Multivalent 3D Display of Glycopolymer Chains for Enhanced Lectin Interaction

Synthetic glycoprotein conjugates were synthesized through the polymerization of glycomonomers (mannose and/or galactose acrylate) directly from a protein macroinitiator. This design combines the multivalency of polymer structures with 3D display of saccharides randomly arranged around a central protein structure. The conjugates were tested for their interaction with mannose binding lectin (MBL), a key protein of immune complement. Increasing mannose number (controlled through polymer chain length) and density (controlled through comonomer feed ratio of mannose versus galactose) result in greater interaction with MBL. Most significantly, mannose glycopolymers displayed in a multivalent and 3D configuration from the protein exhibit dramatically enhanced interaction with MBL compared to linear glycopolymer chains with similar total valency but lacking 3D display. These findings demonstrate the importance of the 3D presentation of ligand structures for designing biomimetic materials

Effect of Branching Density on Avidity of Hyperbranched Glycomimetics for Mannose Binding Lectin

Hyperbranched glycopolymers containing mannose units in the branch point were synthesized through the copolymerization of a mannose inimer and mannose acrylate via atom transfer radical polymerization (ATRP). Incorporating a saccharide residue at the branch point results in a closer analogue to natural branched polysaccharides. Gel permeation chromatography characterization of the polymers qualitatively indicates branching in samples from polymerizations utilizing the mannose inimer. Deprotection of the acetate protecting groups from the hyperbranched mannose polymers yields water-soluble polymers that interact with mannose binding lectin (MBL), a key protein of the innate immunity complement system. MBL interaction increases with increasing polymer molecular weight and increasing branching density. Notably, incorporating mannose into the branching repeat unit also increases the interaction of the glycopolymers with MBL compared with glycopolymers with the same branching density but with no mannose at the branch point

Photodegradable Macromers and Hydrogels for Live Cell Encapsulation and Release

Hydrogel scaffolds are commonly used as 3D carriers for cells because their properties can be tailored to match natural extracellular matrix. Hydrogels may be used in tissue engineering and regenerative medicine to deliver therapeutic cells to injured or diseased tissue through controlled degradation. Hydrolysis and enzymolysis are the two most common mechanisms employed for hydrogel degradation, but neither allows sequential or staged release of cells. In contrast, photodegradation allows external real-time spatial and temporal control over hydrogel degradation, and allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating photodegradbale <i>ortho</i>-nitrobenzyl (<i>o</i>-NB) groups in the macromer backbone. We formed hydrogels from these macromers via redox polymerization and quantified the apparent rate constants of degradation (<i>k</i>_app) of each via photorheology at 370 nm, 10 mW/cm². Decreasing the number of aryl ethers on the <i>o</i>-NB group increases <i>k</i>_app, and changing the functionality from primary to seconday at the benzylic site dramatically increases <i>k</i>_app. Human mesenchymal stem cells (hMSCs) survive encapsulation in the hydrogels (90% viability postencapsulation). By exploiting the differences in reactivity of two different <i>o</i>-NB linkers, we quantitatively demonstrate the biased release of one stem cell population (green-fluoroescent protein expressing hMSCs) over another (red-fluorescent protein expressing hMSCs)

Poly(methyl 6‑acryloyl-β‑d‑glucosaminoside) as a Cationic Glycomimetic of Chitosan

