Recreating the Trabecular Outflow Tissue on Implantable, Micropatterned, Ultrathin, Porous Polycaprolactone Scaffolds.

Glaucoma, where increased intraocular pressure (IOP) leads to damage to the optic nerve and loss of sight, is amongst the foremost causes of irreversible blindness worldwide. In primary open angle glaucoma, the increased IOP is a result of the malfunctioning human trabecular meshwork (HTM) cells' inability to properly regulate the outflow of aqueous humor from the eye. A potential future treatment for glaucoma is to replace damaged HTM cells with a tissue-engineered substitute, thus restoring proper fluid outflow. Polycaprolactone (PCL) is a versatile, biodegradable, and implantable material that is widely used for cell culture and tissue engineering. In this work, PCL scaffolds were lithographically fabricated using a sacrificial process to produce submicron-thick scaffolds with openings of specific sizes and shapes (e.g., grid, hexagonal pattern). The HTM cell growth on gelatin-coated PCL scaffolds was assessed by scanning electron microscopy, tetrazolium metabolic activity assay, and cytoskeletal organization of F-actin. Expression of HTM-specific markers and ECM deposition were assessed by immunocytochemistry and qPCR analysis. Gelatin-coated, micropatterned, ultrathin, porous PCL scaffolds with a grid pattern supported proper HTM cell growth, cytoskeleton organization, HTM-marker expression, and ECM deposition, demonstrating the feasibility of using these PCL scaffolds to tissue-engineer implantable, healthy ocular outflow tissue.

MRI compatible MS2 nanoparticles designed to cross the blood-brain-barrier: providing a path towards tinnitus treatment.

Fundamental challenges of targeting specific brain regions for treatment using pharmacotherapeutic nanoparticle (NP) carriers include circumventing the blood-brain-barrier (BBB) and tracking delivery. Angiopep-2 (AP2) has been shown to facilitate the transport of large macromolecules and synthetic nanoparticles across the BBB. Thus, conjugation of AP2 to an MS2 bacteriophage based NP should also permit transport across the BBB. We have fabricated and tested a novel MS2 capsid-based NP conjugated to the ligand AP2. The reaction efficiency was determined to be over 70%, with up to two angiopep-2 conjugated per MS2 capsid protein. When linked with a porphyrin ring, manganese (Mn2+) remained stable within MS2 and was MRI detectable. Nanoparticles were introduced intracerebroventricularly or systemically. Systemic delivery yielded dose dependent, non-toxic accumulation of NPs in the midbrain. Design of a multifunctional MRI compatible NP platform provides a significant step forward for the diagnosis and treatment of intractable brain conditions, such as tinnitus.

In silico characterization of enantioselective molecularly imprinted binding sites.

A 2-step molecular mechanical and quantum mechanical geometry optimization scheme (MM âž” QM) was used to "computationally imprint" chiral molecules. Using a docking technique, we show the imprinted binding sites to exhibit an enantioselective preference for the imprinted molecule over its enantiomer. Docking of structurally similar chiral molecules showed that the sites computationally imprinted with R- or S-tBOC-tyrosine were able to differentiate between R- and S-forms of other tyrosine derivatives. The cross-enantioselectivity did not hold for chiral molecules that did not share the tyrosine H-bonding functional group orientations. Further analysis of the individual monomer-target interactions within the binding site lead us to conclude that H-bonding functional groups that are located immediately next to the chiral center and therefore spatially fixed relative to the chiral center will have a stronger contribution to the enantioselectivity of the site than those groups separated from the chiral center by 2 or more rotatable bonds. Here, we present our novel approach for computationally imprinting and characterizing enantioselective binding sites. All modeling schemes were designed to minimize the computational expense. In silico analysis of the properties of molecularly imprinted polymer systems will ultimately allow for the fabrication of more sensitive and selective materials.

Polymer-lipid-PEG hybrid nanoparticles as photosensitizer carrier for photodynamic therapy.

