Size Dependency of Selective Cellular Uptake of Epigallocatechin Gallate-modified Gold Nanoparticles for Effective Radiosensitization

The high incidence and mortality of cancer make it a global health issue. However, conventional cancer therapies have several disadvantages, especially serious side effects due to low selective toxicity to cancer cells. Gold nanoparticles (AuNPs) are an excellent drug carrier, enhance drug delivery efficiency, and hold promise for photothermal and radiation therapies. (−)-Epigallocatechin-3-gallate (EGCG) is the major polyphenolic antioxidant constituent of green tea, has a potent antitumor effect, and binds specifically to the 67 kDa laminin receptor, which is overexpressed on the surface of several cancer cell lines such as HeLa and MDA-MB-231 cells. We synthesized EGCG-modified AuNPs (EGCG-AuNPs) using ratios (nEGCG/ngold) from 1:2 to 10:1 and evaluated their size, morphology, stability, antioxidant ability, cytotoxicity, cellular uptake, and uptake mechanisms in vitro in comparison with the conventional AuNPs prepared by using citrate as the reducing agent (citrate-AuNPs). In HeLa cells, EGCG-AuNPs (10:1) (135 nm diameter, sea-urchin-like shape) exhibited the highest cellular uptake. Conversely, EGCG-AuNPs (1:2) (39 nm diameter, spherical shape) were preferentially taken up by MDA-MB-231 cells. Cellular uptake of EGCG-AuNPs toward normal cells (NIH3T3 cells) was found to be in a nonspecific manner, and the amount of uptake was suppressed. X-ray irradiation after cellular uptake of EGCG-AuNPs (1:2) in MDA-MB-231 cells significantly enhanced irradiation-induced cell death. These findings suggest enhanced cellular uptake of EGCG-AuNPs with a 39 nm diameter and their potential use in combinatorial therapeutics of EGCG-AuNPs for breast cancer

Positively charged lysine residues, particularly located at codons 24 and 27, are importnat for the pre-OR residues 23–31 to form a PK-resistant structure in prion-infected N2a cells.

<p>(A) Amino acid sequences of the pre-OR residues 23–31 in mutant proteins. Bold residues indicate substituted residues. (B) Western blotting of N2aC24L1-3 cells transfected with control pcDNA3.1(+) and expression vectors encoding each mutant protein using 3F4 anti-PrP antibodies. The cell lysates were treated with PK at 5 µg/ml. All of the mutant proteins were converted into PK-resistant isoforms in N2aC24L1-3 cells, and all of the mutant isoforms gave rise to doublet non-glycosylated and mono-glycosylated bands. The doublet bands of moPrP(3F4)^ScΔ32–88(K23A), moPrP(3F4)^ScΔ32–88(K24A) and moPrP(3F4)^ScΔ32–88(K27A) were similar in molecular size to those of moPrP(3F4)^ScΔ32–88. However, moPrP(3F4)^ScΔ32–88(K24,27A) gave rise to the doublet band with the upper band migrating very closely to the lower band, similarly to moPrP(3F4)^ScΔ32–88(3K3A). MoPrP(3F4)^ScΔ32–88(K23,24A) and moPrP(3F4)^ScΔ32–88(K23,27A) showed the upper band with an intermediate molecular size. MoPrP(3F4)^ScΔ32–88(3K3R) giving rise to doublet bands with similar molecular size to those of moPrP(3F4)^ScΔ32–88. (C) IBL-N antibodies recognized all of the PK-resistant isoforms except for moPrP(3F4)^ScΔ32–88(3K3A) and moPrP(3F4)^ScΔ32–88(K24,27A).</p

Incidence rate and incubation times in wild-type ddY indicator mice inoculated with serial 10-fold dilutions of RML prions.

<p>Incidence rate and incubation times in wild-type ddY indicator mice inoculated with serial 10-fold dilutions of RML prions.</p

The pre-OR residues 23–31 with a substitution of the proline residues by tryptophan or glycine residues form a PK-resistant structure in prion-infected N2a cells.

