Effect of polymer deposition method on thermoresponsive polymer films and resulting cellular behavior

Poly(N-isopropyl acrylamide) or pNIPAM is a thermoresponsive polymer that is widely studied for use in bioengineering applications. The interest in this polymer lies in the polymer's unique capability to undergo a sharp property change near physiological temperature, which aids in the spontaneous release of biological cells from substrates. Currently, there are many methods for depositing pNIPAM onto substrates, including atom-transfer radical polymerization (ATRP) and electron beam ionization. Each method yields pNIPAM-coated substrates with different surface characteristics that can influence cell behavior. In this work, we compare two methods of pNIPAM deposition: plasma deposition and codeposition with a sol-gel. The resulting pNIPAM films were analyzed for use as substrates for mammalian cell culture based on surface characterization (XPS, ToF-SIMS, AFM, contact angles), cell attachment/detachment studies, and an analysis of exocytosis function using carbon-fiber microelectrode amperometry (CFMA). We find that although both methods are useful for the deposition of functional pNIPAM films, plasma deposition is much preferred for cell-sheet engineering applications because of the films' thermoresponse, minimal change in cell density, and maintenance of supported cell exocytosis function.     

Recent advances in cell micropatterning techniques for bioanalytical and biomedical sciences

Cell micropatterning is an important technique for a wide range of applications, such as tissue engineering, cell-based drug screening, and fundamental cell biology studies. This paper overviews cell patterning techniques based on chemically modified substrates with different degrees of cell adhesiveness. In particular, the focus is on dynamic substrates that change their cell adhesiveness in response to external stimuli, such as heat, voltage, and light. Such substrates allow researchers to achieve an in situ alteration of patterns of cell adhesiveness, which is useful for co-culturing multiple cell types and analyzing dynamic cellular activities. As an example of dynamic substrates, we introduce a dynamic substrate based on a caged compound, where we accomplished a light-driven alteration of cell adhesiveness and the analysis of a single cell's motility.     

Sortase-Mediated Transpeptidation for Site-Specific Modification of Peptides,Glycopeptides, and Proteins

Sortases are a family of transpeptidases found in gram-positive bacteria responsible for covalent anchoring of cell surface proteins to bacterial cell walls. It has been discovered that sortase A (SrtA) of Staphylococcus aureus origin is rather promiscuous and can accept various molecules as substrates. As a result, SrtA has been widely used to ligate peptides and proteins with a variety of nucleophiles, and the ligation products are useful for research in chemical biology, proteomics, biomedicine, etc. This review summarizes the recent applications of SrtA with special emphasis on SrtA-catalyzed ligation of carbohydrates with peptides and proteins.     

Engineering Substrate Topography at the Micro‐ and Nanoscale to Control Cell Function

The interaction of mammalian cells with nanoscale topography has proven to be an important signaling modality in controlling cell function. Naturally occurring nanotopographic structures within the extracellular matrix present surrounding cells with mechanotransductive cues that influence local migration, cell polarization, and other functions. Synthetically nanofabricated topography can also influence cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organization. We review the use of in vitro synthetic cell–nanotopography interactions to control cell behavior and influence complex cellular processes, including stem‐cell differentiation and tissue organization. Future challenges and opportunities in cell–nanotopography engineering are also discussed, including the elucidation of mechanisms and applications in tissue engineering.     

Photocontrol of cell adhesion on amino-bearing surfaces by reversible conjugation of poly(ethylene glycol) via a photocleavable linker

