Application of a double-colour upconversion nanofluorescent probe for targeted imaging of mantle cell lymphoma

Abstract

Upconversion nanoparticles are a new type of fluorescent marker in biomedical imaging that can convert a longer wavelength (such as near-infrared fluorescence) into a shorter wavelength (such as visible light). Mantle cell lymphoma, which is derived from B-cell lymphoma, is a subtype of non-Hodgkin's lymphoma, and the immune phenotype is a mature B-cell phenotype (CD20+, CD5+). To develop the use of nanomaterials as specific markers for the medical imaging of mantle cell lymphoma, we modified the surface of UCNPs by oxidation so that the CD20 or CD5 antibody could covalently attach to the upconversion nanoparticles to form antibody-UCNP conjugates. These antibody-UCNP conjugates were used as fluorescent probes to detect the CD20 or CD5 antigen. Due to the excessive expression of these antigens on the surface of MCL cells and successful strong connection between the antibody and UCNPs, the latter could specifically combine with mantle cell lymphoma cells. Upon near-infrared excitation at 980 nm, cells labelled with UCNPs emitted bright upconversion fluorescence.

Keywords: biological imaging; immune labelling; mantle cell lymphoma; nanoprobe; upconversion fluorescence.

Figure 1. Upconversion imaging of UCNPs

(A) Imaging with no near-infrared laser excitation. (B) Imaging with a near-infrared laser excitation power of 0.7 W. (C) Imaging with a near-infrared laser excitation power of 1 W.

Figure 2. FT-IR spectral analysis of UCNPs

(A) FT-IR spectrum of oxidized UCNPs by H2O2. (B) FT-IR spectrum of UCNPs reacted with NHS and EDC. (C) FT-IR spectrum of UCNPs reacted with antibody.

Figure 3

(A) Viability of Jeko-1 cells when they were cultured with different amounts of UCNPs suspension at 37°C and 5% CO₂ for 24 h, 48 h and 72 h. (B) Imaging of viable cells when Jeko-1 cells were cultured with 160 μl of UCNP suspension for 72 h.

Figure 4. Upconversion fluorescence images of Jeko-1 cells after incubation of cells with a mixture including the UCNP-CD20 antibody conjugates and UCNP-CD5 antibody conjugates for 2 h

(A) Bright field imaging; (B) Dark field fluorescence imaging in the green channel; (C) Dark field fluorescence imaging in the blue channel; (D) Overlap of A and B; (E) Overlap of A and C; (F) Overlap of D and E.

Figure 5. Upconversion fluorescence images of Jeko-1 cells after incubation of the cells with a mixture containing UCNP-CD20 antibody conjugates and UCNP-CD5 antibody conjugates for 2 h

(A) Bright field imaging; (B) Dark field fluorescence imaging in the green channel; (C) Dark field fluorescence imaging in the blue channel; (D) Overlap of A and B; (E) Overlap of the A and C; (F) Overlap of D and E.

Figure 6. Upconversion fluorescence images of SP50B cells after incubation of the cells with a mixture containing UCNP-CD20 antibody conjugates and UCNP-CD5 antibody conjugates for 2 h

(A) Bright field imaging; (B) Dark field fluorescence imaging in the green channel; (C) Dark field fluorescence imaging in the blue channel; (D) Overlap of A and B; (E) Overlap of A and C; (F) Overlap of D and E.

Figure 7. Upconversion fluorescence images of Jeko-1 cells after incubation of the cells with a mixture containing NaYF⁴:Er3⁺ nanoparticle suspension without CD20 antibody and CD5 antibody-NaYF⁴: Yb3⁺/Tm3⁺ nanoparticle suspension for 2 h

(A) Bright field imaging; (B) Dark field fluorescence imaging in the green channel; (C) Dark field fluorescence imaging in the blue channel; (D) Overlap of A, B and C.

Figure 8. Upconversion fluorescence images of Jeko-1 cells after incubation of the cells with a mixture containing CD20 antibody-NaYF₄: Er³⁺ nanoparticle suspension and NaYF₄: Yb³⁺/Tm³⁺ nanoparticle suspension without CD5 antibody for 2 h

(A) Bright field imaging; (B) Dark field fluorescence imaging in the green channel; (C) Dark field fluorescence imaging in the blue channel; (D) Overlap of A, B and C.

Figure 9. Upconversion fluorescence images of Jeko-1 cells after incubation of the cells with a mixture containing NaYF⁴: Er³⁺ nanoparticle suspension and NaYF₄: Yb³⁺/Tm³⁺ nanoparticle suspension without any antibody for 2 h

(A) Bright field imaging; (B) Dark field fluorescence imaging in the green channel; (C) Dark field fluorescence imaging in the blue channel; (D) Overlap of A, B and C.

Figure 10

(A) Upconversion imaging of a mouse that was subcutaneously inoculated with Jeko-1 cells after tail vein injection of UCNP-CD20 antibody suspension for 12 h, 48 h, 96 h, and 126 h. (B) Upconversion imaging of a mouse that was subcutaneously inoculated with Jeko-1 cells after the tail vein injection of the UCNP-CD5 antibody suspension for 12 h, 48 h, 96 h, and 126 h.

Figure 11

(A) Upconversion imaging of a mouse that was subcutaneously inoculated with SP50B cells after the tail vein injection of the UCNP-CD20 antibody suspension for 12 h, 48 h, 96 h, and 126 h. (B) Upconversion imaging of a mouse that was subcutaneously inoculated with SP50B cells after tail vein injection of the UCNP-CD5 antibody suspension for 12 h, 48 h, 96 h, and 126 h.

Figure 12. Upconversion imaging of a mouse that was subcutaneously inoculated with SP50B cells after tail vein injection of the UCNP suspension without any antibody

(A) Imaging without NIR; (B) Upconversion imaging with NIR after tail vein injection of the UCNP suspension without any antibody after 48 h; (C) Upconversion imaging with NIR after tail vein injection of the UCNP suspension without any antibody after 96 h; (D) Upconversion imaging with NIR after tail vein injection of the UCNP suspension without any antibody after 115 h.

Figure 13

(A) Upconversion imaging from the same mouse after changing the laser spot on the mouse tumour. (B) Upconversion imaging of a mouse that was subcutaneously inoculated with Jeko-1 cells after tail vein injection of the UCNP-CD5 antibody suspension for 220 h.

Figure 14

(A, B) HE imaging of tumour metastasis located in the liver. A: Observation using a 10× objective; B: Observation using a 20× objective. C, D: Immunohistochemistry imaging of tumour metastasis located in the liver. (C) Observation using a 10× objective; (D) Observation using a 20× objective. (E) Paraffin-embedded liver tissue imaging without near-infrared laser excitation; (F) Paraffin-embedded liver tissue imaging with near-infrared laser excitation; (G) HE slice imaging of liver tissue without near-infrared laser excitation; (H) HE slice imaging of liver tissue with near-infrared laser excitation.

Figure 15. Upconversion imaging of the HE slice of liver tissue using the Ti-S Eclipse inverted fluorescence microscope with a 980 nm near-infrared laser

(A) Observation using a 10× objective; (B) Observation using a 20× objective.