No abstract

Smart biomaterials have the ability to respond to changes in physiological parameters and exogenous stimuli and continue to impact many aspects of modern medicine. Smart materials can promote promising therapies and improve treatment of debilitating diseases. Here, we describe recent advances in the current state-of-the-art design and application of smart biomaterials in tissue engineering, drug delivery systems, medical devices, and immune engineering.

The disaccharide moiety is responsible for the tumor cell targeting properties of bleomycin (BLM). While the aglycon (deglycobleomycin) mediates DNA cleavage in much the same fashion as bleomycin, it exhibits diminished cytotoxicity in comparison to BLM. These findings suggested that BLM might be modular in nature, composed of tumor-seeking and tumoricidal domains. To explore this possibility, BLM analogues were prepared in which the disaccharide moiety was attached to deglycobleomycin at novel positions, namely, via the threonine moiety or C-terminal substituent. The analogues were compared with BLM and deglycoBLM for DNA cleavage, cancer cell uptake, and cytotoxic activity. BLM is more potent than deglycoBLM in supercoiled plasmid DNA relaxation, while the analogue having the disaccharide on threonine was less active than deglycoBLM and the analogue containing the C-terminal disaccharide was slightly more potent. While having unexceptional DNA cleavage potencies, both glycosylated analogues were more cytotoxic to cultured DU145 prostate cancer cells than deglycoBLM. Dye-labeled conjugates of the cytotoxic BLM aglycons were used in imaging experiments to determine the extent of cell uptake. The rank order of internalization efficiencies was the same as their order of cytotoxicities toward DU145 cells. These findings establish a role for the BLM disaccharide in tumor targeting/uptake and suggest that the disaccharide moiety may be capable of delivering other cytotoxins to cancer cells. While the mechanism responsible for uptake of the BLM disaccharide selectively by tumor cells has not yet been established, data are presented which suggest that the metabolic shift to glycolysis in cancer cells may provide the vehicle for selective internalization.

Two fluorescent amino acids, including the novel fluorescent species biphenyl-phenylalanine, have been incorporated into positions 17 and 115 of dihydrofolate reductase to enable a study of conformational changes associated with inhibitor binding. Unlike most studies involving fluorescently labeled proteins, the fluorophores were incorporated into the amino acid side chains, and both probes (biphenyl-phenylalanine (1) and L-(7-hydroxycoumarin-4-yl)ethylglycine (2)) were smaller than fluorophores typically used for such studies. The DHFR positions were chosen as potentially useful for FRET measurements based on their estimated separation (~17–18 Å), and expected change in distance along the reaction coordinate. Also of interest was the steric accessibility of the two sites: Glu17 is on the surface of DHFR, while Ile115 is within a folded region of the protein. Modified DHFR I (1 at position 17; 2 at position 115) and DHFR II (2 at position 17; 1 at position 115) were both catalytically competent. However, DHFR II, containing the potentially rotatable biphenyl-phenylalanine moiety at sterically encumbered position 115 was significantly more active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected excitation of biphenyl-phenylalanine (1), energy transfer to coumarin 2 and emission at 450 nm. However, energy transfer was substantially more efficient for DHFR II. The effect of inhibitor binding was also measured. Trimethoprim mediated concentration dependent diminution of the emission observed at 450 nm for DHFR II, but not for DHFR I. These findings demonstrate that amino acids containing small fluorophores can be introduced into DHFR with minimal disruption of function, and in a fashion that enables sensitive monitoring of changes in DHFR conformation.