Chitosan, a cationic polysaccharide derived from one of the most abundant natural polymers, chitin, has been investigated extensively for its antimicrobial properties. However, it suffers from the inherent drawbacks of natural products such as batch-to-batch variability, limited supply, contamination, and potential adverse reaction. Additionally, its solubility depends on the degree of deacetylation and pH, as it is only soluble under acidic conditions. As an alternative to chitosan, we synthesized the protected cationic glycomimetic monomer methyl <i>N</i>-Fmoc-6-acryloyl-β-d-glucosaminoside from glucosamine. This monomer retains structural features critical to recapitulating the properties of the chitosan repeat unit, namely, the p<i>K</i>_a of the protonated amine. We optimized the free radical polymerization of methyl <i>N</i>-Fmoc-6-acryloyl-β-d-glucosaminoside and fractionated the resultant poly­(methyl <i>N</i>-Fmoc-6-acryloyl-β-d-glucosaminoside) to obtain a range of molecular weights. Following Fmoc deprotection, the cationic glycopolymers retained 95% of their expected amine content by mass and exhibited a p<i>K</i>_a of 6.61. Poly­(methyl 6-acryloyl-β-d-glucosaminoside) mimicked the molecular weight-dependent bacterial inhibitory property of chitosan in acidic solutions. Importantly, poly­(methyl 6-acryloyl-β-d-glucosaminoside) remained soluble at elevated pH (conditions under which chitosan is insoluble) and maintained its antibacterial activity. Mammalian cell viability in the presence of poly­(methyl 6-acryloyl-β-d-glucosaminoside) at acidic pH is good, although somewhat lower than viability in the presence of chitosan. No cytotoxic effect was observed at neutral pH. These results demonstrate that poly­(methyl 6-acryloyl-β-d-glucosaminoside) is not only a suitable biomimetic for chitosan, but that it can be utilized as an antibacterial agent in a broader range of biologically relevant pHs

Biodegradable Aromatic–Aliphatic Poly(ester–amides) from Monolignol-Based Ester Dimers

Biobased polymers with tunable properties have received increased attention in the literature due to a decline in petroleum reserves. Owing to its low cost, abundance, and aromatic structure, lignin has great potential as a feedstock for value-added polymeric products. In this work, we condensed carboxylic acid precursors with monolignols to generate reactive dimers for polymer synthesis. Three different aromatic ester dimers, each corresponding to a different monolignol, were synthesized and characterized. The dicarboxylic acid dimers were converted to the corresponding diacid chloride in situ with thionyl chloride, and a series of poly­(ester–amides) were synthesized via interfacial polymerization of these diacid chlorides with seven different aliphatic or aromatic diamines. The thermal properties (decomposition, glass transition temperature, and melting temperature) and hydrolytic stability in acidic and neutral aqueous conditions of the resulting polymers were studied

Photoselective Delivery of Model Therapeutics from Hydrogels

Hydrogels are commonly used in biomedical applications to sequester and release therapeutics. Covalently tethering therapeutic agents to a hydrogel through a degradable linkage allows their controlled release, but temporally separating the release of multiple therapeutics from a single hydrogel remains a major challenge. In this report, we use a series of photodegradable <i>ortho</i>-nitrobenzyl (<i>o</i>-NB) groups with varying structures to link model therapeutic agents (fluorescein, rhodamine, and aminomethylcoumarin acetate) to poly­(ethylene glycol) macromers. We polymerized the macromers into hydrogel networks via redox polymerization and quantified the apparent rate constants of degradation (<i>k</i>_app) of each of the photoreleasable compounds. By exploiting differences in reactivity of the different <i>o</i>-NB groups, we are able to create complex, multistage release profiles. We demonstrate the ability to switch between concurrent and biased release of model therapeutics simply by switching wavelengths. We also demonstrate a complex four-stage release profile in which the release of three separate model therapeutics is controlled by varying wavelength, intensity, and exposure time. This is the first report of photoselective release of therapeutics from a hydrogel, allowing user-dictated real-time spatial and temporal control over <i>multiple</i> chemical signals in a cell microenvironment in 2D and 3D

Low-Dose, Long-Wave UV Light Does Not Affect Gene Expression of Human Mesenchymal Stem Cells