<p>(A) Amino acid sequences of the pre-OR residues 23–31 in mutant proteins. Bold residues indicate substituted residues. (B) Western blotting of N2aC24L1-3 cells transfected with control pcDNA3.1(+) and expression vectors encoding each mutant protein using 3F4 anti-PrP antibodies. The cell lysates were treated with PK at 5 µg/ml. All of the mutant proteins were converted into PK-resistant isoforms in N2aC24L1-3 cells, and all of the mutant isoforms, moPrP(3F4)^ScΔ32–88, moPrP(3F4)^ScΔ32–88(2P2A), moPrP(3F4)^ScΔ32–88(2P2W) and moPrP(3F4)^ScΔ32–88(2P2G), gave rise to similar doublet non-glycosylated and mono-glycosylated bands. (C) Since substitution of proline residues into alanine, tryptophan or glycine residues disrupted the IBL-N epitope, the PK-resistant pre-OR residues in these mutant proteins failed to be visualized by IBL-N anti-PrP antibodies.</p

The pre-OR region of PrP^ScΔOR is PK-resistant.

<p>(A) The brain homogenates of terminally ill wild-type and tg(PrPΔOR)/<i>Prnp^0/0</i> mice were treated with PNGase F after digestion with PK, and subjected to immunoblotting with M-20 anti-PrP antibodies. The deglycosylated PK-resistant band of PrP^ScΔOR was higher in molecular size than that of full-length PrP^Sc. Arrows indicates PK-resistant deglycosylated PrPs. (B) The brain homogenates from terminally ill wild-type and tg(PrPΔOR)/<i>Prnp^0/0</i> mice were digested with PK, and subjected to immunoblotting with N-terminus-specific IBL-N anti-PrP antibody. The IBL-N antibodies recognized the PK-resistant PrPs from PrP^ScΔOR but not from full-length PrP^Sc.</p

Incubation times in tg(PrPΔOR)/<i>Prnp^0/0</i> and wild-type mice after infection with RML prions.

<p>Incubation times in tg(PrPΔOR)/<i>Prnp^0/0</i> and wild-type mice after infection with RML prions.</p

Overexpression of PrPΔOR in the brains and spinal cords of tg(PrPΔOR)/<i>Prnp^0/0</i> mice.

<p>The brain and spinal cord homogenates from tg(PrPΔOR)/<i>Prnp^0/0</i> mice and wild-type mice were subjected to Western blotting with SAF61 or SAF32 anti-PrP antibodies. The expression of β-actin was detected in these homogenates as an internal control.</p

Prion titers in the brains and spinal cords of terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> and wild-type mice inoculated with RML prions.

<p>Prion titers in the brains and spinal cords of terminally ill tg(PrPΔOR)/<i>Prnp^0/0</i> and wild-type mice inoculated with RML prions.</p

PK-resistant pre-OR residues 23–31 of PrP^ScΔ32–88 generated in prion-infected N2a cells.

<p>(A) Schematic diagrams of moPrP(3F4) and moPrP(3F4) Δ32–88. Arabic numbers represent the codon numbers. SP, signal peptide; OR, octapeptide repeat; GPI, GPI anchor signal; α, α-helix; β, β-strand. (B, C) Western blotting of N2aC24L1-3 cells transfected with control pcDNA3.1(+), pcDNA3.1-moPrP(3F4), and pcDNA3.1-moPrP(3F4) Δ32–88 using 3F4 (B) or IBL-N anti-PrP antibodies (C). The cell lysates were treated with PK at 5 µg/ml and then subjected to Western blotting. Both moPrP(3F4) and moPrP(3F4) Δ32–88 were converted to the PK-resistant isoforms, moPrP^Sc(3F4) and moPrP^Sc(3F4) Δ32–88, respectively. However, IBL-N anti-PrP antibody reacted only with the PK-resistant fragments of moPrP^Sc(3F4) Δ32–88.</p