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.
ABSTRACT
Polyamidoamine PAMAM dendrimer generation 3 (G3) was modified by attachment of biotin via amide bond and glucoheptoamidated by addition of α-D-glucoheptono-1,4-lacton to obtain a series of conjugates with a variable number of biotin residues. The composition of conjugates was determined by detailed 1-D and 2-D NMR spectroscopy to reveal the number of biotin residues, which were 1, 2, 4, 6, or 8, while the number of glucoheptoamide residues substituted most of the remaining primary amine groups of PAMAM G3. The conjugates were then used as host molecules to encapsulate the 5-aminolevulinic acid. The solubility of 5-aminolevulinic acid increased twice in the presence of the 5-mM guest in water. The interaction between host and guest was accompanied by deprotonation of the carboxylic group of 5-aminolevulinic acid and proton transfer into internal ternary nitrogen atoms of the guest as evidenced by a characteristic chemical shift of resonances in the 1H NMR spectrum of associates. The guest molecules were most likely encapsulated inside inner shell voids of the host. The number of guest molecules depended on the number of biotin residues of the host, which was 15 for non-biotin-containing glucoheptoamidated G3 down to 6 for glucoheptoamidated G3 with 8 biotin residues on the host surface. The encapsulates were not cytotoxic against Caco-2 cells up to 200-µM concentration in the dark. All encapsulates were able to deliver 5-aminolevulinic acid to cells but aqueous encapsulates were more active in this regard. Simultaneously, the reactive oxygen species were detected by staining with H2DCFDA in Caco-2 cells incubated with encapsulates. The amount of PpIX was sufficient for induction of reactive oxygen species upon 30-s illumination with a 655-nm laser beam.
