pH-Triggered Release of Hydrophobic Molecules from Self-Assembling Hybrid Nanoscaffolds

Self-assembling peptide based hydrogels have a wide range of applications in the field of tissue repair and tissue regeneration. Because of its physicochemical properties, (RADA)₄ has been studied as a potential platform for 3D cell culture, drug delivery, and tissue engineering. Despite some small molecule and protein release studies with this system, there is a lack of work investigating the controlled release of hydrophobic compounds (i.e., anti-inflammatory, anticancer, antibacterial drugs, etc.) that are important for many clinical therapies. Attempts to incorporate hydrophobic compounds into self-assembling matrices usually inhibited nanofiber formation, rather resulting in a peptide–drug complex or microcrystal formation. Herein, a self-assembling chitosan/carboxymethyl-β-cyclodextrin nanoparticle system was used to load dexamethasone, which formed within a self-assembling (RADA)₄ nanoscaffold matrix. Nanoparticles dispersed within the matrix were stabilized by the nanofibers within. The in vitro release of dexamethasone from the hybrid system was observed to be pH sensitive. At pH 7, release was observed for more than 8 days, with three distinct kinetic domains in the first 6 days. Data suggest that the deprotonation of chitosan at a solution pH > 6.8 leads to nanoparticle dissociation and ultimately the release of dexamethasone from the hybrid system. This system has the potential to form a multifunctional scaffold that can self-assemble with the ability to control the release of hydrophobic drugs for a wide variety of applications

Understanding the Effect of Secondary Structure on Molecular Interactions of Poly‑l‑lysine with Different Substrates by SFA

Nonspecific adsorption of proteins on biomaterial surfaces challenges the widespread application of engineered materials, and understanding the impact of secondary structure of proteins and peptides on their adsorption process is of both fundamental and practical importance in bioengineering. In this work, poly-l-lysine (PLL)-based α-helices and β-sheets were chosen as a model system to investigate the effect of secondary structure on peptide interactions with substrates of various surface chemistries. Circular dichroism (CD) was used to confirm the presence of both α-helix and β-sheet structured PLL in aqueous solutions and upon adsorption to quartz, where these secondary structures seemed to be preserved. Atomic force microscopy (AFM) imaging showed different surface patterns for adsorbed α-helix and β-sheet PLL. Interactions between PLL of different secondary structures and various substrates (i.e., PLL, Au, mica, and poly­(ethylene glycol) (PEG)) were directly measured using a surface forces apparatus (SFA). It was found that β-sheet PLL films showed higher adsorbed layer thicknesses in general. Adhesion energies of β-sheet versus Au and β-sheet versus β-sheet were considerably higher than that of α-helix versus Au and α-helix versus α-helix systems, respectively. Au and β-sheet PLL interactions seemed to be more dependent on the salt concentration than that of α-helix, while the presence of a grafted PEG layer greatly diminished any attraction with either PLL structure. The molecular interaction mechanism of peptide in different secondary structures is discussed in terms of Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, Alexander-de Gennes (AdG) steric model and hydrogen bonding, which provides important insight into the fundamental understanding of the interaction mechanism between proteins and biomaterials

Molecular Retention Limitations for Prevascularized Subcutaneous Sites for Islet Transplantation

Beta cell replacement therapies utilizing the subcutaneous space have inherent advantages to other sites: the potential for increased accessibility, noninvasive monitoring, and graft extraction. Site prevascularization has been developed to enhance islet survivability in the subcutaneous zone while minimizing potential foreign body immune responses. Molecular communication between the host and prevascularized implant site remains ill-defined. Poly(ethylene oxide)s (PEOs) of various hydrated radii (i.e., ∼11–62 Å) were injected into prevascularized subcutaneous sites in C57BL/6 mice, and the clearance and organ biodistribution were characterized. Prevascularization formed a barrier that confined the molecules compared with the unmodified site. Molecular clearance from the prevascularized site was inversely proportional to the molecular weight. The upper limit in molecular size for entering the vasculature to be cleared was determined to be 35 kDa MW PEO. These findings provide insight into the impact of vascularization on molecular retention at the injection site and the effect of molecular size on the mobility of hydrophilic molecules from the prevascularized site to the host. This information is necessary for optimizing the transplantation site for increasing the beta cell graft survival

