Chiral Recognition of Homochiral Poly (amidoamine) Dendrimers Substituted with R- and S-Glycidol by Keratinocyte (HaCaT) and Squamous Carcinoma (SCC-15) Cells In Vitro

Abstract

The generation 2 and 3 poly(amidoamine) dendrimers (PAMAM G2 and G3) were converted into N-(2,3-dihydroxy)propyl derivatives by the addition of enantiomerically pure S- and R-glycidol. The homochiral dendrimers bind to HaCaT and SCC-15 cell membranes with an R/S glycidol enantioselectivity ratio of 1.5:1, as was quantitatively determined by fluorescence microscopy and visualized by confocal microscopy. Fully substituted G2 and G3 dendrimers were equipped with 32 and 64 N-(2,3-dihydroxy)propyl residues and showed effectively radial symmetry for homochiral derivatives in ¹³C NMR spectrum in contrary to analogs obtained by reaction with rac-glycidol. The sub-stoichiometric derivatives of G2 and G3 were also obtained in order to characterize them spectroscopically. The homochiral dendrimers were labeled with two different fluorescent labels, fluorescein, and rhodamine B, using their isothiocyanates to react with G2 and G3 followed by the addition of S- and R-glycidol. Obtained fluorescent derivatives were deficiently filled with N-(2,3-dihydroxy)propyl substituents due to steric hindrance imposed by the attached label. Nevertheless, these derivatives were used to determine their ability to bind to the cell membrane of human keratinocytes (HaCaT) and squamous carcinoma cells (SCC-15). Confocal microscopy images obtained from cells treated with variously labeled conjugates and fluorescence analysis with fluorescence reader allowed us to conclude that R-glycidol derivatives were bound and entered the cells preferentially, with higher accumulation in cancer cells. The G3 polyamidoamine (PAMAM)-based dendrimers were taken up more efficiently than G2 derivatives. Moreover, S- and R-glycidol furnished dendrimers were highly biocompatible with no toxicity up to 300 µM concentrations, in contrast to the amine-terminated PAMAM analogs.

Keywords: chiral biorecognition; confocal microscopy; glycidol; homochiral dendrimer; polyamidoamine dendrimer; toxicity.

Scheme 1

The scheme of synthesis of glycidol-modified polyamidoamine (PAMAM) G3 (m = 3) and G2 (m = 2) dendrimers (A) and fluorescein or rhodamine B dendrimers substituted by addition of glycidol (B). The isothiocyanates of fluorescein (FITC) and rhodamine B (RBTC) were used to attach one molecule of label by thiourea bond into a dendrimer molecule. The following series of not-labeled and fluorescein-labeled (F) and rhodamine B-labeled (R) derivatives were obtained by stepwise addition of glycidol: G2^9Sgl; G2^14Sgl; G2^22Sgl; G2^32Sgl; G2^16RglF (2a + b = 16, c = 1, d = 0); G2^19SglF (2a + b = 19, c = 1, d = 0); G3^19RglF (2a + b = 24, c = 1, d = 0); G3^35SglF (2a + b = 35, c = 1, d = 0); G2^19RglR (2a + b = 19, c = 0, d = 1); G2^30SglR (2a + b = 30, c = 0, d = 1); G3^45RglR (2a + b = 45, c = 0, d = 1); G3^35SglR (2a + b = 45, c = 0, d = 1).

Figure 1

The ¹H NMR spectra of G2 (A) and G2^Sgl (B) in D₂O. Atom numbering is given in trace (C). Methanol resonance at 3.43 is labeled with an asterisk.

Figure 2

The ¹³C NMR spectra of G2 (A) and G2^Sgl (B) in D₂O. Labeling of resonances according to the scheme shown in Figure 1.

Figure 3

¹H NMR spectra of G2 (A), G2^9Sgl (B), G2^14Sgl (C), G2^22Sgl (D), and G2^32Sgl (E) in D₂O.

Figure 4

¹³C NMR spectra of G2 (A), G2^9Sgl (B), G2^14Sgl (C), G2^22Sgl (D), and G2^32Sgl (E). The carbonyl group resonances are omitted for clarity. The sharp resonance of methanol carbon at 44.3 ppm is labeled with an asterisk.

Figure 5

Cytotoxicity of G3^RglF, G3^SglF, G2^RglF, and G2^SglF after 24 h treatment of human squamous carcinoma cells (SCC-15) and human immortalized keratinocytes (HaCaT). Cell viability is presented as medians of a percent against non-treated control (control expressed as 100%). The whiskers are lower (25%) and upper (75%) quartile ranges. * p ≤ 0.05; Kruskal–Wallis test (against non-treated control).

Figure 6

(A): Time-dependent accumulation of G3^RglF, G3^SglF, G2^RglF, and G2^SglF in immortalized human keratinocytes (HaCaTs) and squamous carcinoma cells (SCC-15). Cells were incubated with 1 µM working solutions of dendrimers for 0.1–4 h and the fluorescence signal was measured with a fluorescence microplate reader at 485 nm/530 nm (exc./em.). Results are expressed as medians of relative fluorescence units, and the whiskers are lower (25%) and upper (75%) quartile ranges. ▼ p ≤ 0.05; Mann–Whitney U-test (S against R enantiomers). (B): Confocal microscopy images of HaCaT and SCC-15 cells incubated for 4 h with 100 nM solutions of dendrimers in the respective pairs (G3^RglR–G3^SglF), (G3^RglF–G3^SglR), (G2^RglR–G2^SglF), (G2^RglF–G2^SglR). White arrows indicate dendrimers bounded with cell membranes. Yellow arrows show the presence of dendrimers in nucleoli.

Figure 7

Concentration-dependent cellular accumulation of G3^RglF, G3^SglF, G2^RglF and G2^SglF in HaCaT and SCC-15 cells after 24 h incubation. Images were obtained with an Olympus IX-83 fluorescence microscope.