abbreviations: HFn, H chain ferritin; M-HFn, ferrimagnetic H-ferritin; TMB, 3,3′,5,5′-tetramethylbenzidine; DAB, 3,3′-diaminobenzidine tetrahydrochloride.](/cms/asset/cda24c36-8f33-4cd1-92fc-4472189f95d5/dijn_a_80025_f0004_c.jpg)
Figure 5 MRI of MDA-MB-231 tumor cells incubated with M-HFn nanoparticles.
Notes: (A) Evaluation of the efficiency of M-HFn nanoparticles in detecting MDA-MB-231 tumor cells by MRI. T2-weighted MR images of (a) tumor cell-only, and tumor cells incubated with (b) M-HFn1000, (c) M-HFn3000, (d) M-HFn5000, and (e) M-HFn7000. T2 values are 122.5±0.7 ms, 110.9±1.5 ms, 99.1±1.9 ms, 90.0±3.1 ms, and 81.2±2.6 ms (P<0.05, n=3). (B) T2-weighted MR images of tumor cells of different concentrations incubated with M-HFn7000 nanoparticles: (g) 0 cells mL−1, (h) 1×104 cells mL−1, (i) 1×105 cells mL−1, (j) 2×105 cells mL−1, and (k) 3×105 cells mL−1. T2 values are 140.4±3.7 ms, 103.2±0.4 ms, 93.2±0.8 ms, 88.8±0.8 ms, and 81.2±2.6 ms (P<0.05, n=3).
Abbreviation: M-HFn, ferrimagnetic H-ferritin; MR, magnetic resonance; MRI, magnetic resonance imaging.
![Figure 5 MRI of MDA-MB-231 tumor cells incubated with M-HFn nanoparticles.Notes: (A) Evaluation of the efficiency of M-HFn nanoparticles in detecting MDA-MB-231 tumor cells by MRI. T2-weighted MR images of (a) tumor cell-only, and tumor cells incubated with (b) M-HFn1000, (c) M-HFn3000, (d) M-HFn5000, and (e) M-HFn7000. T2 values are 122.5±0.7 ms, 110.9±1.5 ms, 99.1±1.9 ms, 90.0±3.1 ms, and 81.2±2.6 ms (P<0.05, n=3). (B) T2-weighted MR images of tumor cells of different concentrations incubated with M-HFn7000 nanoparticles: (g) 0 cells mL−1, (h) 1×104 cells mL−1, (i) 1×105 cells mL−1, (j) 2×105 cells mL−1, and (k) 3×105 cells mL−1. T2 values are 140.4±3.7 ms, 103.2±0.4 ms, 93.2±0.8 ms, 88.8±0.8 ms, and 81.2±2.6 ms (P<0.05, n=3).Abbreviation: M-HFn, ferrimagnetic H-ferritin; MR, magnetic resonance; MRI, magnetic resonance imaging.](/cms/asset/8944b6db-53b5-4a1e-956d-735d281142ed/dijn_a_80025_f0005_c.jpg)
Figure 6 Iron uptake analysis of MDA-MB-231 tumor cells incubated with M-HFn nanoparticles.
Notes: Prussian blue staining of MDA-MB-231 tumor cells incubated for 24 hours with (A) no nanoparticles, (B) M-HFn1000, (C) M-HFn3000, (D) M-HFn5000, and (E) M-HFn7000. (F) Iron contents in single cell are 0.16 pg cell−1, 0.29 pg cell−1, 0.80 pg cell−1, and 1.23 pg cell−1 after incubation with M-HFn1000, M-HFn3000, M-HFn5000, and M-HFn7000, respectively, for 24 hours (statistical comparison of iron contents in single cell with cell-only yielded P=0.014, 0.002, 0.011, and 0.023 for M-HFn1000, M-HFn3000, M-HFn5000, and M-HFn7000, respectively).
Abbreviation: M-HFn, ferrimagnetic H-ferritin.