Polymer-lipid-PEG hybrid nanoparticles were investigated as carriers for the photosensitizer (PS), 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H,23H-porphine (pTHPP) for use in photodynamic therapy (PDT). A self-assembled nanoprecipitation technique was used for preparing two types of core polymers poly(d,l-lactide-co-glycolide) (PLGA) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with lipid-PEG as stabilizer. The resulting nanoparticles had an average particle size of 88.5±3.4nm for PLGA and 215.0±6.3nm for PHBV. Both nanoparticles exhibited a core-shell structure under TEM with high zeta potential and loading efficiency. X-ray powder diffraction analysis showed that the encapsulated pTHPP molecules in polymeric nanoparticles no longer had peaks of free pTHPP in the crystalline state. The pTHPP molecules encapsulated inside the polymeric core demonstrated improved photophysical properties in terms of singlet oxygen generation and cellular uptake rate in a FTC-133 human thyroid carcinoma cell line, compared to non-encapsulated pTHPP. The pTHPP-loaded polymer-lipid-PEG nanoparticles showed better in vitro phototoxicity compared to free pTHPP, in both time- and concentration-dependent manners. Overall, this study provides detailed analysis of the photophysical properties of pTHPP molecules when entrapped within either PLGA or PHBV nanoparticle cores, and demonstrates the effectiveness of these systems for delivery of photosensitizers. The two polymeric systems may have different potential benefits, when used with cancer cells. For instance, the pTHPP-loaded PLGA system requires only a short time to show a PDT effect and may be suitable for topical PDT, while the delayed photo-induced cytotoxic effect of the pTHPP-loaded PHBV system may be more suitable for cancer solid tumors. Hence, both pTHPP-encapsulated polymer-lipid-PEG nanoparticles can be considered promising delivery systems for PDT cancer treatment.

Cationic dendritic starch as a vehicle for photodynamic therapy and siRNA co-delivery.

Cationic enzymatically synthesized glycogen (cESG) is a naturally-derived, nano-scale carbohydrate dendrite that has shown promise as a cellular delivery vehicle owing to its flexibility in chemical modifications, biocompatibility and relative low cost. In the present work, cESG was modified and evaluated as a vehicle for tetraphenylporphinesulfonate (TPPS) in order to improve cellular delivery of this photosensitizer and investigate the feasibility of co-delivery with short interfering ribonucleic acid (siRNA). TPPS was electrostatically condensed with cESG, resulting in a sub-50nm particle with a positive zeta potential of approximately 5mV. When tested in normal ovarian surface epithelial and ovarian clear cell carcinoma cell culture models, encapsulation of TPPS in cESG significantly improved cell death in response to light treatment compared to free drug alone. Dosages as low as 0.16µM TPPS resulted in cellular death upon illumination with a 4.8J/cm2 light dosage, decreasing viability by 96%. cESG-TPPS was then further evaluated as a co-delivery system with siRNA for potential combination therapy, by charge-based condensation of an siRNA directed at reducing expression of manganese superoxide dismutase (Sod2) as a proof of principle target. Simultaneous delivery of TPPS and siRNA was achieved, reducing Sod2 protein expression to 48%, while maintaining the photodynamic properties of TPPS under light exposure and maintaining low dark toxicity. This study demonstrates the versatility of cESG as a platform for dual delivery of small molecules and oligonucleotides, and the potential for further development of this system in combination therapy applications.

Biomimicry Promotes the Efficiency of a 10-Step Sequential Enzymatic Reaction on Nanoparticles, Converting Glucose to Lactate.

For nanobiotechnology to achieve its potential, complex organic-inorganic systems must grow to utilize the sequential functions of multiple biological components. Critical challenges exist: immobilizing enzymes can block substrate-binding sites or prohibit conformational changes, substrate composition can interfere with activity, and multistep reactions risk diffusion of intermediates. As a result, the most complex tethered reaction reported involves only 3 enzymes. Inspired by the oriented immobilization of glycolytic enzymes on the fibrous sheath of mammalian sperm, here we show a complex reaction of 10 enzymes tethered to nanoparticles. Although individual enzyme efficiency was higher in solution, the efficacy of the 10-step pathway measured by conversion of glucose to lactate was significantly higher when tethered. To our knowledge, this is the most complex organic-inorganic system described, and it shows that tethered, multi-step biological pathways can be reconstituted in hybrid systems to carry out functions such as energy production or delivery of molecular cargo.

TGFß2-induced outflow alterations in a bioengineered trabecular meshwork are offset by a rho-associated kinase inhibitor.

