Subject Number : S-16-NM-0010
Support Type : 機器利用
Proposal Title (English) : 3D-cellular aggregates
Username (English) : Jukka Niskanen
Affiliation (English) : University of Helsinki, Finland

1. Summary
The aim was to study the mobility of boron nitride nanotubes (BNNT) in 3D cellular aggregates. The surface of the BNNTs was functionalized with polymers and amino acids, to adjust the charge of the BNNT surface. Thus, the mobility of the BNNTs should be affected.
2. Experimental
LCAM cellular aggregates on agarose-coated 96-well plates were prepared in in Dulbecco’s Modified Eagle Medium and incubated for 3 days at 37 °C in humidified air containing 5% CO2.
BNNTs were dispersed with glycine and cut by sonication.1 Yielding in 2-3 µm long BNNTs dispersible in water. The inner channel was loaded under reduced pressure with rhodamine-6G. Loaded BNNTs were surface functionalized with folic acid (FA-BNNTs), poly(L-lycine) (PLL-BNNTs), and PEG-poly(L-lycine) (PEG-PLL-BNNTs).
The functionalized BNNTs were introduced to cell aggregates on a 96-well plate. Before adding the BNNTs to the well, 75 µL of the medium was exchanged to fresh, followed by the addition of 15 µL (200 µg/mL) BNNT dispersion, resulting in 17 µg/mL BNNTs in each well. The aggregates were fixed and stained after 7 h, 24h, and 48 hours. For analysis of the cellular aggregates Leica microsystems, TCS SP5, confocal fluorescence microscope and Leica microsystems, DMIL-TR/EC3, microscope, were used.
3. Results and Discussion
The presence of BNNTs did not affect the growth of the cell aggregates. The diameters of the aggregates increased with a similar rate to control, up to 48 hours (table 1). After 7 hours of incubation, BNNTs could be observed up to 25 µm above the surface (figure 1). However, the BNNTs were bound to the first layer of cells in the aggregates and did not migrate further into the aggregates. This could be an effect of strong binding of the BNNTs to the first layer of cells. PLL is a cationic polymer, and thus it would bind strongly to the negatively charged cell membranes. Many cancer cells have receptors for folic acid, which will also lead to a strong binding of the BNNTs to the cells.

Table 1. Diameters of the LCAM-aggregates after 7, 24, and 48 hours after being introduced to BNNTs (17 µg/mL).

Figure 1. Confocal microscope overlay images of LCAM aggregates after being incubated with BNNTs for 7 hours, with the rhodamine-loaded BNNTs (red), the cell nuclei in blue (DAPI), and the actin cytoskeleton in green (AlexaFluor 488).
4. Others
Platform staffs kindly provided instructions of how to use instruments.

Reference
1. Niskanen, J.; Zhang, I.; Xue, Y.; Golberg, D.; Maysinger, D.; Winnik, F. M. Boron Nitride Nanotubes as Vehicles for Intracellular Delivery of Fluorescent Drugs and Probes. Nanomedicine 2016, 11 (5), 447–463.
5. Publication/Presentation
N/A
6. Patent
N/A.