<div><p>Light is a non-invasive tool that is widely used in a range of biomedical applications. Techniques such as photopolymerization, photodegradation, and photouncaging can be used to alter the chemical and physical properties of biomaterials in the presence of live cells. Long-wave UV light (315 nm–400 nm) is an easily accessible and commonly used energy source for triggering biomaterial changes. Although exposure to low doses of long-wave UV light is generally accepted as biocompatible, most studies employing this wavelength only establish cell viability, ignoring other possible (non-toxic) effects. Since light exposure of wavelengths longer than 315 nm may potentially induce changes in cell behavior, we examined changes in gene expression of human mesenchymal stem cells exposed to light under both 2D and 3D culture conditions, including two different hydrogel fabrication techniques, decoupling UV exposure and radical generation. While exposure to long-wave UV light did not induce significant changes in gene expression regardless of culture conditions, significant changes were observed due to scaffold fabrication chemistry and between cells plated in 2D versus encapsulated in 3D scaffolds. In order to facilitate others in searching for more specific changes between the many conditions, the full data set is available on Gene Expression Omnibus for querying.</p></div

Experimental groups illustrated.

<p>A single vial of P0 hMSCs were expanded at low density to greater than 25 million cells at P1. These cells were trypsinized and a portion of them replated at 500,000 cells / 75cm² flask for 2D samples. Another portion of those cells was encapsulated in poly(ethylene glycol) diacrylate (MW 4000 Da). Two encapsulation methods were used, radical polymerization with ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), and conjugate addition with a pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) crosslinker. Several samples of each type were created and half from each type were irradiated with a Black Ray UV bench lamp, peak wavelength 365nm.</p

Shape-Changing Photodegradable Hydrogels for Dynamic 3D Cell Culture

Inspired by natural examples of swelling-actuated self-folding, we utilize photodegradable hydrogels as dynamically tunable, shape-changing scaffolds for culturing cells. Poly­(ethylene glycol) diacrylate-based thin films incorporating <i>ortho</i>-nitrobenzyl (<i>o</i>-NB) moieties are transformed from flat 2D sheets to folded 3D structures by exposure to 365 nm UV light. As the UV light is attenuated through the thickness of the gel, a cross-link density gradient is formed. This gradient gives rise to differential swelling and a bending moment, resulting in gel folding. By tuning the UV light dose and the molar ratio of photodegradable to nondegradable species, both the initial degree of folding and the relaxation of tubular structures can be accurately controlled. These self-folding photodegradable gels were further functionalized with a cell-adhesive RGD peptide for both seeding and encapsulation of C2C12 mouse myoblasts. Light-induced folding of RGD functionalized hydrogels from flat sheets to tubular structures was demonstrated 1 or 3 days after C2C12 seeding. The C2C12s remained adhered on the inner walls of folded tubes for up to 6 days after folding. The minimum measured diameter of a tubular structure containing C2C12s was 1 mm, which is similar to the size of muscle fascicles. Furthermore, the viability of encapsulated C2C12s was not adversely affected by the UV light-induced folding. This is the first account of a self-folding material system that allows 2D–3D shape change in the presence of both seeded and encapsulated cells at a user-directed time point of choice

Principle components analysis shows tightly segregated clustering based on culture condition, not by UV exposure.

<p>(alternate viewing angle and axis values available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139307#pone.0139307.s002" target="_blank">S2 Fig</a>). PCA is a statistical analysis tool which reduces the dimensionality of data by determining the key variables resulting in differences seen between samples[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139307#pone.0139307.ref025" target="_blank">25</a>]. Each axis of this PCA map represents a linear combination of expression levels from many thousands of gene transcripts such that, combined, the maximum variation among all data points is achieved on only three axes. The result gives a visual representation of which samples behave similarly to each other by their physical closeness in three dimensions, while including information from many thousands of variables (gene expression levels). This is a bird’s eye view of the entire set of gene array data, for which absolute units and values can be considered arbitrary. The following in-depth analysis of pathway enrichment and specific gene expression provide insight into how each group differs from the others in their gene expression.</p