Dynamic control of cell adhesion on substrates is a useful technology in tissue engineering and basic biology. This paper describes a method for the control of cell adhesion on amino-bearing surfaces by reversible conjugation of an anti-fouling polymer, poly(ethylene glycol) (PEG), via a newly developed photocleavable linker, 1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethyl N-succinimidyl carbonate (1). This molecule has alkyne and succinimidyl carbonate at each end, which are connected by photocleavable 2-nitrobenzyl ester. Under this molecular design, the molecule crosslinked azides and amines, whose linkage cleaved upon application of near-UV light. By using aminosilanised glass and silicon as model substrates, we studied their reversible surface modification with PEG-azide (M(w) = 5000) based on contact angle measurements, ellipsometry, and AFM morphological observations. Protein adsorption and cell adhesion dramatically changed by PEGylation and the following irradiation, which can be used for cellular patterning. Also, the capability of the substrate to change cell adhesiveness by photoirradiation during cell cultivation was demonstrated by inducing cell migration. We believe this method will be useful for dynamic patterning of cells on protein-based scaffolds.     

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity. In this regard, heterogenous sialylation may produce variability in efficacy and limit therapeutic applications. Homogenous sialylation may be achieved through cellular and molecular engineering, both of which have gained traction in recent years. In this paper, we describe the engineering of intracellular glycosylation pathways through targeted disruption and the introduction of carbohydrate active enzyme genes. The focus of this review is on sialic acid-related genes and efforts to achieve homogenous, humanlike sialylation in model hosts. We also discuss the molecular engineering of sialyltransferases and their application in chemoenzymatic sialylation and sialic acid visualization on cell surfaces. The integration of these complementary engineering strategies will be useful for glycoscience to explore the biological significance of sialic acids on cell surfaces as well as the future development of advanced biopharmaceuticals.     

Recent development of temperature‐responsive cell culture surface using poly(N‐isopropylacrylamide)

Poly(N‐isopropylacrylamide) (PIPAAm), which is a well‐known temperature‐responsive polymer, is modified on substrates by various methods. At 37 °C, PIPAAm modified surface is hydrophobic and allows cells to adhere to and proliferate on the surface. By reducing temperature below the lower critical solution temperature of PIPAAm, the surface turns to hydrophilic and allows cells to detach themselves from the surface spontaneously. With this technology, cell sheet engineering is established several years ago. This review focuses on the preparations and characteristics of PIPAAm‐modified surfaces, and discusses the effect of surface properties on cell adhesion and deadhesion. In addition, the recent improvement of PIPAAm‐modified surfaces for cell culture and the clinical applications of cell sheets harvested from the surfaces are also mentioned. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 917–926     

Oxidized Xanthan Gum Crosslinked NOCC: Hydrogel System and Their Biological Stability from Oxidation Levels of the Polymer

Dynamic hydrogel systems from N,O-carboxymethyl chitosan (NOCC) are investigated in the past years, which has facilitated their widespread use in many biomedical engineering applications. However, the influence of the polymer's oxidation levels on the hydrogel biological properties is not fully investigated. In this study, chitosan is converted into NOCC and introduced to react spontaneously with oxidized xanthan gum (OXG) to form several injectable hydrogels with controlled degradability. Different oxidation levels of xanthan gum, as well as NOCC/OXG volume ratios, are trialed. The infrared spectroscopy spectra verify chemical modification on OXG and successful crosslinking. With increasing oxidation levels, more dialdehyde groups are introduced into the OXG, resulting in changes in physical properties including gelation, swelling, and self-healing efficiency. Under different volume ratios, the hydrogel shows a stable structure and rigidity with higher mechanical properties, and a slower degradation rate. The shear-thinning and self-healing properties of the hydrogels are confirmed. In vitro assays with L929 cells show the biocompatibility of all formulations although the use of a high amount of OXG15 and OXG25 limited the cell proliferation capacity. Findings in this study suggested a suitable amount of OXG at different oxidation levels in NOCC hydrogel systems for tissue engineering applications.     

