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. 2012 Nov;29(11):3064-74.
doi: 10.1007/s11095-012-0881-7. Epub 2012 Sep 28.

Formulation and characterization of polyester/polycarbonate nanoparticles for delivery of a novel microtubule destabilizing agent

Vaibhav Mundra  1 , Yan Lu, Michael Danquah, Wei Li, Duane D Miller, Ram I Mahato
Affiliations

Formulation and characterization of polyester/polycarbonate nanoparticles for delivery of a novel microtubule destabilizing agent

Vaibhav Mundra et al. Pharm Res. 2012 Nov.

Abstract

Purpose: Since our newly synthesized potent 5-indolyl derivative, (2-(1 H-Indol-5-yl) thiazol-4-yl) 3, 4, 5-trimethoxyphenyl methanone (LY293), to treat resistant melanoma was hydrophobic, our objective was to synthesize a biodegradable copolymer for formulating this drug into nanoparticles and to determine its anticancer activity and mechanism of action.

Methods: Methoxy poly (ethylene glycol)-b-poly (carbonate-co-lactide) [mPEG-b-P (CB-co-LA)] was synthesized for formulating LY293 into nanoparticles by o/w emulsification and stabilization by solvent evaporation. Particle size, drug release profile, in vitro efficacy in multiple melanoma cells, and mechanism of action of drug-loaded nanoparticles were determined.

Results: LY293-loaded nanoparticles with 170 nm mean size and 2.2 and 4.16% drug loading efficiently inhibited proliferation of A375 and B16F10 cells with IC(50) of 12.5 nM and 25 nM, respectively. LY293 circumvented multidrug resistance and inhibited proliferation of Pgp overexpressing MDA-MB435/LCC6 MDR1 melanoma cells. Upon treatment with LY293-loaded nanoparticles, A375 cells underwent cell cycle arrest in G2/M phase and apoptotic cell death. Immunofluorescence images showed inhibition of tubulin polymerization after treatment with LY293.

Conclusion: LY293-loaded mPEG-b-P (CB-co-LA) nanoparticles showed excellent efficacy and induced apoptosis in melanoma cells. These polyester/polycarbonate-based nanoparticles provided an excellent platform to deliver different poorly soluble drugs to melanoma.

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Figures

Fig. 1
Fig. 1
Synthesis and characterization of 2-(1 H-Indol-5-yl) thiazol-4-yl) 3, 4, 5-trimethoxyphenyl methanone (LY293). (a) Synthesis scheme of LY293; (b) 1H NMR spectrum of LY293 in CDCl3.
Fig. 2
Fig. 2
1H NMR spectra in CDCl3 of (a) 5-methyl-5-benzyloxycarbonyl-1, 3-dioxane-2-one (carbonate monomer) and (b) mPEG114-b-P (CB52-co-LA147) copolymer.
Fig. 3
Fig. 3
mPEG-b-P (CB-co-LA) nanoparticle size distribution and morphology. (a) Particle size measuring using dynamic light scattering; (b) transmission electron micrograph (TEM) obtained using uranyl acetate staining.
Fig. 4
Fig. 4
In vitro drug release of LY293 of 2.5 and 5% drug loaded mPEG-b-P (CB-co-LA) nanoparticles. Results are expressed as the mean±SD (n03).
Fig. 5
Fig. 5
Effect of DMSO solution of LY293 and drug loaded nanoparticles on (a) B16F10 and (b) A375 cell viability after 48 h of treatment. Cell viability was determined by MTT Assay and expressed as the % of control. Results are expressed as the mean±S.D (n04).
Fig. 6
Fig. 6
Effect of paclitaxel and LY293 on cell viability of Pgp overexpressing melanoma cell. Cytotoxicity of paclitaxel (a) and LY293 (b) in MDA-MB435 and MDA-MB435/LCC6 MDR1 cells respectively after 48 h of treatment. Cell viability was determined by MTT Assay and expressed as % of control. Results are expressed as the mean±SD (n04).
Fig. 7
Fig. 7
Effect of LY293 on Pgp activity. (a) Intracellular calcein accumulation in MDA-MB435/LCC6MDR1 after 30 min treatment with Verapamil, LY293 and PTX. Results are expressed as the mean±SE (n03). (b) Fluorescent images depicting dose-dependent increase in intracellular calcein fluorescence in MDA-MB435/LCC6 MDR1 following 30 min treatment with LY293.
Fig. 8
Fig. 8
Effect of DMSO solution of LY293 and nanoparticle formulation of LY293 on A375 cell cycle analysis and apoptosis. (a) Cell cycle distribution after 24 h of treatment with DMSO, LY 293 dissolved in DMSO, blank nanoparticle and drug loaded nanoparticle. *p<0.05 using Student's unpaired t test. (b) Apoptosis of A375 cells after 48 h of treatment expressed as percent sub G1 cells. Results are expressed as the mean±SD (n03). (c) Histogram showing cell cycle distribution.
Fig. 9
Fig. 9
Effect of LY293 treatment on tubulin polymerization. Immunofluorescence images showing tubulin immunostained with rabbit monoclonal anti-tubulin antibodies, followed by Dylight 594 secondary antibody (red color) and nucleus stained with DAPI (blue color). Arrows highlight the difference in cytoskeleton arrangement of cells upon treatment with (a) control, (b) blank NP (c) paclitaxel, (d) mPEG-b-P(CB-co-LA) LY293, and (e) DMSO LY293.
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