Thickness-Tunable Eggshell Membrane Hydrolysate Nanocoating with Enhanced Cytocompatibility and Neurite Outgrowth

Abstract

The eggshell membrane is one of the easily obtainable natural biomaterials, but has been neglected in the biomaterial community, compared with marine biomaterials and discarded as a food waste. In this work, we utilized the ESM hydrolysate (ESMH), which was obtained by the enzymochemical method, as a bioactive functional material for interfacial bioengineering, exemplified by thickness-tunable, layer-by-layer (LbL) nanocoating with the Fe­(III)-tannic acid (TA) complex. [Fe­(III)-TA/ESMH] LbL films, ending with the ESMH layer, showed great cytocompatiblility with HeLa cells and even primary hippocampal neuron cells. More importantly, the films were found to be neurochemically active, inducing the acceleration of neurite outgrowth for the long-term neuron culture. We believe that the ability for building cytocompatible ESMH films in a thickness-tunable manner would be applicable to a broad range of different nanomaterials in shape and size and would be utilized with multimodal functionalities for biomedical applications, such as bioencapsulation, theranostics, and regenerative medicine.

Coffee Melanoidin‐Based Multipurpose Film Formation: Application to Single‐Cell Nanoencapsulation

“…These results confirmed the post‐functionalizations of the CM film, which are highly advantageous over the previous polyphenolic‐based coating strategies in terms of versatility and applicability. The grafting method was also applied to the construction of neurocompatible surfaces via poly‐D‐lysine (PDL) grafting, considering the findings that neuronal developments and branching are intimately related with surface interactions . The grafting capability of the CM film was found to be important in neuronal attachment and development (Figure f, g): in addition to the high viability, we also observed that primary hippocampal neurons developed neurites and branches properly after PDL grafting (Figure S5).…”

“…The post‐functionalizability of the CM film was utilized to fabricate the organic‐inorganic hybrid shell on the yeast cells, mimicking the eggshell structures (Figure a). In this case of shell functionalization and manipulation, the eggshell membrane hydrolysate (ESMH), another nature‐derived, cytocompatible material, was used as both primer and initiator for bioinspired silicification. After ESMH priming, the silicification was performed under the same conditions as those for the gold substrate aforementioned.…”

Single‐Cell Nanoencapsulation of Saccharomyces cerevisiae by Cytocompatible Layer‐by‐Layer Assembly of Eggshell Membrane Hydrolysate and Tannic Acid

“…We recently reported the potential of water-soluble ESMH as a biocompatible material in the construction of cell-material interfaces. [48,49] ESMH can be obtained easily from ESM through treatment with papain (a proteolytic enzyme from papaya) and dithiothreitol (DTT), as reported previously. [48] DTT has been used to reduce the disulfide bonds to thiols in the ESM, which are one of the causes of ESM's water insolubility.…”

“…[48,49] ESMH can be obtained easily from ESM through treatment with papain (a proteolytic enzyme from papaya) and dithiothreitol (DTT), as reported previously. [48] DTT has been used to reduce the disulfide bonds to thiols in the ESM, which are one of the causes of ESM's water insolubility. Briefly, purified ESM was incubated for 4 h at 60 C in the Tris-HCl solution of papain and DTT (pH 7.4), filtered, dialyzed, and freeze-dried.…”

Resveratrol‐β‐Lactoglobulin Composite Nanocoating by Layer‐by‐Layer Assembly with Fe(III)‐Tannic Acid Complex

“…This was a similar deposition pattern to that observed during a bilayer deposition of eggshell membrane hydrolysates and Fe(III)-TA complex. [24] These results imply that proteins on Fe(III)-TA nanofilms would not penetrate whole nanofilm structures but would rather interact with the neighboring Fe(III)-TA layer, although proteins often fall into a diffusing polyelectrolyte. [25] The limited penetration of proteins could be attributed to strong hydrogen bonding cross-links between the hydrogen bonding donors found in TA and hydrogen bonding acceptors from peptide bonds and side chains found in BLG.…”