Synthetic chemoselective rewiring of cell surfaces: generation of three-dimensional tissue structures

Proper cell-cell communication through physical contact is crucial for a range of fundamental biological processes including, cell proliferation, migration, differentiation, and apoptosis and for the correct function of organs and other multicellular tissues. The spatial and temporal arrangements of these cellular interactions in vivo are dynamic and lead to higher-order function that is extremely difficult to recapitulate in vitro. The development of three-dimensional (3D), in vitro model systems to investigate these complex, in vivo interconnectivities would generate novel methods to study the biochemical signaling of these processes, as well as provide platforms for tissue engineering technologies. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. This strategy uses liposome fusion for the delivery of ketone or oxyamine groups to different populations of cells for subsequent cell assembly via oxime ligation. We demonstrate how this method can be used for several applications including, the delivery of reagents to cells for fluorescent labeling and cell-surface engineering, the formation of small, 3D spheroid cell assemblies, and the generation of large and dense, 3D multilayered tissue-like structures for tissue engineering applications.     

Creating Space for Large Acceptors: Rational Biocatalyst Design for Resveratrol Glycosylation in an Aqueous System

Polyphenols display a number of interesting properties but their low solubility limits practical applications. In that respect, glycosylation offers a solution for which sucrose phosphorylase has been proposed as a cost‐effective biocatalyst. However, its activity on alternative acceptor substrates is too low for synthetic purposes and typically requires the addition of organic (co‐)solvents. Here, we describe the engineering of the enzyme from Thermoanaerobacterium thermosaccharolyticum to enable glycosylation of resveratrol as test case. Based on docking and modeling studies, an active‐site loop was predicted to hinder binding. Indeed, the unbolted loop variant R134A showed useful affinity for resveratrol (K_m=185 mM ) and could be used for the quantitative production of resveratrol 3‐α‐glucoside in an aqueous system. Improved activity was also shown for other acceptors, introducing variant R134A as promising new biocatalyst for glycosylation reactions on bulky phenolic acceptors.     

Perspectives for Biotechnological Production and Utilization of Biopolymers: Metabolic Engineering of Polyhydroxyalkanoate Biosynthesis Pathways as a Successful Example

This article provides an overview of biopolymers, classed according to their chemical structures, function and occurrence, the principles of biosynthesis and metabolism in organisms. It will then focus on polyhydroxyalkanoates (PHA) for which technical applications in several areas are currently considered. PHAs represent a complex class of bacterial polyesters consisting of various hydroxyalkanoic acids that are synthesized by bacteria as storage compounds for energy and carbon if a carbon source is present in excess. Poly(3‐hydroxybutyrate), poly(3HB), is just one example. Most other PHAs are only synthesized if pathways exist which mediate between central intermediates of the metabolism or special precursor substrates on one side and coenzyme A thioesters of hydroxyalkanoic acids, which are the substrates of the PHA synthase catalyzing the polymerization, on the other side. During the last decade, basic and applied research have revealed much knowledge about the biochemical and molecular basis of the enzymatic processes for the synthesis of PHAs in microorganisms. The combination of detailed physiological studies, utilization of the overwhelming information provided by the numerous genome sequencing projects, application of recombinant DNA technology, engineering of metabolic pathways or enzymes and molecular breeding techniques applied to plants have provided new perspectives to produce these technically interesting biopolymers by novel or significantly improved biotechnological processes or by agriculture. Some examples for successful in vivo and in vitro engineering of pathways suitable for the synthesis and biotechnological production of PHAs consisting of medium‐chain‐length 3‐hydroxyalkanoic acids and short‐chain‐length hydroxyalkanoic acids will be provided.     

Suppressor capacity of iron nanoparticles biosynthesized using Salvia chloroleuca leaf aqueous extract on methadone-induced cell death in PC12: Formulation a new drug from relationship between the nanobiotechnology and neurology sciences

