# Formulation of Boron Encapsulated Smart Nanocapsules for Targeted Drug Delivery to the Brain
## 3 Results
### Preliminary Cell Studies
#### 3.3.3 Permeation Studies
All spectroscopic characterisation data indicate that the new chemical entities were successfully synthesised. Additionally, the increasing difficulties in purchasing carboranes in the UK have been overcome by the adopted synthetic strategy, which forms the carboranyl cage in situ. Preliminary in vitro evaluation consisted of uptake and toxicological studies. For the uptake studies, compounds 2 and 6 had an uptake of boron into CaCo2 cells after 24 h of 0.6 ppm. Conversely, compound 4 presented an uptake of 1.65 ppm. Comparison of boron accumulation with the control indicates a 20-fold increase in boron concentration into cancer cells compared to control. Similarly, the DLC-synthesized compounds showed an improved uptake potential on U-87 MG cells. These results confirm the high efficiency of DLC dyes in promoting the accumulation of boron into cancer cells. DLCs cause minimal variations in free energy change when crossing biological membranes, hence affording the diffusion of the boronated dye through the plasma membrane and into the cells. As revealed in Figures 20-23, nanovehicles showed the capability to permeate through the cellular monolayer. Boron levels obtained showed that after 6 h of testing, around 1.5, 1.4, and 1 ppm of boron had been able to permeate the cell using compound 2, 4, and 6 derivatives, respectively. After 24 h, there was a significant increase in permeation, as expected, with 2.5, 2.7, and 2.3 ppm of boron able to permeate the cellular monolayer for each compound, respectively. This is expected due to the increase in time, allowing more boron to enter the receiver compartment. These results have indicated that the NP delivery system is capable of permeating a cellular monolayer. Uptake studies were undertaken in conjunction with the permeation studies. U-87 MG cells were plated on a 24 well-plate and were then allowed to grow to a confluence of about 80%. Once this was achieved, the cells were put into the second part of the permeation and uptake assay. Samples were taken at 1, 6, and 24 h to check the amount of boron that was taken up by the cells. The result would then indicate how many nanoparticles have permeated the cell monolayer of CaCo2 and gone to the basolateral compartment. The results below show graphs of formulation made against boron taken up into cells (ppm). Figures 22 and 23 show uptake of boron measured on U-87 MG cells, with results indicating greater amounts of boron were taken up by cells over time. Boron uptake was also measured using an ICP-MS, after 6 h boron uptake registering 1.3, 1.7, and 0.7 ppm for the compounds 2, 4, and 6 derivatives, respectively. The boron uptake into cells increased on average 2-fold after 24 h, indicating the effectiveness of these vehicles to not only permeate the cellular monolayer but also to deliver boron to cancer cells. A similar trend could be seen for the PPy-CHIT NC preparations, shown in Figure 22. The permeation and uptake studies were repeated five times with an average boron concentration calculated from those five separate assays. The neutral red assay examined post-treatment the integrity of the cytoplasm of the cell. The cytoplasm is integral to the cell as it fills each cell enclosed within the cell membrane; they include all the material inside and outside the nucleus. The integrity of the cytoplasm allows for all the processes within the cell to occur. Once this scaffold is broken, the cell's integrity is compromised, and hence the cell is lysed. The positive control used was actinomycin-D, which inhibits transcription within the cell, killing it and giving it a low absorbance. The higher the absorbance values, the healthier the cell appears; this suggests that the cytoplasm could store a higher amount of neutral red dye. When tested on the CaCo2 and U-87 MG cell lines (Figure 19), the synthesized carboranyl DLCs showed a marked difference in cell health, therefore giving high absorbance values. This indicated that these compounds were nontoxic and suitable for use in the BNCT of tumours.