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. 2011:17:2724-37.
Epub 2011 Oct 19.

Enhanced in vitro antiproliferative effects of EpCAM antibody-functionalized paclitaxel-loaded PLGA nanoparticles in retinoblastoma cells

Moutushy Mitra  1 , Ranjita Misra, Anju Harilal, Sanjeeb K Sahoo, Subramanian Krishnakumar
Affiliations

Enhanced in vitro antiproliferative effects of EpCAM antibody-functionalized paclitaxel-loaded PLGA nanoparticles in retinoblastoma cells

Moutushy Mitra et al. Mol Vis. 2011.

Retraction in

Abstract

Background: To specifically deliver paclitaxel (PTX) to retinoblastoma (RB) cells, the anionic surface-charged poly(lactic-co-glycolic acid) (PLGA) NPs loaded with paclitaxel were conjugated with epithelial cell adhesion molecule (EpCAM) antibody for enhancing site-specific intracellular delivery of paclitaxel against EpCAM overexpressing RB cells.

Methods: PTX-loaded PLGA NPs were prepared by the oil-in-water single emulsion solvent evaporation method, and the PTX content in NPs was estimated by the reverse phase isocratic mode of high performance liquid chromatography. Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide chemistry was employed for the covalent attachment of monoclonal EpCAM antibody onto the NP surface. In vitro cytotoxicity of native PTX, unconjugated PTX-loaded NPs (PTX-NPs), and EpCAM antibody-conjugated PTX-loaded nanoparticles (PTX-NP-EpCAM) were evaluated on a Y79 RB cell line by a dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while cellular apoptosis, cysteinyl-aspartic acid protease (caspase)-3 activation, Poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage, and cell-cycle arrest were quantified by flow cytometry. By employing flow cytometry and fluorescence image analyses, the extent of cellular uptake was comparatively evaluated.

Results: PTX-NP-EpCAM had superior antiproliferation activity, increased arrested cell population at the G(2)-M phase, and increased activation of caspase-3, followed by PARP cleavage in parallel with the induction of apoptosis. Increased uptake of PTX-Np-EpCAM by the cells suggests that they were mainly taken up through EpCAM mediated endocytosis.

Conclusions: EpCAM antibody-functionalized biodegradable NPs for tumor-selective drug delivery and overcoming drug resistance could be an efficient therapeutic strategy for retinoblastoma treatment.

