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

Małgorzata Malinga-Drozd,Łukasz Uram,Stanisław Wołowiec,Konrad Wróbel

doi:10.3390/polym13071049

Małgorzata Malinga-Drozd, Łukasz Uram + Show 2 more

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https://doi.org/10.3390/polym13071049

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Journal: Polymers	Publication Date: Mar 27, 2021
Citations: 8	License type: CC BY 4.0

Affiliation: Rzeszów University of Technology, Rzeszów University

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.

Highlights

Polyamidoamine (PAMAM) dendrimers were synthesized in 1985 by Tomalia et al [1].Since that time, PAMAM dendrimers became often explored macromolecular reagents due to their strictly defined molecular weight, radial symmetry, and availability of terminal functional groups on the surface [2]
Human immortalized keratinocytes (HaCaT) were grown in Dulbecco’s modified Eagle’s medium (DMEM) and human squamous carcinoma cells SCC-15 (CRL-1623 ATCC) were cultured in DMEM/F-12 supplemented with hydrocortisone (400 ng/mL)
Polyamidoamine (PAMAM) dendrimers are well soluble in water

Summary

Introduction

Polyamidoamine (PAMAM) dendrimers were synthesized in 1985 by Tomalia et al [1].Since that time, PAMAM dendrimers became often explored macromolecular reagents due to their strictly defined molecular weight, radial symmetry, and availability of terminal functional groups on the surface [2]. Full generation PAMAM dendrimers provide amine groups that can be functionalized with various substituents, including drug molecules. They are continuously tested, especially dendrimers of generation 3, 4, and 5 (G3, G4, G5), as drug carriers and gene delivery systems [3,4]. The toxicity of PAMAM G2-G6 dendrimers is rather low, while 0.5 μM concentration near cell surface already enables to observe cell internalization of these molecules [5]. PAMAM G3, G4, and G5 dendrimers with 32, 64, and 128 amine groups, respectively, were used to covalently bind anticancer drug molecules such as methotrexate [6], daunorubicin [7], or paclitaxel [8]

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