![Figure 6 Iron uptake analysis of MDA-MB-231 tumor cells incubated with M-HFn nanoparticles.Notes: Prussian blue staining of MDA-MB-231 tumor cells incubated for 24 hours with (A) no nanoparticles, (B) M-HFn1000, (C) M-HFn3000, (D) M-HFn5000, and (E) M-HFn7000. (F) Iron contents in single cell are 0.16 pg cell−1, 0.29 pg cell−1, 0.80 pg cell−1, and 1.23 pg cell−1 after incubation with M-HFn1000, M-HFn3000, M-HFn5000, and M-HFn7000, respectively, for 24 hours (statistical comparison of iron contents in single cell with cell-only yielded P=0.014, 0.002, 0.011, and 0.023 for M-HFn1000, M-HFn3000, M-HFn5000, and M-HFn7000, respectively).Abbreviation: M-HFn, ferrimagnetic H-ferritin.](/cms/asset/353af8c3-62e9-4e00-a06e-78e944e3e8e2/dijn_a_80025_f0006_c.jpg)
Figure 7 Representative images of immunohistochemical staining of MDA-MB-231 tumor tissues by incubating with M-HFn nanoparticles (DAB as the substrate).
Notes: (A) Without M-HFn nanoparticle incubation. Incubated with (B) M-HFn1000, (C) M-HFn3000, (D) M-HFn5000, and (E) M-HFn7000. (F) Different density mean shows that the tissue sections treated with M-HFn nanoparticles of larger core sizes enhance the efficiency of immunohistochemical staining (P<0.05).
Abbreviations: M-HFn, ferrimagnetic H-ferritin; DAB, 3,3′-diaminobenzidine tetrahydrochloride.
![Figure 7 Representative images of immunohistochemical staining of MDA-MB-231 tumor tissues by incubating with M-HFn nanoparticles (DAB as the substrate).Notes: (A) Without M-HFn nanoparticle incubation. Incubated with (B) M-HFn1000, (C) M-HFn3000, (D) M-HFn5000, and (E) M-HFn7000. (F) Different density mean shows that the tissue sections treated with M-HFn nanoparticles of larger core sizes enhance the efficiency of immunohistochemical staining (P<0.05).Abbreviations: M-HFn, ferrimagnetic H-ferritin; DAB, 3,3′-diaminobenzidine tetrahydrochloride.](/cms/asset/6a7c99f9-fde8-4cff-b480-27be74a35da1/dijn_a_80025_f0007_c.jpg)
Figure S1 Magnetostatic interaction analysis of M-HFn nanoparticles.
Notes: The Wohlfarth–Cisowski test curves of (A) M-HFn1000, (B) M-HFn3000, (C) M-HFn5000, and (D) M-HFn7000 at 5 K. R values are 0.5, 0.48, 0.44, and 0.40 for M-HFn1000, M-HFn3000, M-HFn5000, and M-HFn7000, respectively.
Abbreviations: M-HFn, ferrimagnetic H-ferritin; IRM, isothermal remanent magnetization; H, applied magnetic field; DCD, direct current field demagnetization; R, magnetostatic interaction parameter.
![Figure S1 Magnetostatic interaction analysis of M-HFn nanoparticles.Notes: The Wohlfarth–Cisowski test curves of (A) M-HFn1000, (B) M-HFn3000, (C) M-HFn5000, and (D) M-HFn7000 at 5 K. R values are 0.5, 0.48, 0.44, and 0.40 for M-HFn1000, M-HFn3000, M-HFn5000, and M-HFn7000, respectively.Abbreviations: M-HFn, ferrimagnetic H-ferritin; IRM, isothermal remanent magnetization; H, applied magnetic field; DCD, direct current field demagnetization; R, magnetostatic interaction parameter.](/cms/asset/b2af598c-c814-4ccf-88ae-2be07e48a3e9/dijn_a_80025_sf0001_b.jpg)
Figure S2 Low- and room-temperature magnetic analysis of M-HFn nanoparticles.
