Development of Liposomes Containing NADH Incorporated Fullerene C70
Authors: Ryuto Koike , Sunaga Haruka , Yamamoto Rie , Nishimura Kotaro , Ikeda Atsushi , Konishi Toshifumi 
Published: December 2024

In recent years, photodynamic therapy (PDT) has been attracting attention as a cancer treatment that is less stressful on the body.

In PDT, the photosensitizer is injected intravenously and accumulates in tumor tissue. The photosensitizer is excited by irradiation of red laser light. It in the excited state gives energy to dissolved oxygen, generating reactive oxygen species (ROS). This is how the photosensitizer attacks cancer cells.

Photofrin is currently used as a photosensitizer in the medical field, but there are three issues with it. (1) Tumor tissue is in a hypoxic environment, so it does not produce enough singlet oxygen (1O2
*), one of the ROS. (2) It does not selectively accumulate in cancer cells. (3) After treatment, the photosensitizer flows in the body, causing a side effect called photosensitivity.

We focused on the fact that C70, a photosensitizer, reacts with NADH to form the C70 anion radical, which reduces dissolved oxygen to produce the superoxide anion radical (O2・－), one of the ROS. Compared to 1O2
*, the O2・－ has slightly less oxidizing power to attack cells, but has a very long lifetime. This led us to believe that it would be possible to obtain sufficient photodynamic activity even in tumor tissue. However, since the absorption wavelength of C70 is not in the wavelength range of red laser light. We decided that 1-1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocy anine（DiD）would compensate for this. Furthermore, we believed that incorporation into liposomes suitable for drug delivery system（DDS）would enhance selective accumulation in cancer cells. Liposomes have the property of accumulating in tumor tissues due to enhanced permeability and retention (EPR) effect by controlling the particle size to less than 200 nm. In addition, the negative charge of the generated O2・－ causes the liposomes formed by the positive charge to collapse and fuse. This causes the liposomes to cease to function and reduces the risk of photosensitivity.

In this study, we prepared monolayer liposomes with a particle diameter of 200 nm, which contain NADH in the inner aqueous phase and a photosensitizer, C70, and a light-harvesting molecule, DiD, in the lipid bilayer.

The prepared liposomes were measured with an absorption spectrophotometer, and NADH, C70, and DiD specific peaks were observed, respectively. Next, the particle diameter was measured by DLS, and it was found that the particle diameter could be controlled to less than 200 nm. Furthermore, the particle diameter after light irradiation was measured, and it was confirmed that the particle diameter was larger. Finally, the liposomes were added to HeLa cells and the cell viability was measured in the absence and irradation of light at various oxygen concentrations. The results showed that there was no change in cell viability when the cells were not exposed to light, and only when the cells were exposed to light did the cell viability decrease to the same extent as that of the Photofrin.

Based on the results of absorption spectrophotometry and DLS measurements, we believe that liposomes containing NADH incorporated C70 could be fabricated below 200 nm. In addition, the change in particle size after light irradiation suggested that disintegration and fusion had occurred. The safety of the liposomes at the cellular level was confirmed by the fact that they did not show toxicity when not exposed to light. And when irradiated with light at various oxygen concentrations, it showed the same level of toxicity as Photofrin, suggesting that the approach using O2・－ is working well.

These findings suggest that liposomes containing NADH incorporated C70 may hold promise as a safe PDT agent that can overcome the challenges of Photofrin.