Anti-EpCAM Functionalized I-131 Radiolabeled Biomimetic Nanocarrier Sodium/Iodide-Symporter-Mediated Breast-Cancer Treatment

Abstract

Currently, breast-cancer treatment has a number of adverse side effects and is associated with poor rates of progression-free survival. Therefore, a radiolabeled anti-EpCAM targeted biomimetic coated nanocarrier (EINP) was developed in this study to overcome some of the treatment challenges. The double emulsion method synthesized the poly(lactic-co-glycolic acid) (PLGA) nanoparticle with Na131I entrapped in the core. The PLGA nanoparticle was coated in human red blood cell membranes and labeled with epithelial cell adhesion molecule (EpCAM) antibody to enable it to target EpCAM overexpression by breast-cancer cells. Characterization determined the EINP size as 295 nm, zeta potential as −35.9 mV, and polydispersity as 0.297. EINP radiochemical purity was >95%. Results determined the EINP efficacy against EpCAM positive MCF-7 breast cancer at 24, 48, and 72 h were 69.11%, 77.84%, and 74.6%, respectively, demonstrating that the EINPs achieved greater cytotoxic efficacy supported by NIS-mediated Na131I uptake than the non-targeted 131INPs and Na131I. In comparison, fibroblast (EpCAM negative) treated with EINPs had significantly lower cytotoxicity than Na131I and 131INPs (p < 0.05). Flow cytometry fluorescence imaging visually signified delivery by EINPs specifically to breast-cancer cells as a result of anti-EpCAM targeting. Additionally, the EINP had a favorable safety profile, as determined by hemolysis.

Keywords: EpCAM; I-131; PLGA; breast cancer; drug delivery; ionizing radiation; nanoparticle.

Figure 1

The fabrication of EINPs. Preparation of red blood cell membrane-derived vesicles: modification of human red blood cell membranes (hRBCm) with DSPE-PEG-Biotin, conjugation of streptavidin-FITC with biotin, and addition of biotin-functionalized anti-EpCAM to streptavidin. Nanoparticle preparation involves encapsulation of the Na¹³¹I into the nanoparticle and the coating of the nanoparticle with the hRBCm-derived vesicles.

Figure 2

Physiochemical properties and characterization of PLGA, sodium iodide-131 (Na¹³¹I), human red blood cell membrane-derived vesicles (hRBCm), Na¹³¹I radiolabeled PLGA nanoparticles (¹³¹INPs), and hRBCm-coated anti-EpCAM functionalized Na¹³¹I radiolabeled PLGA NPs (EINPs). (a) Z-average diameter (nm) after 0, 3, 24, and 96 h incubation. (b) Polydispersity index (PDI) after 0, 3, 24, and 96 h incubation. (c) Zeta potential after 0, 3, 24 and 96 h incubation. (d) Transmission electron micrographs of EINPs (samples were stained negatively with uranyl acetate; scale bar = 100 nm). (e) EINPs membrane protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (f) Western blot EINPs and hRBCm protein analysis. (g) Radiochemical purity (RCP). (h) EINPs radioactive stability in 1× PBS and serum at 37 °C. [Bars show the mean ± SD (n = 3)].

Figure 3

In vitro therapeutic responses of EINPs. (a) In vitro cytotoxicity using MTT assay of EINPs at dosages of 0, 0.37, 1.85, 3.70, 5.55, and 7.40 MBq after 24 h incubation. Denote ns = no statistically significant difference in efficacy of destroying cancer cells between 3.70, 5.55, and 7.40 MBq (p > 0.05). (b) Radioactive encapsulation efficiency after 0, 3, 6, 12, and 24 h incubation. (c) In vitro proliferation (% of control) using MTT assay of EINPs compared to control, PLGA, Na¹³¹I, and ¹³¹INPs treatment of MCF-7 breast-cancer cells, after 24, 48 and 72 h incubation. [Results represent mean ± SD (n = 3)]. * the significance between Na¹³¹I and EINPs at 72 h incubation (p < 0.05).

Figure 4

Co-localization of EINPs upon cellular binding/uptake. (a) EINPs were synthesized using PLGA cores, and their membranes were modified with anti-EpCAM labeled with FITC (green channel). DAPI was used to stain the nucleus (blue channel). Software was used to deconvolve all channels to remove out-of-focus fluorescent signals [10× images, scale bar = 200 μm]. (b) Quantification of the mean fluorescence intensities of EINPs on fibroblast and MCF-7 cells [bars show the mean ± SD (n = 3)]. * indicates a statistically significant difference between fibroblast and MCF-7 treated with EINPs (p < 0.05).