Self-Assembling Peptide Nanoscaffold That Activates Human Mast Cells

Engineering biomaterials to manipulate the immune response to elicit specific therapeutic outcomes is a burgeoning field of research. Mast cells play a distinct and central role in the innate immune response, and are characterized by their rapid release of a myriad of proinflammatory mediators in response to stimulation. These mediators are central to protective actions such as wound healing, angiogenesis, and host defense against pathogens and animal venoms. Considering that mast cells are widely distributed in tissues that interface with the external environment, and are loaded with large amounts of preformed protective compounds, they are ideal targets for novel immunotherapies. Here we report that, by using an engineered nanoscaffold, human mast cells can be contact activated in cell and primary human skin tissue culture using a specific receptor–ligand mechanism. The IgE independent PAMP-12 peptide activates human mast cells through the recently identified Mas-related G-protein coupled receptor member X2 (MRGPRX₂) receptor. The PAMP-12 motif was conjugated, via a glycine spacer, with the self-assembling peptide (RADA)₄ and mixed with unmodified (RADA)₄ to form a nanofiber matrix; mast cell activation was influenced directly by this ratio. Moreover, conjugating the PAMP-12 motif within the matrix was shown to only activate local, tissue-resident mast cells. The result of <i>ex vivo</i> human skin tissue tests confirmed that the engineered nanoscaffold successfully activated skin-resident mast cells by contact. Thus, this nanoscaffold design may provide a new platform to modulate localized mast cell functions thereby facilitating their protective role in the skin

ThT fluorescence emission spectra of crystallin proteins in 0:2:1, 1:2:1, 5:2:1 and 10:2:1 ratios incubated at room temperature for 10 hours (solid lines) and exposed to UV-B radiation for 10 hours (dotted lines).

<p><b>Inset graph</b> represents the relative intensity difference between UV-B treated and untreated samples. The data represents the average of 3 independent experiments and error bars represent SD. *: <i>P</i><0.01, ns: Not Significant.</p

UV-B induced fibrillization of crystallin protein mixtures

<div><p>Environmental factors, mainly oxidative stress and exposure to sunlight, induce the oxidation, cross-linking, cleavage, and deamination of crystallin proteins, resulting in their aggregation and, ultimately, cataract formation. Various denaturants have been used to initiate the aggregation of crystallin proteins <i>in vitro</i>. All of these regimens, however, are obviously far from replicating conditions that exist <i>in vivo</i> that lead to cataract formation. In fact, it is our supposition that only UV-B radiation may mimic the observed <i>in vivo</i> cause of crystallin alteration leading to cataract formation. This means of inducing cataract formation may provide the most appropriate <i>in vitro</i> platform for in-depth study of the fundamental cataractous fibril properties and allow for testing of possible treatment strategies. Herein, we showed that cataractous fibrils can be formed using UV-B radiation from α:β:γ crystallin protein mixtures. Characterization of the properties of formed aggregates confirmed the development of amyloid-like fibrils, which are in cross-β-pattern and possibly in anti-parallel β-sheet arrangement. Furthermore, we were also able to confirm that the presence of the molecular chaperone, α-crystallin, was able to inhibit fibril formation, as observed for ‘naturally’ occurring fibrils. Finally, the time-dependent fibrillation profile was found to be similar to the gradual formation of age-related nuclear cataracts. This data provided evidence for the initiation of fibril formation from physiologically relevant crystallin mixtures using UV-B radiation, and that the formed fibrils had several traits similar to that expected from cataracts developing <i>in vivo</i>.</p></div

UV-B induced fibrillization of crystallin protein mixtures - Fig 5

<p><b>(A)</b> Electron micrographs of crystallin fibrils at different ratios (from top to bottom; 0:2:1, 1:2:1, 5:2:1 and 10:2:1) and different times of aggregation (from left to right; 0, 5 and 10 hours of UV-B exposure and 10 hours RT control). <b>(B)</b> Surface area distribution of fibrils in each sample. The bars represent the average of at least three images per representative samples. Error bars represent SD.</p