Members of the transforming growth factor beta (TGFß) cytokine family have long been associated with affecting several cellular functions, including cell proliferation, differentiation and extracellular matrix (ECM) turnover. Of particular interest to this work, TGFß2 has been linked to most types of glaucomas as a potential fibrotic agent that can cause elevation of intraocular pressure (IOP). Given that the trabecular meshwork (TM) provides most of aqueous humor outflow resistance in the eye, an in vitro bioengineered human TM (HTM) model has been created and validated by analyzing effects of TGFß2 on transcellular pressure changes and outflow facility. These changes were correlated with several biological alterations induced by this cytokine, including ECM production and overexpression of HTM-marker myocillin. Furthermore, this TM model has been used to extend current knowledge of gene expression of cytokines involved in TGFß-induced ECM turnover over time. In particular, the ability for a ROCK-inhibitor to diminish the effect of TGFß on TM was demonstrated. This work supports the notion that anti-fibrotic activities of ROCK-inhibitors could counteract the elevation of IOP and increased strain observed in glaucomatous TM.

Computational investigation of stoichiometric effects, binding site heterogeneities, and selectivities of molecularly imprinted polymers.

A series of quantum mechanical (QM) computational optimizations of molecularly imprinted polymer (MIP) systems were used to determine optimal monomer-to-target ratios. Imidazole- and xanthine-derived target molecules were studied. The investigation included both small-scale models (3-7 molecules) and larger-scale models (15-35 molecules). The optimal ratios differed between the small and larger scales. For the larger models containing multiple targets, binding-site surface area analysis was used to quantify the heterogeneity of these sites. The more fully surrounded sites had greater binding energies. No discretization of binding modes was seen, furthering arguments for continuous affinity distribution models. Molecular mechanical (MM) docking was then used to measure the selectivities of the QM-optimized binding sites. Selectivity was also shown to improve as binding sites become more fully encased by the monomers. For internal sites, docking consistently showed selectivity favoring the molecules that had been imprinted via QM geometry optimizations. The computationally imprinted sites were shown to exhibit size-, shape-, and polarity-based selectivity. Here we present a novel approach to investigate the selectivity and heterogeneity of imprinted polymer binding sites, by applying the rapid orientation screening of MM docking to the highly accurate QM-optimized geometries. Modeling schemes were designed such that no computing clusters or other specialized modeling equipment would be required. Improving the in silico analysis of MIP system properties will ultimately allow for the production of more sensitive and selective polymers.

Gallic Acid Is an Antagonist of Semen Amyloid Fibrils That Enhance HIV-1 Infection.

Recent in vitro studies have demonstrated that amyloid fibrils found in semen from healthy and HIV-infected men, as well as semen itself, can markedly enhance HIV infection rates. Semen fibrils are made up of multiple naturally occurring peptide fragments derived from semen. The best characterized of these fibrils are SEVI (semen-derived enhancer of viral infection), made up of residues 248-286 of prostatic acidic phosphatase, and the SEM1 fibrils, made up of residues 86-107 of semenogelin 1. A small molecule screen for antagonists of semen fibrils identified four compounds that lowered semen-mediated enhancement of HIV-1 infectivity. One of the four, gallic acid, was previously reported to antagonize other amyloids and to exert anti-inflammatory effects. To better understand the mechanism by which gallic acid modifies the properties of semen amyloids, we performed biophysical measurements (atomic force microscopy, electron microscopy, confocal microscopy, thioflavin T and Congo Red fluorescence assays, zeta potential measurements) and quantitative assays on the effects of gallic acid on semen-mediated enhancement of HIV infection and inflammation. Our results demonstrate that gallic acid binds to both SEVI and SEM1 fibrils and modifies their surface electrostatics to render them less cationic. In addition, gallic acid decreased semen-mediated enhancement of HIV infection but did not decrease the inflammatory response induced by semen. Together, these observations identify gallic acid as a non-polyanionic compound that inhibits semen-mediated enhancement of HIV infection and suggest the potential utility of incorporating gallic acid into a multicomponent microbicide targeting both the HIV virus and host components that promote viral infection.

Targeted fibrillar nanocarbon RNAi treatment of acute kidney injury.

