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Preparation and characterization of liposomal-pegilated calix[4]arenes nanoparticles as drug delivery systems for curcumin Elena Drakalska 1*, Denitsa Momekova 1, Liudmil Antonov 2, Desislava Budurova 3, Nikolay Lambov 1, Stanislav Rangelov 3. – Medical University of Sofia, Faculty of Pharmacy – Bulgarian Academy of Sciences, Institute of Organic Chemistry 3 – Bulgarian Academy of Sciences, Institute of Polymers 1 2 * - [email protected] INTRODUCTION Curcumin, the constituent of Curcuma longa, is considered a very promising anticancer agent due to its potent and pleiotropic antineoplastic activity and low nonspecific toxicity to normal cells (1). However the clinical realization of its potential has been limited due to its poor aqueous solubility (11ng/ml) and very low systemic bioavailability. A possible aproach to overcome these limitations is the design of nanosized vechicles of curcumin. The present work reports the preparation, characterization and in vitro evaluation of antineoplastic activity of curcumin loaded calixarenes-in liposomes nanoparticles. EXPERIMENTAL Solubility studies The solubility of curcumin was determined in aqueous complexation media containing different amount of pegilated tert-butylcalix[n]arenes (0-10 mg/ml) (n=4 or 8 aril units). The hydrophobic properties of curcumin allow it to be incorporated into the liposomal bilayer. We found that the optimal curcumin to phospholipids ration is 0,25:1 (mol:mol). In order to increase the entrapment capacity of curcumin into the liposomes we encapsulate calixarene-solubilized curcumin within the liposomes’ aqueous cavity. The basic physico-chemical and technological characteristics of liposomes are given in table 1. Quantitative analysis of curcumin encapsulation For qantitative determination of loaded curcumin a validated UV-VIS spectroscopic method was used (=427nm). Cytotoxicity assay 18 16 14 12 10 8 6 4 2 0 100 1000 Diameter (nm) Solubility of curcumin (mol/ml) 0,025 0,020 0,015 0,010 0,005 0,000 0,00 0,02 0,04 0,06 0,08 0,10 0,12 PEG-Calix[n]arenes concentration (mol/ml) Figure 1. Phase-solubility diagrams of curcumin in the presence of varying concentrations of pegilated calyx[n]arenes. 145.5 1.25 - 21,8 2,5 95 % 40% 10000 16 14 12 10 8 6 4 2 0 b Figure 2. Size distribution of PEGCX-4 free (a) and PEG-CX-4 curcumin loaded liposomes (b). 100 1000 10000 Diameter (nm) IC50 (μmol/L) (n=8) KG-1a RPMI-8226b Curcumin (DMSO solution) 13,45 2,31 2,89 0,77 Liposomal-PEG-CX-4 curcumin 2,19 0,71 0,59 0,21 PEG-CX-4 curcumin complexes 8,70 1,44 2,22 0,79 G1 и G2 G1 S - фаза S G2 Sub-G1 Apoptoic (Sub-G1) Control Sub-G1 Sub-G1 Sub-G1 CURC (½ IC50) 34% apoptoic cells 0,030 25% Formulations By forming water soluble complexes the PEG-CX-4 and PEGCX-8 proved to drastically increase the water solubility of curcumin from 0,25 ng/ml up to 200g/ml. The pegilated calix[8]aren is proved to show higher solubilizing capacity for curcumin as compared with the pegilated calix[4]aren probably due to its larger inner cavity (Fig.1). The solubility enhancement factor () of curcumin in the presence of the lowest concentration (0,03 mol/ml) of PEG-CX-4 or PEG-CX8 was found to be 800 % and 1691 % respectively. 0,035 98 % a RESULTS 0,040 Entrapment capacity (%) Cytotoxic effect and pro-apoptotic activity of free curcumin and curcumin loaded into liposomes or PEG-CX-4 complexes was investigated on two human cancer cell lines. IC50 values are presented on Fig. 3 and table 2 The cytotoxicity of free and loaded curcumin was evaluated by using the MTT-dye reduction assay in panel of human cancer cell lines. The tested compounds were applied in concentration range 6.5 to 25M for 72 h. PEG-Calix[4]arene PEG-Calix[8]arene Encapsulation efficacy (%) 133.1 1.15 Zetapotential (mV) - 26,3 7,5 Intensity (%) The size and size distribution patterns of curcumin loaded liposomes were investigated by ZetaSizer NanoZS (Malvern Instruments)The parameters were evaluated from measurements in the scattering angle of 173°, at 25°C. PEG-CX-4 free liposomes PEG-CX-4 loaded liposomes Intensity (%) Preparation of liposomes: Liposomes were prepared by modified thin film hydration method and extrusion trough polycarbonate membrane filters with pore size of 100nm. Dynamic light scattering 0,045 Size (nm) Formulation CURC-LIPO (½ IC50) 27% apoptoic cells CURC CX-4-PEG (½ IC50) 21 % apoptoic cells Figure 3. Pro-apoptotic activity of free curcumin, liposomal-PEG-CX-4 (CURCLIPO)curcumin and PEG-CX-4 curcumin complexes (CURC-CX-4-PEG) after 24 hours treatment. CONCLUSION: Thus on the grounds of the excellent in vitro biocompatibility profile and the favorable physicochemical and drug loading characteristics of the tested liposomal nanoparticles, and their ability to retain the instirsnic pharmacological properties of encapsulated drug they could be considered promising drug delivery platforms for lipophilic curcumin. Acknowledgements: Financial support from National Science Fund of Bulgaria, Grant Nr: ДФНИ 501/25