Preparation and Characterization of Biomimetic ß-Lens Crystallins Using Single-Chain Polymeric Nanoparticles.

Presbyopia, the inability to focus at arm's length, and cataracts that cloud vision are associated primarily with changes in the mechanical and optical properties of the lens. The optical properties, particularly the refractive index, of the human lens originate from the cytoplasm of the lens fiber, which contains a highly concentrated solution (∼40%) of globular proteins referred to as α, ß, and γ crystallins, of which ß is the most abundant. In this study, we focus on the synthesis and characterization of a ß-crystallin biomimetic in an effort to understand and develop treatments for presbyopia and cataract. Polyacrylamide was used as a protein analogue. The side chains were endowed with aromatic and acidic functionality. Acrylic acid was incorporated into the copolymer and cross-linked with diamines to form nanoparticles. The composition and cross-linking condition of the biomimetic copolymers were optimized to match the hydrodynamic radius (Rh), refractive index, size, density, and intrinsic and dynamic viscosities with those of ßhigh lens crystallins. The refractive indices and densities of the nanoparticles' dispersion at different concentrations matched that of ßhigh lens crystallins, and the viscosity of the nanoparticles approached that of ßhigh lens crystallins. The biocompatibility findings for primary porcine retinal pigment epithelial (ppRPE) cells and porcine lens epithelial (pLE) cells showed both cell types tolerated up to 30 mg/mL of nanoparticles. These materials have the potential for use as replacements for the crystallins in developing an accommodating intraocular lens nanocomposite hydrogel that closely replicates the natural autofocusing ability of the original.

Novel mode of ISG15-mediated protection against influenza A virus and Sendai virus in mice.

UNLABELLED: ISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15(-/-) mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replication in vivo. A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting the virus burden. In order to better understand the function of ISG15 in vivo, we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15(-/-) mice. We found that ISG15 protects mice from virus induced lethality by a conjugation-dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugation-dependent, but nonantiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15 in vivo. IMPORTANCE: It has previously been demonstrated that ISG15(-/-) mice are more susceptible to a number of viral infections. Since ISG15 is one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. Although a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protection in vivo. In these studies we have found that while ISG15(-/-) mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus, in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15 in vivo.

Synthesis and Characterization of Injectable Sulfonate-Containing Hydrogels.

Sulfonate-containing hydrogels are of particular interest because of their tunable mechanical and swelling properties, as well as their biological effects. Polysulfonate copolymers were synthesized by reacting 2-acrylamido-2-methylpropanesulfonic acid (AMPS), acrylamide (AM), and acrylic acid (AA). We found that the incorporation rate of sulfonate-containing monomer and the molecular weight of the copolymer were significantly enhanced by increasing the ionic strength of the solution. We introduced thiol groups by modifying the pendant carboxylates or copolymerizing along with a disulfide-containing monomer. The thiol-containing copolymers were reacted with a 4-arm acrylamide-terminated poly(ethylene glycol) via a thiol-ene click reaction, which was mediated by a photoinitiator, a redox initiator, or a base-catalyzed Michael-Addition. We were able to tailor the storage modulus (33-1800 Pa) and swelling capacity (1-91 wt %) of the hydrogel by varying the concentration of the copolymers. We determined that the injectable sulfonate-containing hydrogels were biocompatible up to 20 mg/mL, as observed by an electric cell-substrate impedance sensing (ECIS) technique, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using three different cell lines: human retinal pigment epithelial cells (ARPE-19), fibroblasts (NIH 3T3), and Chinese hamster ovary cells (CHO).

Mice lacking the ISG15 E1 enzyme UbE1L demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza B virus infection.

ISG15 functions as a critical antiviral molecule against influenza virus, with infection inducing both the conjugation of ISG15 to target proteins and production of free ISG15. Here, we report that mice lacking the ISG15 E1 enzyme UbE1L fail to form ISG15 conjugates. Both UbE1L(-/-) and ISG15(-/-) mice display increased susceptibility to influenza B virus infection, including non-mouse-adapted strains. Finally, we demonstrate that ISG15 controls influenza B virus infection through its action within radioresistant stromal cells and not bone marrow-derived cells. Thus, the conjugation of ISG15 to target proteins within stromal cells is critical to its activity against influenza virus.

Bioinspired Fibrillary Hydrogel with Controlled Swelling Behavior: Applicability as an Artificial Vitreous.

The vitreous humor of the eye is mainly composed of fibrillary collagen and semiflexible hyaluronic acid (HA). To mimic this macromolecular composition of the vitreous, we previously developed an injectable two-component hydrogel composed of a fibrillary gellan and a semiflexible polyelectrolyte, poly[methacrylamide-co-(methacrylic acid)], both endowed with thiol cross-linkers. We optimized the hydrogel formulations for optical, physical, mechanical, and transport properties approximating those of the vitreous. Here, we studied 11 hydrogel formulations with varying concentrations of each component, and, as expected, we found that they all swelled in physiological solution. The two formulations that most closely matched the vitreous properties were investigated further. Judged against nonsurgical control and silicone oil, a clinically accepted vitreous replacement, both hydrogel formulations were biocompatible in rabbits for 30 days. Both hydrogels maintained optical clarity, physiological intraocular pressure, and intact retinal layers that displayed normal electroretinography. The swelling behavior of the gel led us to postulate that the native vitreous may also exhibit controlled swelling, where ionic HA's swelling capacity is restricted by fibrillary collagen. In conclusion, the two hydrogels merit further in vivo evaluation as an artificial vitreous for an extended duration and additionally in mini-pigs for their similarity to human eyes in size.

