Mobility of lysozyme in poly(l-lysine)/hyaluronic acid multilayer films

Abstract

The spatial and temporal control over presentation of protein-based biomolecules such as growth factors and hormones is crucial for in vitro applications to mimic the complex in vivo environment. We investigated the interaction of a model protein lysozyme (Lys) with poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films. We focused on Lys diffusion as well as adsorption and retention within the film as a function of the film deposition conditions and post-treatment. Additionally, an effect of Lys concentration on its mobility was probed. A combination of confocal fluorescence microscopy, fluorescence recovery after photobleaching, and microfluidics was employed for this investigation. Our main finding is that adsorption of PLL and HA after protein loading induces acceleration and reduction of Lys mobility, respectively. These results suggest that a charge balance in the film to a high extent governs the protein-film interaction. We believe that control over protein mobility is a key to reach the full potential of the PLL/HA films as reservoirs for biomolecules depending on the application demand.

A “Cell‐Friendly” Window for the Interaction of Cells with Hyaluronic Acid/Poly‐l‐Lysine Multilayers

“…This long storage before cell seeding will ensure that no PLL can be released to the bulk and presented to cells, thus affecting cellular adhesion and probably other behavior of cells. PLL as well as proteins loaded into the HA/PLL multilayers are present in the multilayers as fast diffusing (weekly bound, can release the film being at equilibrium with the bulk) and immobile (strongly bound, not at equilibrium with the bulk) fractions . So extensive washing and long storage will eliminate the fast diffusing fraction and only the strongly bound PLL fraction unable to leave the multilayers will remain.…”

“…However, more deep assessment of the effect of the chemical modification of the multilayers is required for better evaluation of the impact of the chemical cues such as fibrionectin adsorption to the multilayers. This is an issue to be considered in our future research based on a set of physicochemical instruments and approaches for the characterization of the multilayers we currently employ such as probing the molecular diffusion into the multilayers and imaging …”

Fluorescence Recovery after Photobleaching in Ultrathin Polymer Films

“…Recovery is limited by the reaction rate if, 1/ k off ≪ w 2 /4 D where, 1/ k off is the rate at which flta‐molecule unbinds . The FRAP experiment takes few minutes to an hour, hence, the recovery is analyzed for an unbinding time scale of ≈100 s and ≈20 min corresponding to k off = 0.01, 0.001 s −1 , respectively . Other reaction parameters such as the forward reaction constant k on and the total number of receptor sites σ m are held constant as their change only shifts the equilibrium time and the total number of bound flta‐molecule, respectively .…”

Probing Bio-Molecules across Polyelectrolyte Multilayers

“…The recovery or the change in the fluorescence intensity of the ROI contains the information about the diffusion of molecules, which are labelled with fluorophores and diffusion coefficients can be extracted by fitting this data to various mathematical models. Existing literature contains a number of examples where FRAP has been employed to study bio-molecular transport in PEMs [8][9][10][11]. In fact, FRAP is the most used technique for studying the lateral molecular transport across PEM [9,10].…”

“…In fact, FRAP is the most used technique for studying the lateral molecular transport across PEM [9,10]. Very recently, FRAP has been utilized to study the interaction and loading behaviour of Lysozyme protein and Human Serum Albumin (HSA) across the polymeric stack of Poly(L-lysine) (PLL) and hyaluronic acid (HA) [8,9]. Due to the exponential layer growth in this system, this PEM stack is thick enough to be explored for excellent loading.…”