RNA interference has tremendous yet unrealized potential to treat a wide range of illnesses. Innovative solutions are needed to protect and selectively deliver small interfering RNA (siRNA) cargo to and within a target cell to fully exploit siRNA as a therapeutic tool in vivo. Herein, we describe ammonium-functionalized carbon nanotube (fCNT)-mediated transport of siRNA selectively and with high efficiency to renal proximal tubule cells in animal models of acute kidney injury (AKI). fCNT enhanced siRNA delivery to tubule cells compared to siRNA alone and effectively knocked down the expression of several target genes, includingTrp53,Mep1b,Ctr1, andEGFP A clinically relevant cisplatin-induced murine model of AKI was used to evaluate the therapeutic potential of fCNT-targeted siRNA to effectively halt the pathogenesis of renal injury. Prophylactic treatment with a combination of fCNT/siMep1band fCNT/siTrp53significantly improved progression-free survival compared to controls via a mechanism that required concurrent reduction of meprin-1ß and p53 expression. The fCNT/siRNA was well tolerated, and no toxicological consequences were observed in murine models. Toward clinical application of this platform, fCNTs were evaluated for the first time in nonhuman primates. The rapid and kidney-specific pharmacokinetic profile of fCNT in primates was comparable to what was observed in mice and suggests that this approach is amenable for use in humans. The nanocarbon-mediated delivery of siRNA provides a therapeutic means for the prevention of AKI to safely overcome the persistent barrier of nephrotoxicity during medical intervention.

Debridement, antibiotics and implant retention in early periprosthetic joint infection.

INTRODUCTION: Periprosthetic joint infection (PJI) is a devastating complication in hip arthroplasty surgery. Debridement, antibiotics (AB) and implant retention (DAIR) is recommended in early PJI in association with stable implants. The aim of this study was to evaluate the success rate of DAIR in early PJI (<4 weeks) and to identify factors predicting the outcome. METHODS: This cohort study included a consecutive series of 35 patients (median age 74 years, 25 women, 26 primary arthroplasties) treated with DAIR for an early PJI in a regional hospital. RESULTS: 28 patients (80%) had their infection eradicated. DAIR-only eradicated the PJI in 22 (63%) patients with a median follow-up of 50 (24-84) months. In 17 (49%) patients, oral AB had been given prior to intraoperative cultures, which delayed first debridement with average 6 days and delayed hospital stay. Primary surgery for a hip fracture increased the risk of DAIR-failure. Surgical experience did not affect the outcome. 17% (n = 6) of the patients sustained a secondary infection during their hospital stay; the majority was beta-lactam resistant coagulase negative Staphylococcus aureus. CONCLUSIONS: The success rate of DAIR was inferior to pervious controls from experienced revision centers. Hip fracture patients should be informed about the increased risk of DAIR treatment failure. In order not to delay surgery, empirically based oral AB should not be administered prior to deep cultures. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02087020.

Bioengineered glaucomatous 3D human trabecular meshwork as an in vitro disease model.

Intraocular pressure (IOP) is mostly regulated by aqueous humor outflow through the human trabecular meshwork (HTM) and represents the only modifiable risk factor of glaucoma. The lack of IOP-modulating therapeutics that targets HTM underscores the need of engineering HTM for understanding the outflow physiology and glaucoma pathology in vitro. Using a 3D HTM model that allows for regulation of outflow in response to a pharmacologic steroid, a fibrotic state has been induced resembling that of glaucomatous HTM. This disease model exhibits HTM marker expression, ECM overproduction, impaired HTM cell phagocytic activity and outflow resistance, which represent characteristics found in steroid-induced glaucoma. In particular, steroid-induced ECM alterations in the glaucomatous model can be modified by a ROCK inhibitor. Altogether, this work presents a novel in vitro disease model that allows for physiological and pathological studies pertaining to regulating outflow, leading to improved understanding of steroid-induced glaucoma and accelerated discovery of new therapeutic targets. Biotechnol. Bioeng. 2016;113: 1357-1368. © 2015 Wiley Periodicals, Inc.

Development and characterization of bio-derived polyhydroxyalkanoate nanoparticles as a delivery system for hydrophobic photodynamic therapy agents.

