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

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.


Materials and Methods
Chemical vapor deposition grown SWCNTs were purified by steam treatment. The chemicals and solvents were obtained from commercial suppliers and used without further purification.
The solvents used for synthesis were analytical grade. When anhydrous conditions were required, high quality commercial dry solvents were used. Water was purified using a Millipore filter system MilliQ®. When stated, suspensions were sonicated in a water bath (20 W, 40 kHz). For CNT filtration, PTFE membranes from Millipore were employed. Dialysis of CNT compounds was carried out employing membrane with MWCO 12000-14000 Da, purchased from Spectrum Laboratories, Inc. The UV-Vis analyses were performed on a Varian Cary 5000 spectrophotometer and the Kaiser test was performed according to reported procedures. 1,2 Thermogravimetric analysis (TGA) was performed using about 300 µg of sample on a TGA1 (Mettler Toledo) apparatus from 30 °C to 900 °C with a ramp of 10 °C·min -1 under N2 using a flow rate of 50 ml/min and platinum pans. The estimation of the degree of functionalization by TGA was done by taking weight loss values at 650 °C.
Transmission electron microscopy (TEM) was performed on a Hitachi H7500 microscope (Tokyo, Japan) with an accelerating voltage of 80 kV and equipped with an AMT Hamamatsu camera (Tokyo, Japan). To prepare the TEM grids, the CNTs were dispersed in a mixture of deionized water and ethanol (1:1) at a concentration of 50 µg/ml using water bath sonication.
Ten microliters were deposited onto a carbon-coated copper TEM grid (Formvar/Carbon 300 Mesh; Cu from Delta Microscopies), which was dried under ambient conditions.

Synthesis of SWCNT-NH2 (1)
In a flame-dried Schlenk tube, pristine SWCNTs (10 mg) were dispersed in dry o- For the deprotection, functionalized CNTs (5 mg) were dispersed in EtOH (10 ml) by sonicating for 10 min, and afterwards treated with hydrazine hydrate (0.5 ml). The dispersion was stirred at r.t. for 2 h, and then diluted with EtOH (10 ml) and filtered (0.1 µm). The recovered CNTs were washed with MeOH (x2) and acetone (x2), and finally dried under vacuum. The free amine loading assessed by the Kaiser test is 104 μmol/g.
The Pht-protected precursor of conjugate 1 was characterized by TGA performed under inert atmosphere, confirming the occurrence of the functionalization (weight loss of the phthalimide conjugate is 6.8% at 650 °C, corresponding to a molar loading of 246 µmol/g).

Apoptosis in RAW macrophages
Supplementary Figure S2. Percentage of apoptotic 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 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.