(T1230-04-21) In Vitro Cytotoxicity and Biodistribution Study of Oxaliplatin-Loaded Multivesicular Liposomal Depot

Purpose: In this study, oxaliplatin-loaded multivesicular liposome (OX MVL) was prepared by double-emulsion method to prolong the release time of oxaliplatin. In vitro cytotoxicity study on HCT116 was conducted. Ex vivo biodistribution and MVL degradation were observed by IVIS.

Methods: OX MVL were prepared using the ‘water-in-oil-in-water’ emulsification procedure previously described by Abuzar et al. Cytotoxic studies were determined in human colon adenocarcinoma cells HCT116. Cytotoxicity assay was conducted using EZ-Cytox WST-1 assay kit. The cells were exposed for 24 and 72 h for 2 groups (OX MVL and OX solution). The absorbance was measured by a microplate reader (Infinite M200 Pro, Tecan, Austria) at 490 nm. Biodistribution of MVL was evaluated in BALB/c mice by IVIS (FOBI; CELLGENTEK, Korea). Group 1 was treated with dye-labeled MVL with Rhodamine DHPE and Cy 5.5 NHS ester and Group 2 was treated with 5% dextrose solution with Cy5.5 NHS ester. Integrated density was calculated according to: Integrated density=Intensity unit (IU)x Area

Results: In vitro cytotoxicity study of OX MVL and OX solution was conducted on HCT116. At 24h, IC50 of OX MVL exceeded 200 μM whereas IC50 of OX 5% solution was 113.98 μM. The IC50 value for OX MVL against HCT116 cell lines was found to be higher than that of OX 5% dextrose solution. On the other hand, at 72 h, IC50 of OX MVL was 8.37 μM, IC50 of OX 5% dextrose solution was 9.62 μM, and IC50 values at 72 h of both OX MVL and OX 5% dextrose solution were observed to be the similar. The biodistribution imaging studies were conducted in BALB/c mice. Two organs, the liver and spleen exhibited different integrated density of Cy 5.5 NHS ester in the MVL treatment compared to solution treatment. In the liver, the intensity gradually decreased from 1 h to 168 h for MVL, while the intensity for solution decreased sharply to 6.97% of the intensity at 1 h, maintaining the intensity after 24 h. The integrated density of Rhodamine DHPE remained highest in the liver from 1 h to 168 h. Unlike the liver, other organs showed a high integrated density at 1 h but the density started to drastically decrease at 24 h. The biodistribution imaging studies were conducted in BALB/c mice. Two organs, the liver and spleen exhibited different integrated density of Cy 5.5 NHS ester in the MVL treatment compared to solution treatment. In the liver, the intensity gradually decreased from 1 h to 168 h for MVL, while the intensity for solution decreased sharply to 6.97% of the intensity at 1 h, maintaining the intensity after 24 h. The integrated density of Rhodamine DHPE remained highest in the liver from 1 h to 168 h. Unlike the liver, other organs showed a high integrated density at 1 h but the density started to drastically decrease at 24 h.

Conclusion: OX MVL showed less cytotoxicity compared to OX solution. Biodistribution images of BALB/c mice showed that OX MVL remained in the liver and spleen for more than 5 days while OX solution was excreted after 1 hour at the same inspection sites.

References: Abuzar, Sharif Md, et al. "Preparation and evaluation of intraperitoneal long-acting oxaliplatin-loaded multi-vesicular liposomal depot for colorectal cancer treatment." Pharmaceutics 12.8 (2020): 736.

Acknowledgements:

Funding: This work was supported by the Mid-Career Researcher Program (No. NRF-2021R1A2C2008834) and Basic Research Infrastructure Support Program (University-Centered Labs-2018R1A6A1A03023718) through the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT)
We thank the Yonsei Center for Research Facilities (YCRF, Seoul, Republic of Korea) and CELLGENTEK (Gyeonggi, Republic of Korea) for the providing analytical support. Thanks go to Editage (www.editage.co.kr) for English language editing.


Cell viability of OX-MVL and the OX 5% dextrose solution: (A) 24 hours after treatment and (B) 72 hours after treatment. Ez-cytox P < .05 (*) and P < .01 (**) was considered statistically significant. Results are expressed as mean ± SD (n=3).


Ex vivo fluorescence imaging of isolated major organs from Balb/C mice (n = 45) collected at different time points (1h, 24h (1d), 72h (3d), 120h (5d), 168h (7d)) post treating with an intraperitoneal injection of Cy5.5 solution (Red) and Rhodamine dye-loaded MVL (Green). Abbreviations: H: heart, Lu: lung, Li: liver, S: Spleen, K: Kidney.