Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells - PubMed (original) (raw)

Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells

Fatemeh Talaei et al. Int J Nanomedicine. 2011.

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Abstract

Thiolated chitosan has high transfection and mucoadhesive properties. We investigated the potential of two recently synthesized polymers: NAC-C (N-acetyl cysteine-chitosan) and NAP-C (N-acetyl penicillamine-chitosan) in anticancer drug delivery targeting epidermal growth factor receptor (EGFR). Doxorubicin (DOX) and antisense oligonucleotide (ASOND)-loaded polymer nanoparticles were prepared in water by a gelation process. Particle characterization, drug loading, and drug release were evaluated. To verify drug delivery efficiency in vitro experiments on a breast cancer cell line (T47D) were performed. EGFR gene and protein expression was analyzed by real time quantitative polymerase chain reaction and Western blotting, respectively. A loading percentage of 63% ± 5% for ASOND and 70% ± 5% for DOX was achieved. Drug release data after 15 hours showed that ASOND and DOX were completely released from chitosan-based particles while a lower and more sustained release of only 22% ± 8% was measured for thiolated particles. In a cytosol simulated release medium/reducing environment, such as found intracellularly, polymer-based nanoparticles dissociated, liberating approximately 50% of both active substances within 7 hours. ASOND-loaded polymer nanoparticles had higher stability and high mucoadhesive properties. The ASOND-loaded thiolated particles significantly suppressed EGFR gene expression in T47D cells compared with ASOND-loaded chitosan particles and downregulated EGFR protein expression in cells. This study could facilitate future investigations into the functionality of NAP-C and NAC-C polymers as an efficient ASOND delivery system in vitro and in vivo.

Keywords: T47D breast cancer cells; antisense oligonucleotide; controlled release; doxorubicin; epidermal growth factor receptor; nanoparticles; thiolated chitosan.

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Figures

Figure 1

Figure 1

Presumptive structures of (A) NAC-chitosan and (B) NAP-chitosan conjugates. Abbreviations: NAC**,** N-acetyl cysteine; NAP, N-acetyl penicillamine.

Figure 2

Figure 2

The effect of different concentration (0.25, 0.5, 1, 2 mg/mL) of polymers (NAC-C, NAP-C, LMWC) on T47D cancer cell proliferation 48 hours after treatment compared with positive control (RPMI + 10% FBS) and negative control (FBS-free RPMI). The data are mean ± SD of three experiments in four wells. In ANOVA tests all the data were compared to controls demonstrating a significant difference (P < 0.0001). Note: *Significantly different from all other groups. Abbreviations: ANOVA, analysis of variance; NAC-C, N-acetyl cysteine-chitosan; NAP-C, N-acetyl penicillamine-chitosan; LMWC, low-molecular-weight chitosan; RPMI, Roswell Park Memorial Institute medium; FBS, fetal bovine serum; SD, standard deviation.

Figure 3

Figure 3

Representative SEM micrograph of nanoparticles formulated via sulfate gelation with Na2SO4. (A) chitosan nanoparticles, (B) thiolated chitosan (NAP-C) nanoparticles, (C) DOX-NAC-C nanoparticles, (D) ASOND-NAC-C nanoparticles demonstrate a spherical, uniform shape with a particle size of 150–300 nm. Abbreviations: DOX, doxorubicin; NAC-C, N-acetyl cysteine-chitosan; NAP-C, N-acetyl penicillamine-chitosan; ASOND, antisense oligonucleotide; SEM, scanning electron microscope.

Figure 4

Figure 4

MTS assay measured the effect of polymer nanoparticles (0.5 mg/mL) containing ASOND and DOX (25 × 10−7 M) on T47D cancer cell proliferation 24 hours after treatment. The data are mean ± SD of three separate experiments in four wells. In ANOVA tests all the data were compared with controls (RPMI + 10% FBS- and FBS-free RPMI). **Notes: ***Significantly different from FBS-free RPMI; #RPMI + 10% FBS is significantly different from all the other treatments (P < 0.0001). Abbreviations: ASOND, antisense oligonucleotide; DOX, doxorubicin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, NAC-C, N-acetyl cysteine-chitosan; NAP-C, N-acetyl penicillamine-chitosan; LMWC, low-molecular-weight chitosan; RPMI, Roswell Park Memorial Institute medium; FBS, fetal bovine serum; SD, standard deviation.

Figure 5

Figure 5

Percentage release of ASOND and doxorubicin from 5 mg of drug-loaded nanoparticles during 15 hours in PBS and cytosol simulated medium using DTT. (A) Percentage release of ASOND, (B) Percentage release of doxorubicin. The release data are mean ± SD for each formulation at different time points indicated (n = 4). Abbreviations: ASOND**,** antisense oligonucleotide; DOX, doxorubicin; NAC-C, N-acetyl cysteine-chitosan; NAP-C, N-acetyl penicillamine-chitosan; LMWC, low-molecular- weight chitosan; RPMI, Roswell Park Memorial Institute medium; FBS, fetal bovine serum; PBS, phosphate buffered saline; DTT, threo-1,4-dimercapto-2,3- butandiol; SD, standard deviation.

Figure 6

Figure 6

Real-time PCR analysis of EG FR gene expression in T47D cells subjected to different treatments. The results were normalized to beta-actin gene expression and the relative expression of EGFR was presented as percent knock down of EGFR. FBS-free RPMI treatment was chosen as control, which presented no knock down of EGFR and all the other cell treatments were compared with this control. All data are presented as percent knock down of EGFR and are significantly different to control. **Notes: ***Difference to control or free polymers (gray bars) within each group separated by indicator lines; #significant difference of each formulation containing ASOND or DOX compared to free DOX or ASOND (P < 0.05); data are mean SD (n = 3). Abbreviations: ASOND, antisense oligonucleotide; DOX, doxorubicin; EGFR, epidermal growth factor receptor; NAC-C, N-acetyl cysteine-chitosan; NAP-C, N-acetyl penicillamine-chitosan; LMWC, low-molecular-weight chitosan; RPMI, Roswell Park Memorial Institute medium; FBS, fetal bovine serum; PBS, phosphate buffered saline; SD, standard deviation.

Figure 7

Figure 7

Analysis of EGFR protein expression in T47D cells. (A) Western blot analysis of EGFR expression in T47D cells through different treatments; (B) immunocytochemical analysis of EGFR protein expression in T47D cells. (1) FBS-free RPMI; (2) ASOND; (3) DOX; (4) NAC-C; (5) ASOND + NAC-C; (6) DOX + NAC-C;, (7) NAP-C; (8) ASOND + NAP-C; (9) DOX + NAP-C; (10) Chitosan; (11) ASOND + chitosan; (12) DOX + chitosan. The brown stain signifies higher expression of this protein which is observed in cytoplasm and on the membrane of the cells and the blue stain indicates the nuclei of the cell. Note: *Significantly different from FBS-free RPMI treated cells as controls (mean ± SD; n = 3). Abbreviations: ASOND, antisense oligonucleotide; DOX, doxorubicin; EGFR, epidermal growth factor receptor; NAC-C, N-acetyl cysteine-chitosan; NAP-C, N-acetyl penicillamine-chitosan; LMWC, low-molecular-weight chitosan; RPMI, Roswell Park Memorial Institute medium; FBS, fetal bovine serum; PBS, phosphate buffered saline; SD, standard deviation.

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