FITC-CRD13 dendrimer (un)locked in liposomal vesicles characterized by size exclusion chromatography and dynamic light scattering methods

Authors: Petra Kohútová 1    Zuzana Garaiová 1    Milan Zvarík 1    Veronika Šubjaková 1    Sylwia Michlewska 2,3    Maksim Ionov 2    Iveta Waczulikova 1    Javier de la Mata 4,5,6    Maria Bryszewska 2    Tibor Hianik 1   
1 Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia    2 Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland    3 Laboratory of Microscopic Imaging and Specialized Biological Techniques, Faculty of Biology and Environmental Protection, University of Lodz, Poland    4 Inorganic Chemistry Department, University Alcala, Alcala de Henares, Spain    5 Ramón y Cajal Health Research Institute (IRYCIS), IRYCIS, Spain    6 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain   
Year: 2021
Section: Biophysics, physics, mathematical modeling, biostatistics
Abstract No.: 2246
ISBN: ISBN 978-80-972360-7-6

Different types of nanoparticles were synthetized and studied either as a drug itself or as a drug delivery system aiming to combine benefits of individual particles. In this work we focused on the encapsulation of fluorescently labelled ruthenium dendrimers (FITC-CRD13) possessing anticancer properties [1] into the lipid vesicles composed of DMPC/DMPG/Cholesterol. FITC-CRD13 dendrimers (10 µM) were encapsulated into the liposomes by passive loading during the hydration step of lipid film (t = 1.5 h, T = 40°C) in sodium-phosphate buffer (10 mM, pH 7.4) upon the continuous shaking. Subsequently, the prepared vesicles were extruded through 400nm polycarbonate membrane and characterized by 1) absorption and fluorescence spectra of nanoparticles using size exclusion chromatography (SEC-HPLC) on a 30 nm size pore column; and 2) size measurements using dynamic light scattering (DLS). Absorbance at 220 nm and fluorescence at 495/520 nm was used for detection of liposomes and FITC-CRD13 dendrimers, respectively.  Firstly, liposomes were eluted at 3.35 min. Fluorescence intensity measured along the elution profile centred at this time point was monitored for dendrimers being locked in liposomes. The coelution, thus encapsulation was less extensive because the subsequent peaks in dendrimer elution profile were observed, indicating the presence of non-entrapped material sized around 70 nm at the later retention times. Hydrodynamic diameter of purified dendrimer-liposomal sample that coeluted at 3.35 min was 210±10 nm with polydispersity index 0.1 reflecting an uniform sample with respect to the particle size. The obtained results can serve for optimization of the encapsulation protocol and as a base for further studies of the dendrimer-liposomal drug delivery platform in the cancer treatment.

This work has been financially supported by Science Grant Agency VEGA, project No. 1/0756/20; by Agency for Promotion Research and Development, project No. SK-PL-18-0080 and SK-BY-RD-19-0019; by KEGA, project No. 041UK-4/2020 and by NAWA International Academic Partnership Programme EUROPARTNER.
[1] Michlewska, S. et al., 2017. Ruthenium metallodendrimers with anticancer potential in an acute promyelocytic leukemia cell line (HL60), European Polymer Journal. Elsevier Ltd, 87, pp. 39–47. doi: 10.1016/j.eurpolymj.2016.12.011.