Nonlinear multiphoton photo-cross-linking and photopolymerization of proteins and polymers in solution have been used to direct the three-dimensional assembly of micron scale objects. Two aspects of fabricated proteinatious matrixes are examined in this paper: the efficiency of protein photopolymerization and the application of fabricated matrixes as sustained release devices. The efficiency of photoactivated cross-linking of the proteins bovine serum albumin and fibrinogen, using rose bengal, have been determined and found to vary with photosensitizer concentration. This concentration dependence suggests that the mechanism for protein cross-linking is a direct hydrogen transfer between an amino acid residue of the protein and the dye molecule itself. A comparison of the surface structure of single and multiple protein oligomers is undertaken and shown to vary significantly depending on fabrication materials. Alkaline phosphatase bioactivity, upon entrapment in a protein structure, is maintained. The properties of fabricated protein matrixes as sustained release devices is also examined. The rates of diffusion of fluorescently labeled dextrans (10 and 40 kDa) from an optically fabricated BSA matrix vary with molecular weight and are linear with cross-link density. The half-life of release of 10 kDa dextran-TMR from a BSA micron scale structure is less than or equal to 6 min while 40 kDa dextran-TMR halflife of release is 25 min. Finally, rhodamine 610, a typical drug size molecule, was entrapped in an acrylamide structure, and its release is found to be diffusion-limited with half-lives of 10-31 min, depending on cross-link density.
We report the synthesis and optical characterization of two new photoactivators and demonstrate their use for multiphoton excited three-dimensional free-form fabrication with proteins. These reagents were developed with the goal of cross-linking Type 1 collagen. This cross-linking process produces structures on the micron and submicron size scales. A rose bengal diisopropyl amine derivative combines the classic photoactivator and co-initiator system into one molecule, reducing the reaction kinetics and increasing cross-linking efficiency. This derivative was successful at producing stable structures from collagen, whereas rose bengal alone was not effective. A benzophenone dimer connected by a flexible diamine tether was also synthesized. This activator has two photochemically reactive groups and is highly efficient in cross-linking bovine serum albumin and Type 1 collagen to form stable, robust structures. This approach is more flexible in terms of cross-linking a variety of proteins than by traditional benzophenone photochemistry. The photophysical properties vary greatly from that of benzophenone, with the appearance of a new, lower energy absorption band (lambda max approximately 370 nm in water) and broad, visible emission band (approximately 500 nm maximum). This absorption band is highly solvatochromic, suggesting it arises, at least in part, from a charge transfer interaction. Collagens are typically difficult to cross-link photochemically, and the results here suggest that these two new activators will be suitable for cross-linking other forms of collagen and additional proteins for biomedical applications such as the de novo assembly of biomimetic tissue scaffolds.
We report the design, development, and characterization of a sensitive, time-resolved fluorescence spectrometer capable of measuring fluorescence spectra and transient decays simultaneously, with data acquisition times less than 1 s. The spectrometer, a portable fluorescence lifetime spectrometer (FLS), was designed to be compatible with both laboratory and clinical research studies on biological systems, and was applied to the study of several biological fluorophores in vitro and human tissue in vivo. The instrument consisted of a nitrogen laser pumping a dye laser for excitation from 337.1 nm through the near infrared, a quartz fiber-optic probe for remote light delivery and collection, and amplified detectors for rapid spectral and temporal detection from 350 to 800 nm. The spectral resolution of the FLS was determined to be 3 nm, which is sufficient for accurately detecting the broad spectral bands associated with biological fluorophores. The FLS was able to detect 5×10−7 M fluorescein dye concentrations with spectral signal-to-noise ratios (SNRs) of 29. Time-resolved detection with the FLS had a dynamic range of approximately three decades with a SNR of 200. Using fluorescence lifetime standards, the FLS was determined to be capable of accurately resolving fluorophore lifetimes from hundreds of picoseconds to tens of nanoseconds in duration, with an ultimate temporal resolution of 360 ps.
Tissue autofluorescence has been explored as a potential method of noninvasive pre-neoplasia (pre-malignancy) detection in the lung. Here, we report the first studies of intrinsic cellular autofluorescence from SV40 immortalized and distinct tobacco-carcinogen-transformed (malignant) human bronchial epithelial cells. These cell lines are useful models for studies seeking to distinguish between normal and pre-neoplastic human bronchial epithelial cells. The cells were characterized via spectrofluorimetry and confocal fluorescence microscopy. Spectrofluorimetry revealed that tryptophan was the dominant fluorophore. No change in tryptophan emission intensity was observed between immortalized and carcinogen-transformed cells. Confocal autofluorescence microscopy was performed using a highly sensitive, spectrometer-coupled instrument capable of limiting emission detection to specific wavelength ranges. These studies revealed two additional endogenous fluorophores, whose excitation and emission characteristics were consistent with nicotinamide adenine dinucleotide (NADH) and flavins. In immortalized human bronchial epithelial cells, the fluorescence of these species was localized to cytoplasmic granules. In contrast, the carcinogen-transformed cells showed an appreciable decrease in the fluorescence intensity of both NADH and flavins and the punctate, spatial localization of the autofluorescence was lost. The observed autofluorescence decrease was potentially the result of changes in the redox state of the fluorophores. The random cytoplasmic fluorescence pattern found in carcinogen-transformed cells may be attributed to changes in the mitochondrial morphology. The implications of these results to pre-neoplasia detection in the lung are discussed.
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