Photocrosslinkable Poly Research Articles

Highly expansive DNA hydrogel films prepared with photocrosslinkable poly(vinyl alcohol)

Deoxyribonucleic acid (DNA) has a lot of characteristic features, e.g., anionic polyelectrolyte, molecular recognition and condensed π electron. Therefore, it is expected to be applied as functional materials in not only biological but also physical fields. In this report, we attempted to prepare water-insoluble DNA films by mixing DNA and photolinkable polymer, aiming at the fabrication of bio-functionalized hydrogel films. For this purpose, photocrosslinkable polyvinyl alcohol (azide-unit pendant water-soluble photopolymer; AWP) was used. An aqueous DNA and AWP mixture was cast onto glass substrates. Irradiation of UV light on it for certain minutes provided a hydrogel film by photocrosslinking between DNA and AWP. When the formed hydrogel film was soaked in pure water for 1 day, large expansion of the film was observed. The expanded film was shrunk by soaking it in aqueous NaCl or CTAB solutions. The expansion and shrinkage mechanism of the film was attributed to the penetration of water into the gap between the film and the substrate due to osmotic pressure. This phenomenon can be applied to the fabrication of gel devices, e.g., a novel biosensor and drug delivery system.

A digital micro‐mirror device‐based system for the microfabrication of complex, spatially patterned tissue engineering scaffolds

Our ability to create precise, pre-designed, spatially patterned biochemical and physical microenvironments inside polymer scaffolds could provide a powerful tool in studying progenitor cell behavior and differentiation under biomimetic, three-dimensional (3D) culture conditions. We have developed a simple and fast, layer-by-layer microstereolithography system consisting of an ultra-violet light source, a digital micro-mirror masking device, and a conventional computer projector, that allows fabrication of complex internal features along with precise spatial distribution of biological factors inside a single scaffold. Photo-crosslinkable poly(ethylene glycol) diacrylates were used as the scaffold material, and murine bone marrow-derived cells were successfully encapsulated or seeded on fibronectin-functionalized scaffolds. Fluorescently-labeled polystyrene microparticles were used to show the capability of this system to create scaffolds with complex internal architectures and spatial patterns. We demonstrate that precisely controlled pore size and shapes can be easily fabricated using a simple, computer-aided process. Our results further indicate that multi-layered scaffolds with spatially distributed factors in the same layer or across different layers can be efficiently manufactured using this technique. These microfabricated scaffolds are conducive for osteogenic differentiation of marrow-derived stem cells, as indicated by efficient matrix mineralization.

Laser‐layered microfabrication of spatially patterned functionalized tissue‐engineering scaffolds

Understanding cell behavior inside complex, three-dimensional (3D) microenvironments with controlled spatiotemporal patterning of physical and biochemical factors would provide significant insights into the basic biology of organ development and tissue functions. One of the fundamental limitations in studying such behavior has been the inability to create patterned microenvironments within 3D scaffold structures. Here a simple, layer-by-layer stereolithography (SL) method that can precisely pattern ligands, extracellular-matrix (ECM) components, and growth factors, as well as controlled release particles inside a single scaffold, has been developed. The process also allows fabrication of predesigned internal architectures and porosities. Photocrosslinkable poly(ethylene glycol) dimethacrylate (PEGDMA) was used as the basic structural component of these microfabricated scaffolds. PEG acrylates, covalently modified with the cell adhesive peptide arginine-glycine-aspartic acid (RGD) or the ECM component heparan sulfate, was incorporated within the scaffolds to facilitate cell attachment and to allow spatial sequestration of heparan-binding growth factors. Fluorescently labeled polymer microparticles and basic fibroblast growth factor (FGF-2) were chosen to illustrate the capability of SL to spatiotemporally pattern scaffolds. The results demonstrate that a precise, predesigned distribution of single or multiple factors within a single 3D structure can be created, and specific internal architectures can be fabricated. Functionalization of these scaffolds with RGD is demonstrated, and heparan sulfate allows efficient cell attachment and spatial localization of growth factors. Such patterned scaffolds might provide effective systems to study cell behavior in complex microenvironments and could eventually lead to engineering of complex, hybrid tissue structures through predesigned, multilineage differentiation of a single stem-cell population.

