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FLIm and raman spectroscopy for investigating biochemical changes of bovine pericardium upon genipin cross-linking
Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm-1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
The chemistry of cross-linked polymeric vesicles and their functionalization towards biocatalytic nanoreactors
Self-assembly of amphiphilic block copolymers into polymersomes continues to be a hot topic in modern research on biomimetics. Their well-known and valued mechanical strength can be increased even further if they are cross-linked. These additional bonds prevent a collapse or disassembly of the polymersomes and open the way towards smart nanoreactors. A variety of chemistries have been applied to obtain the desired cross-linked polymersomes, and therefore, the chemical approaches performed over time will be highlighted in this mini-review. Due to the large number of studies, a selected set of photo-cross-linked and pH-sensitive polymersomes will be specifically highlighted. This system has proven to be a very potent candidate for the formation of nanoreactors and drug delivery systems, and even for the formation of functional multicompartment cell mimics. [Figure not available: see fulltext.]. © 2020, The Author(s).
Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications
Casein is the major protein fraction in milk, and its cross-linking has been a topic of scientific interest for many years. Enzymatic cross-linking has huge potential to modify relevant techno-functional properties of casein, whereas non-enzymatic cross-linking occurs naturally during the storage and processing of milk and dairy products. Two size separation techniques were applied for characterisation of these reactions: gel electrophoresis and size exclusion chromatography. This review summarises their separation principles and discusses the outcome of studies on cross-linked casein from the last ~20 years. Both methods, however, show limitations concerning separation range and are applied mainly under denaturing and reducing conditions. In contrast, field flow fractionation has a broad separation range and can be easily applied under native conditions. Although this method has become a powerful tool in polymer and nanoparticle analysis and was used in few studies on casein micelles, it has not yet been applied to investigate cross-linked casein. Finally, the principles and requirements for absolute molar mass determination are reviewed, which will be of increased interest in the future since suitable calibration substances for casein polymers are scarce.