Mobility of Green Fluorescent Protein in Hydrogel‐Based Drug‐Delivery Systems Studied by Anisotropy and Fluorescence Recovery After Photobleaching

Abstract

Modified hydroxyethyl starch is photo-crosslinked in the presence of a green fluorescent protein (GFP) (mTagGFP) to obtain loaded hydrogels as model for a drug-delivery system. An important factor for the protein release is the crosslinking density since a dense network should lead to hindered diffusion. To obtain information on the rotational and translational diffusion of GFP in the hydrogel, mTagGFP is analyzed by fluorescence anisotropy and fluorescence recovery after photo-bleaching experiments using two-photon excitation. The mTagGFP shows a viscosity-retarded rotational and strongly hindered translational diffusion, depending on the polymer concentration. A comparison of anisotropy studies with mTagGFP-loaded microparticles and hydrogel disks allows the polymer concentration to be determined for the microparticles, which has been previously unknown.

Measuring the effects of α1‐antitrypsin polymerisation on the structure and biophysical properties of the endoplasmic reticulum

“…However, the physical nature of the hydrogel has complex effects on the movement of proteins, since it can both hinder diffusion by acting as a partial barrier, but also speed the movement of large molecules through size exclusion effects. Existing techniques, such as FRAP and fluorescence anisotropy have been used to probe the effects of some physical parameters of hydrogels, such as polymer density and cross‐linking, on rotational and translational diffusion of model proteins, but important questions remain [Bertz et al., ; Rapp et al., ]. To what extent, for example, is protein mobility affected by changes to ER microviscosity rather than through effects on protein binding or crowding?…”

Evidence of superdiffusive nanoscale motion in anionic polymeric hydrogels: Analysis of PGSE- NMR data and comparison with drug release properties

“…Among the experimental techniques able to monitor solute diffusion in hydrogels, [55][56][57][58][59][60][61] pulse gradient spin echo NMR (PGSE-NMR) has proven especially valuable, as it offers the possibility to investigate the translational dynamics of small molecules non-invasively and quantitatively, [36,62,63] also in presence of nanoscale confinement. Importantly, this technique can be applied to drug-loaded hydrogels in thermal equilibrium, with no need of drug concentration gradients.…”

Fluorescence anisotropy (polarization): from drug screening to precision medicine

“…Due to the versatility of FA and the availability of high quality polarizers, stable detectors, and excitation sources, this method has found widespread use in diverse biological applications, from probing the cellular microenvironment 9, 10 and monitoring cell signaling pathways, 11, 12 to 2D and 3D imaging, 13–17 temperature mapping, 18 and evaluation of drug delivery systems. 19 Introduced in 1970s and with the first dedicated instruments in 1980s, FA has become a standard way of quantitatively measuring biomarkers (first clinical utility of FA), elucidating the mechanism of drug action, and screening potential drug candidates. 20, 21 In the last decade, a variety of FA designs and formats have been utilized to study enzymes, 22–24 as well as protein-protein 25 and protein-DNA interactions, with the goal of developing drugs.…”