Fast, multi-responsive microgels based on photo-crosslinkable poly(2-(dimethylamino)ethyl methacrylate)

Multi-responsive microgels based on poly(2-( N, N-dimethylamino)ethyl methacrylate) were developed and their properties were investigated. The primary goal of this research was to speed up the stimulus-response time of the hydrogels to a level usable for actuator applications, by reducing the diffusion distance of water. The gels were prepared by a UV induced photodimerization of a copolymer of 2-(dimethylamino)ethyl methacrylate and 4-cinnamoyl-phenyl methacrylate. Patterning studies showed that these materials can be used as photo-resist materials with high resolution at short exposure times. They showed lower critical solution temperature behavior in water, as well as pH dependent solubility and swelling ratios. While 1 mm thick gels showed response times to temperature and pH-changes of several hours, Si-supported microgels of 300 nm thickness had response times in the range of only a few seconds. The copolymer was prepared by free radical copolymerization, and the reactivity ratios were determined with the extended Kelen Tudos method. Spin-coating of this copolymer on Si supports and subsequent UV-irradiation yielded microgels of variable thickness (200 nm–15 μm), which was determined by confocal scanning laser microscopy. Surface plasmon resonance spectroscopy measurements demonstrated the fast, stimuli-responsive swelling behavior, while differential scanning calorimetry gave insight into the morphology of the networks.

Towards the protein phosphatase-based biosensor for microcystin detection

A colorimetric test for the detection of microcystins based on immobilised protein phosphatase (PP) has been developed. A PP2A produced by molecular engineering has been used and its performance has been compared to those of commercial PP2A and PP1. Covalent immobilisation of the enzyme using glutaraldehyde, encapsulation by sol–gel and entrapment with photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) have been compared, the latter method providing the highest immobilisation yields. Screen-printed carbon electrodes (SPEs), Maxisorp microtiter wells and Ultrabind modified polyethersulfone affinity membranes have been used as immobilisation supports. Whilst the highest immobilisation yields were obtained with microtiter wells, the highest operational and storage stabilities were achieved with carbon SPEs and membranes, respectively. The immobilisation of PP by PVA-SbQ provided a means to preserve the enzymatic activity, which decreased at fast rates when the enzyme was kept in solution. The colorimetric test using p-nitrophenyl phosphate has demonstrated that the immobilised enzyme is able to recognise both microcystin variants (MC-LR and MC-RR), although optimisation work should be performed to achieve appropriate limits of detection. With the purpose to develop an electrochemical biosensor, several phosphorylated substrates have been used. Promising results have been achieved with the commercial enzymes and α-naphtyl phosphate, p-aminophenol phosphate and catechol monophosphate as enzyme substrates, guaranteeing the viability of the electrochemical approach.

Stabilization of photosynthetic materials.

Isolated photosynthetic materials have a relatively short active lifetime that limits their effective use. To circumvent this limitation, various immobilization techniques have been designed to improve their stability both under storage and working conditions. The immobilization methods are identified either as chemical or physical methods. In this chapter, two immobilization methods frequently used are described; a physical method based on the entrapment of photosynthetic materials in photo-crosslinkable poly(vinylalcohol) polymer bearing styrylpyridinium groups (PVA-SbQ) and a chemical method where the photosynthetic materials are immobilized by co-reticulation in an albumin-glutaraldehyde crosslinked matrix (BSA-Glu). Different immobilization procedures in relation with various photosynthetic materials are mentioned.

A photo-crosslinked poly(vinyl alcohol) hydrogel growth factor release vehicle for wound healing applications.

The objective of this study was to develop and evaluate a hydrogel vehicle for sustained release of growth factors for wound healing applications. Hydrogels were fabricated using ultraviolet photo-crosslinking of acrylamide-functionalized nondegradable poly(vinyl alcohol) (PVA). Protein permeability was initially assessed using trypsin inhibitor (TI), a 21 000 MW model protein drug. TI permeability was altered by changing the solids content of the gel and by adding hydrophilic PVA fillers. As the PVA content increased from 10% to 20%, protein flux decreased, with no TI permeating through 20% PVA hydrogels. Further increase in model drug release was achieved by incorporating hydrophilic PVA fillers into the hydrogel. As filler molecular weight increased, TI flux increased. The mechanism for this is most likely an alteration in protein/gel interactions and transient variations in water content. The percent protein released was also altered by varying protein loading concentration. Release studies conducted using growth factor in vehicles with hydrophilic filler showed sustained release of platelet-derived growth factor (PDGF-beta,beta) for up to 3 days compared with less than 24 hours in the controls. In vitro bioactivity was demonstrated by doubling of normal human dermal fibroblast numbers when exposed to growth factor-loaded vehicle compared to control. The release vehicle developed in this study uses a rapid and simple fabrication method, and protein release can be tailored by modifying solid content, incorporating biocompatible hydrophilic fillers, and varying protein loading concentration.

