Cellular uptake of glucoheptoamidated poly(amidoamine) PAMAM G3 dendrimer with amide-conjugated biotin, a potential carrier of anticancer drugs

Abstract

In search for soluble derivatives of PAMAM dendrimers as potential carriers for hydrophobic drugs, the conjugates of PAMAM G3 with biotin, further converted into glycodendrimer with d-glucoheptono-1,4-lactone, were prepared. Polyamidoamine dendrimer (PAMAM) of third generation, G3 was functionalized with four biotin equivalents covalently attached to terminal amine nitrogens via amide bond G3^4B. The remaining 28 amine groups were blocked by glucoheptoamide substituents (gh) to give G3^4B28gh or with one fluorescein equivalent (attached by reaction of G3^4B with fluorescein isothiocyanate, FITC) via thiourea bond as FITC followed by exhaustive glucoheptoamidation to get G3^4B27gh1F. As a control the G3 substituted totally with 32 glucoheptoamide residues, G3^gh and its fluorescein labeled analogue G3^31gh1F were synthesized. The glucoheptoamidation of PAMAM G0 dendrimer with glucoheptono-1,4-lactone was performed in order to fully characterize the ¹H NMR spectra of glucoheptoamidated PAMAM dendrimers and to control the derivatization of G3 with glucoheptono-1,4-lactone. Another two derivatives of G3, namely G3^4B28gh1F' and G3^32ghF', with ester bonded fluorescein were also obtained. Biological properties of obtained dendrimer conjugates were estimated in vitro with human cell lines: normal fibroblast (BJ) and two cancer glioblastoma (U-118 MG) and squamous carcinoma (SCC-15), including cytotoxicity by reduction of XTT and neutral red (NR) assays. Cellular uptake of dendrimer conjugates was evaluated with confocal microscopy. Obtained results confirmed, that biotinylated bioconjugates have always lower cytotoxicity and 3-4 times higher cellular uptake than non-biotinylated dendrimer conjugates in all cell lines. Comparison of various cell lines revealed different dose-dependent cell responses and the lower cytotoxicity of examined dendrimer conjugates for normal fibroblasts and squamous carcinoma, as compared with much higher cytotoxic effects seen in glioblastoma cell line. Synthetized multi-functional conjugate (G3^4B27gh1F) is a promising candidate as biocompatible vehicle for hydrophobic molecules used in anticancer therapy.

Keywords: Anticancer vehicle; Biotin bioconjugate; Cellular uptake; Confocal microscopy; In vitro; Normal and cancer cells; PAMAM G3 dendrimer; Synthesis; Toxicity; d-Glucoheptono-1,4-lactone.