Since malignancies have long been the second most common cause of death, it is important to explore new approaches to the treatment of cancer patients. In this context, we focused on a novel dual nanoparticle-based drug delivery platform. Specifically, we combined a fluorescently labelled carbosilane-ruthenium dendrimer FITC-CRD13 possessing anticancer properties [1] with liposomes and investigated its cytotoxic effect on adhesive breast (MCF-7) and colon (HT-29) cancer cells with expected minimal effect on the viability of healthy human fibroblasts (BJ). Liposomes were prepared by a hydration method using zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC; 15mM). FITC-CRD13 dendimer (0.15mM) was added during the hydration step of the lipid film. In order to reduce the size of liposomes and remove unencapsulated dendrimers, prepared samples were extruded through a 400 nm polycarbonate membrane and centrifuged thereafter.
Subsequently, the effect of DMPC+FITC-CRD13 liposomes (0.5; 1; 2.5; 5; 10 µM; presumed dendrimer concentration) on the viability of selected cell lines was investigated using MTT assay [2]. Non-treated cells as 100% viability reference (negative control) and methanol (10µM, positive control) were used.
Treatment of the cells with DMPC+FITC-CRD13 liposomes along the whole tested concentration range caused a viability drop to or below 80% for HT-29 and MCF-7 cancer cell lines. On the other hand, in BJ cell line, the viability was maintained at 80% or more. This suggests a selective effect of DMPC+FITC-CRD13 liposomes on the cell viability, with a desirable toxicity towards cancer cells. For all three tested cell lines, a larger decrease in cell viability upon the 3h incubation time in comparison to 24h, was observed. Concentration dependent relation between the applied DMPC+FITC-CRD13 and viability of cells was not significant, probably as a result of narrow concentration range chosen.
Obtained preliminary results will be used for further protocol optimization. Stability of DMPC+FITC-CRD13 liposomes as well as the effect of lipid composition should be tested.
This work was supported by the Slovak Research and Development Agency, APVV (Projects No. SK-PL-18-0080, APVV-14-0267, SK-BY-RD-19-0019), VEGA 1/0756/20, KEGA 041UK-4/2020 and by PL-SK 2019–2020 bilateral project of NAWA PPN/BIL/2018/1/00150; Polish National Agency for Academic Exchange, NAWA, PPI/APM/2018/1/00007/U/001.
[1] Michlewska et al. 2019: Synthesis and Characterization of FITC Labelled Ruthenium Dendrimer as a Prospective Anticancer Drug; In Biomolecules 2019, 9, 411, doi:10.3390/biom9090411.
[2] Chonco L. et al.: Carbosilane dendrimer nanotechnology outlines of the broad HIV blocker profile; In Journal of Controlled Release, 2012, 161, 949–958, doi:10.1016/j.jconrel.2012.04.050.
Prospects for further research
Hello Simon,
Thank you for the interesting contribution - there is no doubt that development of efficient and safe drug delivery systems for tumor targeting is of utmost importance. The combination of liposomes and dendrimers represents one of the possible ways to overwhelm toxic effects of anticancer drugs without compromising their activity towards tumor cells.
Your results however point to a very important issue of sufficient anticancer activity of the encapsulated compound at a known (or reasonably well estimated) amount. So I have a question - is it possible to make a reasonable estimate of the amount of entrapped dendrimers? Can you also be more specific about experiments planned in the near future? What other cell lines do you plan on working with? Do you consider also some changes in lipid composition?
I wish you all the best in your endeavours.
iwa
Re: Prospects for further research
Hello Iveta,
Thank you for your comments and questions.
1. We where thinking about estimating the final amount of entrapped liposomes using STED-microscopy and measuring the fluorescence of FITC-CRD-13 entrapped in the liposomes.
2. For the future we plan further experiments on healthy and cancer cells, to prove the cytotoxic effect of CRD-13 on cancer cells. Also we would like to try wider range of concentrations od CRD-13 to find out, which concentration is the most toxic for cancer cells and least toxic for healthy cells.
3. We plan to work with the same cell line, plus we would like to add some more adhesive (HeLa) and suspension (CEM-SS) cancer lines, if the further experiments go well.
4. We were thinking about adding some cholesterol to the membrane structure, to make the liposomal membrane more stiff and stable.
Interesting contribution
Hello Simon,
it is the interesting contribution,
why did you add dendrimers during the hydration step of the lipid film?
Re: Interesting contribution
Hello Lucia
Thank you for your question. From previous experiments we estimated that liposome-CRD-13 complex is more stable when CRD-13 is added during the hydration step, in comparison when CRD-13 was added with the organic solvent the complex proved to be less stable. Because of this fact, we decided to add CRD-13 during hydration step.
nice work
Hi Šimon,
can you explain why size of DMPC is larger than size of DMPC+FITC-CRD13?
Re: nice work
Hello Daniela,
Thank you for your question. Previous work of Michlewska S. et al. (2019) indicates, that CRD-13 interacts with both hydrophobic and hydrophilic part of the membrane of liposomes. Therefore we assume that these interactions are the reason for lower stability of DMPC+FITC-CRD-13 complex and their smaller size, in comparison to pure DMPC liposomes.