Evaluation of the immunological profile of antibody-functionalized metal-filled single-walled carbon nanocapsules for targeted radiotherapy

Abstract

This study investigates the immune responses induced by metal-filled single-walled carbon nanotubes (SWCNT) under in vitro, ex vivo and in vivo settings. Either empty amino-functionalized CNTs [SWCNT-NH₂ (1)] or samarium chloride-filled amino-functionalized CNTs with [SmCl₃@SWCNT-mAb (3)] or without [SmCl₃@SWCNT-NH₂ (2)] Cetuximab functionalization were tested. Conjugates were added to RAW 264.7 or PBMC cells in a range of 1 μg/ml to 100 μg/ml for 24 h. Cell viability and IL-6/TNFα production were determined by flow cytometry and ELISA. Additionally, the effect of SWCNTs on the number of T lymphocytes, B lymphocytes and monocytes within the PBMC subpopulations was evaluated by immunostaining and flow cytometry. The effect on monocyte number in living mice was assessed after tail vein injection (150 μg of each conjugate per mouse) at 1, 7 and 13 days post-injection. Overall, our study showed that all the conjugates had no significant effect on cell viability of RAW 264.7 but conjugates 1 and 3 led to a slight increase in IL-6/TNFα. All the conjugates resulted in significant reduction in monocyte/macrophage cell numbers within PBMCs in a dose-dependent manner. Interestingly, monocyte depletion was not observed in vivo, suggesting their suitability for future testing in the field of targeted radiotherapy in mice.

Figure 1. Molecular structures of SWCNT-NH₂ (1), SmCl₃@SWCNT-NH₂ (2) and SmCl₃@SWCNT-mAb (3).

The left-side open end of compounds 2 and 3 is meant to visually indicate the extended length of the nanotubes.

Figure 2. Electron microscopy characterization of empty and SmCl₃ filled SWCNT conjugates.

Low resolution TEM images of pristine SWCNTs (a), Pht-protected SWCNT-NH₂ 1 (b) and SWCNT-NH₂ 1 (c). (d,g) HRTEM image of SmCl₃@SWCNT precursor and corresponding EDX spectrum. (e,h) HAADF-STEM image of SmCl₃@SWCNT-NH₂ (2) showing the bright lines arising from the filling material and EDX spectrum showing the nitrogen signal associated with the attached amino functional groups. (f,i) Sm and Au EDX line profiles acquired in STEM mode showing the location of the filling compound and the gold nanoparticles (AuNPs) in SmCl₃@SWCNT-mAb (3) after being immunostained with a secondary antibody conjugated with AuNPs.

Figure 3. Viability of RAW 264.7 macrophages.

Cells were incubated with the three SWCNT conjugates for 24 h at increasing concentrations (1, 10, 25, 50 and 100 μg/ml). DMSO (20%) was used as a positive control of death. Cell viability was determined with Annexin V/Propidium iodide staining and quantified by flow cytometry and no significant differences were observed for all compounds after 24 h of incubation (n = 3). Values are expressed as mean ± SD. **p < 0.01 with respect to untreated cells.

Figure 4. Activation of RAW 264.7 macrophages.

RAW 264.7 cells were incubated with the SWCNT conjugates for 24 h at increasing concentrations (1, 10, 25, 50 and 100 μg/ml), then stained for CD86+ expression. LPS combined with IFN-γ was used as a positive control of activation. Mean fluorescence intensity of CD86+ cells was measured to express degree of cell activation, following treatments with the conjugates. No significant differences were observed for any of the treatments. Values are expressed as mean ± SD (n = 3). *p < 0.05 with respect to untreated cells.

Figure 5. Cytokine production by RAW 264.7 macrophages.

RAW 264.7 macrophages were incubated with the SWCNT conjugates for 24 h at increasing concentrations (1, 10, 25, 50 and 100 μg/ml), followed by measurements of IL-6 (top) and TNFα (bottom) cytokine production by ELISA. Values are expressed as mean ± SD (n = 3). The absence of bars indicates negligible levels. **p < 0.01 with respect to untreated cells.

Figure 6. Effect of treatments with conjugates on PBMC viability and subpopulation cell numbers.

PBMCs were incubated with the SWCNT conjugates for 24 h at increasing concentrations (1, 10, 25, 50 and 100 μg/ml), followed by flow cytometry measurements via propidium iodide staining to determine cell viability. Further staining was performed for CD3 (T lymphocytes, L_T), CD19 (B lymphocytes, L_B) or CD14 (monocytes/macrophages) to determine cell numbers within each of the subpopulation by flow cytometry. DMSO was used as a positive control. Percentage cell viability (bars) (PI staining) and number of positive events (black lines) are shown for L_T CD3+, L_B CD19+ and monocytes/macrophages CD14+ (from top to bottom). Values are expressed as mean ± SD (n = 3). **p < 0.01 with respect to untreated cells (% live cells). ^#p < 0.05 with respect to untreated cells (events vs. untreated).

Figure 7. Cytokine production by PBMCs.

PBMCs were incubated with the SWCNT conjugates for 24 h at increasing concentrations (1, 10, 25, 50 and 100 μg/ml), followed by measurements of IL-6 (top) and TNFα (bottom) cytokine production by ELISA. Values are expressed as mean ± SD (n = 3). The absence of bars indicates negligible levels. **p < 0.01 with respect to untreated cells.

Figure 8. Percentage of monocyte/macrophage population within PBMCs after tail vein injection of the conjugates in mice.

C57Bl6 mice were injected via the tail vein with 150 μl of one of the conjugates (1 mg/ml), PBS (negative control), LPS (3 mg/kg) (positive control). Whole blood was collected at 1, 7 and 13 days post-injection and cells were stained for CD3e (L_T), CD45R/B220 (L_B) or CD11b (monocytes/macrophages) expression. Percentage of CD11b+ cells (monocytes/macrophages) in PBMCs was calculated. Values are expressed as mean ± SD (n = 4). **p < 0.01 with respect to the other samples.