Synthesis, Internalization and Visualization of N-(4-Carbomethoxy) Pyrrolidone Terminated PAMAM [G5:G3-TREN] Tecto(dendrimers) in Mammalian Cells.

Tecto(dendrimers) are well-defined, dendrimer cluster type covalent structures. In this article, we present the synthesis of such a PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer). This tecto(dendrimer) exhibits nontraditional intrinsic luminescence (NTIL; excitation 376 nm; emission 455 nm) that has been attributed to three fluorescent components characterized by different fluorescence lifetimes. Furthermore, it has been shown that this PAMAM [G5:G3-(TREN)]-N-(4-carbomethoxy) pyrrolidone terminated tecto(dendrimer) is able to form a polyplex with double stranded DNA, and is nontoxic for HeLa and HMEC-1 cells up to a concentration of 10 mg/mL, even though it accumulates in endosomal compartments as demonstrated by its unique NTIL emission properties. Many of the above features would portend the proposed use of this tecto(dendrimer) as an efficient transfection agent. Quite surprisingly, transfection activity could not be demonstrated in HeLa cells, and the possible reasons are discussed in the article.

Molecular Mechanisms of Antitumor Activity of PAMAM Dendrimer Conjugates with Anticancer Drugs and a Monoclonal Antibody.

Taxanes are considered fundamental drugs in the treatment of breast cancer, but despite the similarities, docetaxel (doc) and paclitaxel (ptx) work differently. For this reason, it is interesting to identify mechanisms of antitumor activity of PAMAM dendrimer conjugates that carry docetaxel or paclitaxel and monoclonal antibody trastuzumab, specifically targeted to cells which overexpressed HER-2. For this purpose, the impact on the level of reactive oxygen species, the mitochondrial membrane potential, cell cycle distribution and the activity of caspases-3/7, -8 and -9 of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined and compared with free docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. Moreover, apoptosis and necrosis were studied using flow cytometry and confocal microscopy, respectively. Our studies show the complexity of the potential mechanism of cytotoxic action of PAMAM-drug-trastuzumab conjugates that should be sought as a resultant of oxidative stress, mitochondrial activation of the caspase cascade and the HER-2 receptor blockade.

Sugar Modification Enhances Cytotoxic Activity of PAMAM-Doxorubicin Conjugate in Glucose-Deprived MCF-7 Cells - Possible Role of GLUT1 Transporter.

PURPOSE: In order to overcome the obstacles and side effects of classical chemotherapy, numerous studies have been performed to develop the treatment based on targeted transport of active compounds directly to the site of action. Since tumor cells are featured with intensified glucose metabolism, we set out to develop innovative, glucose-modified PAMAM dendrimer for the delivery of doxorubicin to breast cancer cells. METHODS: PAMAM-dox-glc conjugate was synthesized and characterized by 1H NMR, FT-IR, size and zeta potential measurements. The drug release rate from conjugate was evaluated by dialysis under different pH conditions. The expression level of GLUT family receptors in cells cultured in full and glucose-deprived medium was evaluated by quantitative real-time RT-PCR and flow cytometry. The cytotoxicity of conjugate in presence or absence of GLUT1 inhibitors was determined by MTT assay. RESULTS: We showed that PAMAM-dox-glc conjugate exhibits pH-dependent drug release and increased cytotoxic activity compared to free drug in cells cultured in medium without glucose. Further, we proved that these cells overexpress transporters of GLUT family. The toxic effect of conjugate was eliminated by the application of specific GLUT1 inhibitors. CONCLUSION: Our findings revealed that the glucose moiety plays a crucial role in the recognition of cells with high expression of GLUT receptors. By selectively blocking GLUT1 transporter we showed its importance for the cytotoxic activity of PAMAM-dox-glc conjugate. These results suggest that PAMAM-glucose formulations may constitute an efficient platform for the specific delivery of anticancer drugs to tumor cells overexpressing transporters of GLUT family.