Metallic nanoparticles have gained significant attention in the area of biomedical technology. Because of its high surface area, metallic nanoparticles are being widely used in various fields including the medical and engineering sciences. One of the valuable applications of metallic nanoparticles especially copper, zinc, and iron nanoparticles is increasing the physiological function of central nervous system. Besides, Iranian people are using the Salvia chloroleuca for neuroprotective properties. In the present research, iron nanoparticles were biosynthesized by S. chloroleuca leaf aqueous extract as reducing and stabilizing agents. Also, we revealed the protective effect of FeNPs in methadone-treated PC12 cells. FeNPs were characterized and analyzed using common nanotechnology techniques including FT-IR, UV–Vis. spectroscopy; EDS, TEM, and FE-SEM. TEM and FE-SEM images revealed a uniform spherical morphology for FeNPs. In the biological part of the current study, the both treatments of FeNPs significantly (p ≤ 0.01) reduced the cell cytotoxicity and cell death index as well as increased the cell viability and cell proliferation in methadone-treated PC12 cells. In these treatments, mitochondrial membrane potential significantly (p ≤ 0.01) increased compared to methadone-induced PC12 cells. DPPH free radical scavenging test was did to evaluate the antioxidant potentials of FeCl₃, S. chloroleuca, and FeNPs. DPPH test indicated similar antioxidant activities for S. chloroleuca, FeNPs, and butylated hydroxytoluene. In current experiment, we concluded that iron nanoparticles biosynthesized by S. chloroleuca leaf aqueous extract suppressed methadone-induced cell death in a dose-dependent manner in PC12 cells.     

Multi‐Colored Fibers by Self‐Assembly of DNA,Histone Proteins,and Cationic Conjugated Polymers

The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering.     

Multi‐Colored Fibers by Self‐Assembly of DNA,Histone Proteins,and Cationic Conjugated Polymers

The development of biomolecular fiber materials with imaging ability has become more and more useful for biological applications. In this work, cationic conjugated polymers (CCPs) were used to construct inherent fluorescent microfibers with natural biological macromolecules (DNA and histone proteins) through the interfacial polyelectrolyte complexation (IPC) procedure. Isothermal titration microcalorimetry results show that the driving forces for fiber formation are electrostatic and hydrophobic interactions, as well as the release of counterions and bound water molecules. Color‐encoded IPC fibers were also obtained based on the co‐assembly of DNA, histone proteins, and blue‐, green‐, or red‐ (RGB‐) emissive CCPs by tuning the fluorescence resonance energy‐transfer among the CCPs at a single excitation wavelength. The fibers could encapsulate GFP‐coded Escherichia coli BL21, and the expression of GFP proteins was successfully regulated by the external environment of the fibers. These multi‐colored fibers show a great potential in biomedical applications, such as biosensor, delivery, and release of biological molecules and tissue engineering.     

Polymethacrylate Networks as Substrates for Cell Culture

Summary: methacrylate networks have a long history of applications in medical technology and much is known of their non-fouling properties. However, in recent times it has become clear that the swollen nature of these materials may provide some advantages if they are used as scaffolds in tissue engineering. In general however these hydrogels are resistant to protein adsorption and human cells do not easily adhere. In this work we provide an overview of several strategies that are designed to improve the cell-adhesive properties of hydrogels while maintaining their useful properties, mainly ease of diffusion of nutrients and growth factors. We describe our early attempts at modifying hydrogels based on 2,3-propandiol -1-methacrylate, with either hydrophobic units or acid groups. Modification with lauryl methacrylate produced an improvement but acid modification failed to provide surfaces that were conducive to cell culture. Much better scaffolds were prepared by amination of epoxy functional 2,3-propandiol-1-methacrylate networks. Optimized materials in this class were shown to be good substrates for the co-culture of bovine keratocytes with human corneal epithelial cells. We also describe the synthesis and biological properties of methacrylate conetworks, which phase separate during synthesis to give porous amphiphilic materials. Optimization of these materials produces materials that perform as well as tissue culture plastic so that confluent sheets of human dermal fibroblasts can be produced using standard culture techniques.     