Synthesis and characterization of in situ forming anionic hydrogel as vitreous substitutes.

The natural vitreous is a biological hydrogel consisting primarily of a collagen and anionic hyaluronate. It is surgically removed in many ocular diseases and replaced with fluids, gases, or silicone oils. We have been interested in developing synthetic hydrogels as vitreous substitutes. In this study, we combined the stiffness and hydrophobicity of polymethacrylamide (PMAM) and the anionic nature of polymethacrylate (PMAA) to make copolymers that would mimic the natural vitreous. We used bis-methacryloyl cystamine (BMAC) to introduce thiol groups for reversible crosslink. The Mn of copolymers ranged from ∼100 k to ∼200 k Da (polydisperisty index of 1.47-2.63) and their composition as determined by titration, 1 H NMR and disulfide test were close to the feed ratio. The reactivities of monomers were as follows: MAM > MAA ∼ BMAC. Copolymers with higher MAA contents gelled faster, swelled more, and had higher storage modulus (1.5 to 100 Pa) comparable to that of the natural vitreous. We evaluated the biocompatibility of copolymers by electric cell-substrate impedance sensing (ECIS) using human retinal pigment epithelial cells, primary porcine retinal pigmented epithelial cells, human microvascular endothelial cells adult dermis, and a fibroblast line 3T3. The biocompatibility decreases as the content of BMAC increases. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 977-988, 2017.

Biomimetic hydrogel with tunable mechanical properties for vitreous substitutes.

UNLABELLED: The vitreous humor of the eye is a biological hydrogel principally composed of collagen fibers interspersed with hyaluronic acid. Certain pathological conditions necessitate its removal and replacement. Current substitutes, like silicone oils and perfluorocarbons, are not biomimetic and have known complications. In this study, we have developed an in situ forming two-component biomimetic hydrogel with tunable mechanical and osmotic properties. The components are gellan, an analogue of collagen, and poly(methacrylamide-co-methacrylate), an analogue of hyaluronic acid; both endowed with thiol side groups. We used response surface methodology to consider seventeen possible hydrogels to determine how each component affects the optical, mechanical, sol-gel transition temperature and swelling properties. The optical and physical properties of the hydrogels were similar to vitreous. The shear storage moduli ranged from 3 to 358Pa at 1Hz and sol-gel transition temperatures from 35.5 to 43°C. The hydrogel had the ability to remain swollen without degradation for four weeks in vitro. Three hydrogels were tested for biocompatibility on primary porcine retinal pigment epithelial cells, human retinal pigment epithelial cells, and fibroblast (3T3/NIH) cells, by electric cell-substrate impedance sensing system. The two-component hydrogels allowed for the tuning and optimizing of mechanical, swelling, and transition temperature to obtain three biocompatible hydrogels with properties similar to the vitreous. Future studies include testing of the optimized hydrogels in animal models for use as a long-term substitute, whose preliminary results are mentioned. STATEMENT OF SIGNIFICANCE: Although hydrogels are researched as long-term vitreous substitute, none have advanced sufficiently to reach clinical application. Our work focuses on the development of a novel two component in situ forming hydrogel that bio-mimic the natural vitreous. Our thiol-containing copolymers can be injected as an aqueous solution into the vitreous cavity wherein, at physiological temperature, the rigid component will instantaneously form a physical gel imbedding the random coil copolymer. Upon subsequent oxidation, the two components will form disulfide cross-links and a stable reversible hydrogel capable of providing osmotic pressure to reattach the retina. It may be left in the eye permanently or easily removed by injection of a simple reducing agent to cleave the disulfide bonds, rather than surgery. This contribution is significant because it is expected to provide patients with a much better quality of life by improving surgical outcomes, creating much less post-operative burden, and reducing the need for secondary surgeries.

Investigating triazine-based modification of hyaluronan using statistical designs.

Hyaluronan (HA) and its derivatives have been extensively researched for many biomedical applications. To precisely tailor the property of HA by derivatizing it to a pre-determined extent is challenging, yet critical. In this paper, we used 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) to derivatize HA via a triazine-based coupling reaction. Using a fractional factorial (FF) design, we observed that water content in the solvent, and molar ratios of CDMT and NaHCO3 to the carboxylate were the significant factors controlling the derivatization. We investigated how the effect of each factor changes as reaction conditions change. Moreover, by altering the amount of CDMT and NaHCO3, we developed a cubic regression model for precise control of the extent of derivatization using a response surface methodology (RSM) with a D-optimal design. No spurious peaks were detected by (1)H NMR spectrum and only 10% decrease of molecular weight of the derivatized HA was determined by GPC. The HA with 6% modification was relatively biocompatible up to 15 mg/mL.