In this study, we developed and investigated nanoparticles of biologically-derived, biodegradable polyhydroxyalkanoates (PHAs) as carriers of a hydrophobic photosensitizer, 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H, 23H-porphine (pTHPP) for photodynamic therapy (PDT). Three PHA variants; polyhydroxybutyrate, poly(hydroxybutyrate-co-hydroxyvalerate) or P(HB-HV) with 12 and 50% HV were used to formulate pTHPP-loaded PHA nanoparticles by an emulsification-diffusion method, where we compared two different poly(vinyl alcohol) (PVA) stabilizers. The nanoparticles exhibited nano-scale spherical morphology under TEM and hydrodynamic diameters ranging from 169.0 to 211.2 nm with narrow size distribution. The amount of drug loaded and the drug entrapment efficiency were also investigated. The in vitro photocytotoxicity was evaluated using human colon adenocarcinoma cell line HT-29 and revealed time and concentration dependent cell death, consistent with a gradual release pattern of pTHPP over 24 h. This study is the first demonstration using bacterially derived P(HB-HV) copolymers for nanoparticle delivery of a hydrophobic photosensitizer drug and their potential application in PDT.

A sacrificial process for fabrication of biodegradable polymer membranes with submicron thickness.

A new sacrificial molding process using a single mask has been developed to fabricate ultrathin 2-dimensional membranes from several biocompatible polymeric materials. The fabrication process is similar to a sacrificial microelectromechanical systems (MEMS) process flow, where a mold is created from a material that can be coated with a biodegradable polymer and subsequently etched away, leaving behind a very thin polymer membrane. In this work, two different sacrificial mold materials, silicon dioxide (SiO2 ) and Liftoff Resist (LOR) were used. Three different biodegradable materials; polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and polyglycidyl methacrylate (PGMA), were chosen as model polymers. We demonstrate that this process is capable of fabricating 200-500 nm thin, through-hole polymer membranes with various geometries, pore-sizes and spatial features approaching 2.5 µm using a mold fabricated via a single contact photolithography exposure. In addition, the membranes can be mounted to support rings made from either SU8 or PCL for easy handling after release. Cell culture compatibility of the fabricated membranes was evaluated with human dermal microvascular endothelial cells (HDMECs) seeded onto the ultrathin porous membranes, where the cells grew and formed confluent layers with well-established cell-cell contacts. Furthermore, human trabecular meshwork cells (HTMCs) cultured on these scaffolds showed similar proliferation as on flat PCL substrates, further validating its compatibility. All together, these results demonstrated the feasibility of our sacrificial fabrication process to produce biocompatible, ultra-thin membranes with defined microstructures (i.e., pores) with the potential to be used as substrates for tissue engineering applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1192-1201, 2016.

A biomimetic Schlemm's canal inner wall: A model to study outflow physiology, glaucoma pathology and high-throughput drug screening.

Glaucoma is a disease that damages the optic nerve, frequently leading to blindness. Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, which is expected to affect 80 million people by 2020, causing bilateral blindness in over 10 million individuals. Because pathological changes to Schlemm's canal (SC) may account for significant resistance to outflow, there is considerable interest in characterizing and evaluating the Schlemm's canal as a target for glaucoma therapeutics. In conventional, two-dimensional culture, human Schlemm's canal (HSC) cells lose spatial, mechanical and biochemical cues, resulting in altered gene expression and cell signaling than observed in vivo, compromising the clinical relevance of data obtained from such systems. Here, we report, for the first time, that 3D culture of HSC cells on microfabricated scaffolds with defined physical and biochemical cues, rescued expression of key HSC markers, VE-cadherin and PECAM1, and mediated pore formation, crucial for the Schlemm's canal regulation of IOP. We demonstrated that following treatment with the glaucopathogenic agent, TGF-ß2, HSC cells undergo an endothelial-mesenchymal transition, which together with the increase in extracellular matrix (ECM) proteins might account for the decrease in outflow facility observed in patients with high TGF-ß2 levels in their aqueous humor. We also demonstrated that unlike 2D cultures, 3D cultures of HSC cells are amenable to gene transfer. Thus, our data imply that 3D culture of HSC cells may be used as a platform to advance our understanding of HSC physiology and pathology and as a model for high-throughput drug and gene screening.

Chemically Modified Dendritic Starch: A Novel Nanomaterial for siRNA Delivery.