Zein‐Based Electrospun Fibers Containing Bioactive Glass with Antibacterial Capabilities

Zein, a natural protein from corn, has important applications in food and pharmaceutical industries due the fact that it is biodegradable and biocompatible. However, due its relatively low mechanical properties and water solubility, many inorganic compounds (e.g., bioactive glasses [BGs]) have been used in combination with zein to obtain composite materials with improved mechanical properties. Such inorganic additions provide further biological functionality to zein. In this work, fiber mats of zein with incorporation of BG and copper doped BG particles are successfully obtained by electrospinning. At first the electrospinnability of the blends is assessed, then the morphological and chemical characterization of the mats is done. Degradation study in cell culture medium (Dubelcco’s modified Eagle’s medium) reveals a sufficient strength of the fibers, which in turn is necessary for in vitro cellular studies. Cell culture studies using MG‐63 and C2C12 cells show promising results, demonstrating increased cell proliferation and growth for fiber mats containing both types of BGs. Also, evaluation with Staphylococcus aureus and Escherichia coli bacteria confirms the antibacterial activity of the scaffolds containing copper. The presence of Cu thus imparts antibacterial properties without influencing cell behavior. The developed electrospun fibers represent a novel scaffold system for tissue engineering applications.     

A Review of the Recent Developments in the Bioproduction of Polylactic Acid and Its Precursors Optically Pure Lactic Acids

Lactic acid (LA) is an important organic acid with broad industrial applications. Considered as an environmentally friendly alternative to petroleum-based plastic with a wide range of applications, polylactic acid has generated a great deal of interest and therefore the demand for optically pure l- or d-lactic acid has increased accordingly. Microbial fermentation is the industrial route for LA production. LA bacteria and certain genetic engineering bacteria are widely used for LA production. Although some fungi, such as Saccharomyces cerevisiae, are not natural LA producers, they have recently received increased attention for LA production because of their acid tolerance. The main challenge for LA bioproduction is the high cost of substrates. The development of LA production from cost-effective biomasses is a potential solution to reduce the cost of LA production. This review examined and discussed recent progress in optically pure l-lactic acid and optically pure d-lactic acid fermentation. The utilization of inexpensive substrates is also focused on. Additionally, for PLA production, a complete biological process by one-step fermentation from renewable resources is also currently being developed by metabolically engineered bacteria. We also summarize the strategies and procedures for metabolically engineering microorganisms producing PLA. In addition, there exists some challenges to efficiently produce PLA, therefore strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are also discussed.     

Recent Advances in DNA-Based Cell Surface Engineering for Biological Applications

Due to its excellent programmability and biocompatibility, DNA molecule has unique advantages in cell surface engineering. Recent progresses provide a reliable and feasible way to engineer cell surfaces with diverse DNA molecules and DNA nanostructures. The abundant form of DNA nanostructures has greatly expanded the toolbox of DNA-based cell surface engineering and gave rise to a variety of novel and fascinating applications. In this review, we summarize recent advances in DNA-based cell surface engineering and its biological applications. We first introduce some widely used methods of immobilizing DNA molecules on cell surfaces and their application features. Then we discuss the approaches of employing DNA nanostructures and dynamic DNA nanotechnology as elements for creating functional cell surfaces. Finally, we review the extensive biological applications of DNA-based cell surface engineering and discuss the challenges and prospects of DNA-based cell surface engineering.     

Near‐Infrared Luminescence Sensing of Glutamic Acid,Aspartic Acid,and Their Dipeptides with Tris(β‐diketonato)lanthanide Probes

A series of tris(β‐diketonato)lanthanides with Yb³⁺, Eu³⁺, and Nd³⁺ centers were characterized as luminescent sensing probes specific to glutamic acid, aspartic acid, and their dipeptides, which are important substrates involved in nervous systems, taste receptors, and other biological systems. In particular, tris(6,6,7,7,8,8,8‐heptafluoro‐2,2‐dimethyloctane‐3,5‐dionato)ytterbium(III) exhibited a near‐infrared emission around 980 nm in response to these biological substrates. Near‐infrared‐emissive complexes have several advantages over common luminescent probes; therefore, the proposed lanthanide complexes have potential analytical applications in proteomics, metabolics, food science, astrobiology, and related technologies.