Notes: Hysteresis loops of M-HFn nanoparticles measured at (A) 300 K and (B) 5 K. (C) Low-field (1.5 mT) magnetization curves as a function of temperature measured after ZFC and FC treatments of the M-HFn nanoparticles.
Abbreviations: M-HFn, ferrimagnetic H-ferritin; ZFC, zero-field cooling; FC, field cooling; M, magnetization; H, magnetic field; Tb, blocking temperature.
![Figure S2 Low- and room-temperature magnetic analysis of M-HFn nanoparticles.Notes: Hysteresis loops of M-HFn nanoparticles measured at (A) 300 K and (B) 5 K. (C) Low-field (1.5 mT) magnetization curves as a function of temperature measured after ZFC and FC treatments of the M-HFn nanoparticles.Abbreviations: M-HFn, ferrimagnetic H-ferritin; ZFC, zero-field cooling; FC, field cooling; M, magnetization; H, magnetic field; Tb, blocking temperature.](/cms/asset/71d20fd0-68ca-4425-9d6d-97e0a9b39b4a/dijn_a_80025_sf0002_b.jpg)
![Figure S2 Low- and room-temperature magnetic analysis of M-HFn nanoparticles.Notes: Hysteresis loops of M-HFn nanoparticles measured at (A) 300 K and (B) 5 K. (C) Low-field (1.5 mT) magnetization curves as a function of temperature measured after ZFC and FC treatments of the M-HFn nanoparticles.Abbreviations: M-HFn, ferrimagnetic H-ferritin; ZFC, zero-field cooling; FC, field cooling; M, magnetization; H, magnetic field; Tb, blocking temperature.](/cms/asset/7cfb8d1c-e77e-48a9-b0d9-fddb6972d06b/dijn_a_80025_sf0002a_b.jpg)
Figure S3 Analysis of transverse relaxivity of M-HFn nanoparticles by linear mapping iron concentration and 1/T2.
Notes: Transverse relaxivity (r2) analysis of (A) M-HFn1000, (B) M-HFn3000, (C) M-HFn5000, and (D) M-HFn7000. Slope of line represents r2.
Abbreviations: M-HFn, ferrimagnetic H-ferritin; T2, transverse relaxation times.
![Figure S3 Analysis of transverse relaxivity of M-HFn nanoparticles by linear mapping iron concentration and 1/T2.Notes: Transverse relaxivity (r2) analysis of (A) M-HFn1000, (B) M-HFn3000, (C) M-HFn5000, and (D) M-HFn7000. Slope of line represents r2.Abbreviations: M-HFn, ferrimagnetic H-ferritin; T2, transverse relaxation times.](/cms/asset/41796c51-7d11-46e8-9c76-1549c08c2685/dijn_a_80025_sf0003_b.jpg)
Figure S4 Analysis of longitudinal relaxivity of M-HFn nanoparticles by linear mapping iron concentration and 1/T1.
Notes: Longitudinal relaxivity (r1) analysis of (A) M-HFn1000, (B) M-HFn3000, (C) M-HFn5000, and (D) M-HFn7000. Slope of line represents r1.
Abbreviations: M-HFn, ferrimagnetic H-ferritin; T1, longitudinal relaxation times.
![Figure S4 Analysis of longitudinal relaxivity of M-HFn nanoparticles by linear mapping iron concentration and 1/T1.Notes: Longitudinal relaxivity (r1) analysis of (A) M-HFn1000, (B) M-HFn3000, (C) M-HFn5000, and (D) M-HFn7000. Slope of line represents r1.Abbreviations: M-HFn, ferrimagnetic H-ferritin; T1, longitudinal relaxation times.](/cms/asset/8269d8ea-5b98-4818-9906-e8dd5b47e973/dijn_a_80025_sf0004_b.jpg)