Photoresponsive peptide and polypeptide systems 15*: Synthesis of photo-crosslinkable poly(amino acid)s by watery process and its application as a reinforcement for polyion complex fibers

The present article described the synthesis of the coumarin-containing polypeptides using the water-soluble active ester method and the reinforcement of the polyion complex (PIC) fibers by the photochemical cross-linking between the polypeptide-pendant coumaryloxyacetyl groups. Two kinds of cationic polypeptides, poly(α,L-lysine) (PLL) and poly(α,L-ornithine) (PLO) were treated with (4-hydroxyphenyl)dimethyl sulfonium methyl sulfate ester in water to incorporate the photoreactive coumaryloxyacetyl groups into the side chain amino groups of the PLL and PLO. The contents of the photoreactive groups in the produced coumarin-containing polypepides were controlled by the molar ratios of the amino groups to the active ester. The resulting coumarin-containing cationic polypeptides were spun into the PIC fibers with the anionic polysaccharide gellan in water. The tensile strengths of the coumarin-containing PIC fibers were superior to those of the intact PLL- and PLO-gellan PIC fibers. The mechanical strength of the coumarin-containing PIC fibers further increased upon light-irradiation. From these results, we concluded that the hydrophobic interactions and the covalent cross-linking due to the photochemical dimerization between the coumaryloxyacetyl groups significantly contributed to reinforcement of the PIC fibers. The present results afford a new methodology to the PIC reinforcement that was achieved by the polymer modification reaction in watery process.

FT-IR Imaging of Patterned Photocrosslinkable Poly(Vinyl Cinnamate)

The use of photocrosslinkable polymers and photomasks was proposed as a means of reducing the number of raw materials necessary to create a chemical sensor array. To test this hypothesis, poly(vinyl cinnamate) was crosslinked to various degrees with the use of a photomask, exposed to a binary solvent vapor of benzonitrile and dimethyl formamide, and examined by FT-IR imaging. The results illustrate that FT-IR imaging is effective for visualizing the patterning of the crosslink density of a polymer film as well as the temporal and spatial sorption patterns of different solvent vapors. The FT-IR images show that the decrease in sorption capacity with crosslink density is solvent-specific, which suggests that photocrosslinkable polymers can be used to reduce the number of raw materials needed to create a chemical sensor array. The potential disadvantage of using glassy polymers as chemical sensors is also demonstrated by the temporal FT-IR images of solvent desorption.

Networks of photocrosslinked poly(meth)acrylates in linear poly(vinyl chloride)

AbstractFive different multifunctional acrylic monomers (trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol tetraacrylate, and dipentaerythritol pentaacrylate) were photopolymerized alone or in a matrix of linear poly(vinyl chloride) (PVC) with 2,2‐dimethyl‐2‐hydroxyacetophenone as a photoinitiator. The course of photopolymerization was estimated with Fourier transform infrared spectroscopy. The amount of insoluble gel formed during photopolymerization was determined gravimetrically. The crosslinked polymerization of pure monomers was much faster than that in the presence of PVC. However, the efficiency of the reaction was higher when it was carried out in a PVC blend because of the higher mobility of the propagating macroradicals. The influence of the monomer structure and functionality on the polymerization course was examined. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 3725–3734, 2002

Synthesis and characterization of photocrosslinkable, degradable poly(vinyl alcohol)-based tissue engineering scaffolds

Hydrogels have many advantages that make them prime candidates for tissue engineering applications: high water content, tissue-like elasticity, and relative biocompatibility. We aim to tissue engineer heart valves using a hydrogel scaffold based on poly(vinyl alcohol) (PVA), and the design parameters for a suitable tissue engineering scaffold are quite stringent. In this research, we develop degradable and photocrosslinkable poly(lactic acid)-g-PVA multifunctional macromers that can be reacted in solution to form degradable networks. The mass loss profiles and bulk properties of the resulting scaffolds are easily tailored by modifying the structure of the starting macromers. Specifically, altering the number of lactide repeat units per crosslinking side chain, percent substitution, molecular weight of PVA backbone, and macromer solution concentration, the rate of mass loss from these degradable networks is controlled. In addition, by increasing the network's hydrophobicity, valve interstitial cell adhesion is improved.