Nanostructured starches are naturally derived nanomaterials that can be chemically modified to allow for the introduction of functional groups, enhancing their potential for drug delivery and other biotechnology applications. In this proof of concept study, we investigate chemically modified, enzymatically synthesized glycogen (ESG) nanodendrites as a biodegradable, biocompatible, siRNA delivery system. Commercially available ESG was modified using glycidyltrimethylammonium chloride (GTMA), introducing quaternary ammonium groups via an epoxide ring opening reaction. This cationic ESG (cESG) electrostatically bound siRNA and successfully knocked down protein expression in an in vitro ovarian clear cell carcinoma model. The construct exhibited sustained siRNA delivery for up to 6 days while exhibiting less toxicity than a common liposome-based siRNA delivery reagent, Lipofectamine RNAiMAX. These promising results set the stage for the use of dendritic starch as a cost-effective, easily modifiable nanoscale delivery system for a diverse range of cargo including nucleic acids and therapeutic compounds.

Development of nanoscale approaches for ovarian cancer therapeutics and diagnostics.

Ovarian cancer is the deadliest of all gynecological cancers and the fifth leading cause of death due to cancer in women. This is largely due to late-stage diagnosis, poor prognosis related to advanced-stage disease, and the high recurrence rate associated with development of chemoresistance. Survival statistics have not improved significantly over the last three decades, highlighting the fact that improved therapeutic strategies and early detection require substantial improvements. Here, we review and highlight nanotechnology-based approaches that seek to address this need. The success of Doxil, a PEGylated liposomal nanoencapsulation of doxorubicin, which was approved by the FDA for use on recurrent ovarian cancer, has paved the way for the current wave of nanoparticle formulations in drug discovery and clinical trials. We discuss and summarize new nanoformulations that are currently moving into clinical trials and highlight novel nanotherapeutic strategies that have shown promising results in preclinical in vivo studies. Further, the potential for nanomaterials in diagnostic imaging techniques and the ability to leverage nanotechnology for early detection of ovarian cancer are also discussed.

Manganese enhanced magnetic resonance imaging (MEMRI): a powerful new imaging method to study tinnitus.

Manganese enhanced magnetic resonance imaging (MEMRI) is a method used primarily in basic science experiments to advance the understanding of information processing in central nervous system pathways. With this mechanistic approach, manganese (Mn(2+)) acts as a calcium surrogate, whereby voltage-gated calcium channels allow for activity driven entry of Mn(2+) into neurons. The detection and quantification of neuronal activity via Mn(2+) accumulation is facilitated by "hemodynamic-independent contrast" using high resolution MRI scans. This review emphasizes initial efforts to-date in the development and application of MEMRI for evaluating tinnitus (the perception of sound in the absence of overt acoustic stimulation). Perspectives from leaders in the field highlight MEMRI related studies by comparing and contrasting this technique when tinnitus is induced by high-level noise exposure and salicylate administration. Together, these studies underscore the considerable potential of MEMRI for advancing the field of auditory neuroscience in general and tinnitus research in particular. Because of the technical and functional gaps that are filled by this method and the prospect that human studies are on the near horizon, MEMRI should be of considerable interest to the auditory research community. This article is part of a Special Issue entitled .

Recreating a human trabecular meshwork outflow system on microfabricated porous structures.

Glaucoma is the leading cause of irreversible blindness, resulting from an increase in intraocular pressure (IOP). IOP is the only modifiable risk factor of glaucoma and is controlled by the outflow of the aqueous humor through the human trabecular meshwork (HTM). Currently, the lack of a proper in vitro HTM model impedes advances in understanding outflow physiology and discovering effective IOP-lowering anti-glaucoma therapeutics. Therefore, we designed and constructed an in vitro HTM model using micropatterned, porous SU-8 scaffolds, which support cells to recapitulate functional HTM morphology and allow the study of outflow physiology. The pore size of SU-8 scaffolds, surface coating, cell seeding density, and culture duration were evaluated for HTM cell growth. The bioengineered HTM was characterized by F-actin staining and immunocytochemistry of HTM markers. A stand-alone perfusion chamber with an integrated pressure sensing system was further constructed and used for the investigation of the outflow facility of the bioengineered HTM treated with latrunculin B-an IOP lowering agent. Cells in the in vitro model exhibited HTM-like morphology, expression of α-smooth muscle actin, myocilin, and αß-crystallin, outflow characteristics and drug responsiveness. Altogether, we have developed an in vitro HTM model system for understanding HTM cell biology and screening of pharmacological or biological agents that affect trabecular outflow facility, expediting discovery of IOP-lowering, anti-glaucoma therapeutics.

Biomimicry enhances sequential reactions of tethered glycolytic enzymes, TPI and GAPDHS.

Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.