Figure 5

In vitro cell-surface immunofluorescence. (a) EINPs were fabricated with PLGA cores and their membranes modified using anti-EpCAM labeled with FITC (green channel) and DiD (red channel). The morphology of the cancer cell was imaged in bright-field (grey channel). All channels were imaged and fluorescent signals measured by flow cytometer [scale bar = 10 µm]. (b) The scatter plot of anti-EpCAM fluorescence intensities on MCF-7 cells treated with control, PLGA, Na¹³¹I, ¹³¹INPs, and EINPs on MCF-7 cells. (c) Quantification of mean fluorescence intensities of control, PLGA, Na¹³¹I, ¹³¹INPs, and EINPs on MCF-7 cells. (d) Relative EpCAM surface immunofluorescence (fold change of control) [bars represent mean ± SD (n = 3)].

Figure 6

Two-Dimensional (2D) in vitro live/dead upon cellular binding/uptake (a) In vitro live/dead cell imaging of control, PLGA, Na¹³¹I, ¹³¹INPs, and EINPs. Fibroblast and MCF-7 cells were treated with PLGA at a fixed concentration of 10 mg/mL and Na¹³¹I, ¹³¹INPs, and EINPs activity 3.70 MBq for 72 h. Two-color fluorescence live (green channel) and dead (red channel) enabled the evaluation of live and dead cells to determine cell viability and cytotoxicity. Additionally, to eliminate out-of-focus fluorescent signals, all the channels were deconvolved by software [10× images, scale bar = 200 µm]. (b) Cytotoxicity quantification by dead/live mean fluorescence intensity of fibroblast (fold change of control) of control, PLGA, Na¹³¹I, ¹³¹INPs, and EINPs. (c) Cytotoxicity quantification by dead/live mean fluorescence intensity of MCF-7 (fold change of control) of control, PLGA, Na¹³¹I, ¹³¹INPs, and EINPs. [bars represent mean ± SD (n = 3)]. * the significance between Na¹³¹I and EINPs of fibroblast (p < 0.05). ** the significance between Na¹³¹I and EINPs of MCF-7 (p < 0.05).

Figure 7

Three-dimensional (3D) human tumor spheroid in vitro live/dead cell imaging. (a) In vitro live/dead MCF-7 cell tumor spheroid imaging of control, PLGA, Na¹³¹I, ¹³¹INPs, and EINPs (I-131 activity of 3.70 MBq). Two-color fluorescence, live (green channel) and dead (red channel), enabled assessment of live and dead cells to evaluate cell viability. Scale bar = 200 µm. (b) Mean fluorescence intensity (fold change of control). (c) In vitro cytotoxicity study using CellTiter-Glo^® 3D cell assay of control, PLGA, Na¹³¹I, ¹³¹INPs, and EINPs against 3D human MCF-7 spheroids. Results represent mean ± SD (n = 3).

Figure 8

EINPs penetration in three-dimensional human (3D) MCF-7 tumor spheroid. (a) MCF-7 (EpCAM+) human spheroid fluorescence images treated with EINPs (green channel). The MCF-7 cell nucleus was stained with DAPI (blue channel). Scale bar = 200 µm. (b) Quantification of mean fluorescence intensity (fold change from initial 24 h) of EINPs penetration of MCF-7 spheroids with incubation times of 24, 48 and 72 h. Bars represent mean ± SD (n = 3).

Figure 9

Analysis of nanoparticle hemolytic properties in vitro: blood compatibility was incubated with 8 × 10⁹ red blood cells/mL at 37 °C. (a) Red blood cells were pelleted down, and the supernatant was analyzed for lysed hemoglobin (DMSO as positive control), PLGA, Na¹³¹I, ¹³¹INPs, and EINPs after 0, 3, 6, 12, 24, 36, 48, and 80 h incubation. (b) Quantitative hemolytic interpretation of supernatant was analyzed for lysed hemoglobin. Data are given as mean ± SD (n = 3). Denote ns = no statistically significant difference in the hemolytic properties between PLGA, Na¹³¹I, ¹³¹INPs, and EINPs (p > 0.05).