Therapeutic Micro and Nanotechnology Section: Microwell Array Photoimprint Lithography: Micropost Sensors and Guidance of PC12 Neurite Processes

Microwell array photoimprint lithography (PIL) is demonstrated by fabricating a patterned chemical sensing layer that is suitable for use with cultured biological cells and inverted epifluorescence microscopes. A microwell array is a ∼50,000-count, coherent fiber-optic bundle whose distal face was etched chemically. Individual microwells had ∼3 μm depths and ∼8 μm widths. The microwell array PIL process patterned an array of ∼50,000 individual polymeric micropost sensors on a glass coverslip. Individual microposts were ∼3 μm tall and ∼8 μm wide. O2-sensitive micropost array sensors (MPASs) were fabricated using a ruthenium complex encapsulated in a gas permeable photopolymerizable siloxane. pH-sensitive MPASs were fabricated using a fluorescein conjugate encapsulated in a photocrosslinkable poly(vinyl alcohol)-based polymer. PO2 and pH were quantitated by acquiring MPAS luminescence images with an epifluorescence microscope/charge coupled device imaging system. The advantage of a patterned MPAS layer relative to a planar sensing layer is the ability to direct the growth of biological cells. Preliminary data are presented whereby nerve growth factor-differentiated rat pheochromocytoma (PC12) cells grew neurite-like processes that extended along paths defined by the micropost architecture.

Soft and hard tissue response to photocrosslinked poly(propylene fumarate) scaffolds in a rabbit model.

The treatment of large cranial defects may be greatly improved by the development of precisely formed bone tissue engineering scaffolds. Such scaffolds could be constructed by using UV laser stereolithography to photocrosslink a linear, biodegradable polymer into a three-dimensional implant. We have previously presented a method to photocrosslink the biodegradable polyester, poly(propylene fumarate) (PPF). To ensure the safety and effectiveness of this technique, the soft and hard tissue response to photocrosslinked PPF scaffolds of different pore morphologies was investigated. Four classes of photocrosslinked PPF scaffolds, constructed with differing porosities (57-75%) and pore sizes (300-500 or 600-800 microm), were implanted both subcutaneously and in 6.3-mm-diameter cranial defects in a rabbit model. The rabbits were sacrificed at 2 and 8 weeks, and the implants were analyzed by light microscopy, histological scoring analysis, and histomorphometric analysis. Results showed the PPF scaffolds elicit a mild tissue response in both soft and hard tissues. Inflammatory cells, vascularization, and connective tissue were observed at 2 weeks; a decrease in inflammatory cell density and a more organized connective tissue were observed at 8 weeks. Scaffold porosity and scaffold pore size were not found to significantly affect the observed tissue response. Evidence of scaffold surface degradation was noted both by histology and histomorphometric analysis. Bone ingrowth in PPF scaffolds implanted into cranial defects was <3% of the defect area. The results indicate that photocrosslinked PPF scaffolds are biocompatible in both soft and hard tissues and thus may be an attractive platform for bone tissue engineering.

Competitive sorption of water vapor and CO2 in photocrosslinked PVCA film for a capacitive‐type humidity sensor

AbstractThe sorption behavior of water vapor and CO2 gas in photocrosslinked poly(vinyl cinnamate) (PVCA) film was examined at 30°C under atmospheric pressure. Both the water sorption isotherm and the CO2 sorption isotherm obtained with quartz crystal microbalance (QCM) method obeyed the simple Langmuir's equation. Water vapor/CO2 mixed‐gas sorption isotherms were also obtained. Total amount of sorbed mixed gases was clearly influenced by the partial pressure of water vapor (pw) and CO2 gas (pc) in the atmosphere. A modified Langmuir's equation based on a dual‐site model was employed for predicting the competitive adsorption isotherm, and the isotherm was clearly described by the equation. The theoretically estimated amount of adsorbed water at the constant pw decreased slightly with increasing pc. The effect of this phenomenon on the sensitivity of the capacitive‐type relative humidity sensor was examined. As expected, the electrical capacitance of the sensor at the constant relative humidity decreased because of the coexistence of CO2 gas. However, the influence was quite small in the CO2 concentration range in the ordinary environment. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 401–407, 2002