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Figures

Figure 1
Figure 1
Schematic presentation of epithelial cell adhesion molecule conjugated to paclitaxel-loaded nanoparticles. The free carboxyl groups of poly(lactic-co-glycolic acid) (PLGA) are covalently conjugated to amine groups of antibody through ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) chemistry. Abbreviations: COOH represents carboxylic functional group; NH2 represents amine functional group; CO NH represents amide functional group.
Figure 2
Figure 2
This figure shows the size distribution of the paclitaxel loaded poly(lactic-co-glycolic acid) (PLGA). Nanoparticles in terms of the diameter in nanometers (272±1.6) as estimated by photon correlation spectroscopy.
Figure 3
Figure 3
This figure shows the measurement of zeta potential (surface charge) of paclitaxel loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles measured by photon correlation spectroscopy.
Figure 4
Figure 4
Characterization of paclitaxel loaded nanoparticles by transmission electron microscopy (TEM). TEM was used to analyze the size and size distribution of the paclitaxel loaded nanoparticles.
Figure 5
Figure 5
Characterization of paclitaxel loaded nanoparticles by scanning electron microscopy (SEM). SEM was used for imaging and to study the surface morphology of the paclitaxel loaded nanoparticles.
Figure 6
Figure 6
The line graph shows the cumulative percentage release of paclitaxel drug into the phosphate buffer solution (PBS) buffer from the paclitaxel loaded nanoparticles over a period of time (in days). Error bars represents that experiments were performed in triplicates.
Figure 7
Figure 7
Dose and time-dependent cytotoxicity of paclitaxel (PTX) and PTX loaded nanoparticles (NPs) in Y79 cells. A and B: Different concentrations of PTX either as solution or PTX encapsulated in NPs or Epithelial cell adhesion molecule antibody-conjugated PTX-NPs were added to the wells with medium. The extent of growth inhibition was measured at 48 h and at day 5 by the (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Inhibition was calculated with respect to respective controls. Experiments were performed in triplicates and the data are represented as mean±standard error (*p<0.001).
Figure 8
Figure 8
Apoptotic effects of paclitaxel loaded nano-conjugates on Y79 cells. Flow cytometry analysis showing the effect of control (A), native paclitaxel (PTX; B), PTX-loaded nanoparticles (PTX-NP; C), and PTX-NP conjugated with epithelial cell adhesion molecule antibody (PTX-NP-EpCAM; D) treatment on Y79 cells after 48 h incubation. The Y79 cells treated with PTX-NP-EpCAM showed significantly higher (p<0.001) early and intermediary apoptotic events compared to PTX-NP or native PTX. Abbreviation: PI represents propidium iodide.
Figure 9
Figure 9
Analysis of caspase-3 expression in paclitaxel loaded nano-conjugates. Flow cytometry analysis showing caspase-3 expression in Y79 cells after the treatment with control (A); native paclitaxel (PTX; B); PTX-loaded nanoparticles (PTX-NP; C), and PTX-NP conjugated with epithelial cell adhesion molecule antibody (PTX-NP-EpCAM; D). Increased caspase expression was noted in Y79 cells treated with PTX-NP-EpCAM compared to PTX-NP or native PTX.
Figure 10
Figure 10
Analysis of Poly (adenosine diphosphate-ribose) polymerase expression in paclitaxel loaded nano-conjugates. Flow cytometry analysis showing PARP activation in Y79 cells after the treatment with control (A); native paclitaxel (PTX; B); PTX-loaded nanoparticles (PTX-NP; C), and PTX-NP conjugated with epithelial cell adhesion molecule antibody (PTX-NP-EpCAM; D). Increased PARP activation was noted in Y79 cells treated with PTX-NP-EpCAM compared to PTX-NP or native PTX.
Figure 11
Figure 11
Cell cycle analysis of Y79 cells treated with paclitaxel loaded nano-conjugates. Increased G2-M arrest was observed by flow cytometry analysis in Y79 cells treated with paclitaxel-loaded nanoparticles conjugated with epithelial cell adhesion molecule antibody (PTX-NP-EpCAM) at 48 h compared to paclitaxel-loaded nanoparticles (PTX-NP) or native paclitaxel (PTX). A: Untreated Y79 cells showing 3.8% of G2-M phase after doublet discrimination. B: Y79 cells treated with native paclitaxel showing increased G2-M cells (12.2%). C: Y79 cells treated with PTX-NP showing 17.8% of G2-M phase. D: Y79 cells treated with PTX-NP-EpCAM showing 21.7% G2-M phase.
Figure 12
Figure 12
Flow cytometry analysis of uptake of nanoparticles by Y79 cells Flow cytometry analysis showed significantly higher uptake of coumarin encapsulated nanoparticles conjugated with epithelial cell adhesion molecule antibody (COU-NP-EpCAM) by Y79 cells compared to that of unconjugated coumarin encapsulated nanoparticles.” EpCAM negative cell line HeLa cells were used as negative control which showed relatively less uptake (mean intensity -1144.44) compared to EpCAM positive Y79 cells (mean intensity -2090.80).
Figure 13
Figure 13
Microscopic analysis of uptake of nanoparticles by Y79 cells. Fluorescent microscopic analysis showing the uptake of native coumarin (A-D), coumarin encapsulated nanoparticles (COU-NP; E-H), and coumarin encapsulated nanoparticles conjugated with epithelial cell adhesion molecule antibody (COU-NP-EpCAM; K-N) at 4 h (A, E, K), day 1 (B, F, L), day 2 (C, G, M), and day 5 (D, H, N). The Y79 cells showed increased COU-NP-EpCAM uptake compared to unconjugated COU-NP at all time points. Increased coumarin-NP retention was observed in the Y79 cells even at day 5 when compared to free coumarin.
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