DEVELOPMENT OF A DISPOSABLE BIOSENSOR FOR THE DETECTION OF METAM-SODIUM AND ITS METABOLITE MITC

A disposable monoenzymatic sensor for the reliable determination of dithiocarbamate fungicides was developed based on aldehyde dehydrogenase inhibition. Aldehyde dehydrogenase was immobilized on the surface of a disposable screen-printed carbon-paste electrode by using a photocross linkable poly-(vinylalcohol) bearing styrylpyridinium groups. Electrochemical oxidation of NADH was carried out at a potential as low as 0V vs Ag/AgCl using Meldola’s Blue as mediator. The sensor was used for the determination of metam-sodium, a soil fumigant, and its main metabolite MITC. It was shown that metam-sodium did not inhibit AlDH while MITC could be detected at levels of 100 ppb. The sensor thus allowed to discrimate between metam-sodium and its toxic metabolite MITC.

The effects of scaffold thickness on tissue engineered cartilage in photocrosslinked poly(ethylene oxide) hydrogels

The thickness of human articular cartilage has been reported to vary from less than 0.5 up to 7 mm. Hence, tissue engineered cartilage scaffolds should be able to span the thickness of native cartilage to fill defects of all shapes and sizes. In this study, we demonstrate the potential for using photopolymerization technology to encapsulate chondrocytes in poly(ethylene oxide) hydrogels, which vary in thickness from 2 to 8 mm. Chondrocytes, encapsulated in an 8 mm thick, photocrosslinked hydrogel and cultured in vitro for 6 weeks, remained viable and produced cartilaginous tissue throughout the construct comparable to a 2 mm thick gel as seen both histologically and biochemically. In addition, the total collagen and glycosaminoglycan contents per wet weight of the 8 mm thick cell-polymer construct were 0.13±0.01 and 0.25±0.03%, respectively, and did not vary significantly as a function of spatial position in the construct. The histological evidence and the biochemical content were similar in all constructs of varying thickness. The results suggest that photocrosslinked hydrogels are promising scaffolds for tissue engineering cartilage as cell viability is readily maintained; uniform cell seeding is easy to achieve; and the biochemical content of the extracellular matrix is not compromised as the scaffold thickness is increased from 2 to 8 mm.

Synthesis and properties of photocross-linked poly(propylene fumarate) scaffolds

The photocross-linking of poly(propylene fumarate) (PPF) to form porous scaffolds for bone tissue engineering applications was investigated. PPF was cross-linked using the photoinitiator bis(2,4,6-trimethylbenzoyl) phenylphosphine oxide (BAPO) and exposure to 30 min of long wavelength ultraviolet (UV) light. The porous photocross-linked PPF scaffolds (6.5 mm diameter cylinders) were synthesized by including a NaCl porogen (70, 80, and 90 wt% at cross-linking) prior to photocross-linking. After UV exposure, the samples were placed in water to remove the soluble porogen, revealing the porous PPF scaffold. As porogen leaching has not been used often with cross-linked polymers, and even more rarely with photoinitiated cross-linking, a study of the efficacy of this strategy and the properties of the resulting material was required. Results show that the inclusion of a porogen does not significantly alter the photoinitiation process and the resulting scaffolds are homogeneously cross-linked throughout their diameter. It was also shown that porosity can be generally controlled by porogen content and that scaffolds synthesized with at least 80 wt% porogen possess an interconnected pore structure. Compressive mechanical testing showed scaffold strength to decrease with increasing porogen content. The strongest scaffolds with interconnected pores had an elastic modulus of 2.3 ± 0.5 MPa and compressive strength at 1% yield of 0.11 ± 0.02 MPa. This work has shown that a photocross-linking/porogen leaching technique is a viable method to form porous scaffolds from photoinitiated materials.

Amperometric determination of pyruvate, phosphate and urea using enzyme electrodes based on pyruvate oxidase-containing poly(vinyl alcohol)/polyion complex-bilayer membrane

An amperometric pyruvate-sensing electrode was prepared by immobilizing pyruvate oxidase (PyOx) on a polyion complex membrane. First, aqueous solutions of poly- l-lysine and poly(4-styrenesulfonate) were successively placed on a mercaptopropionic acid-modified gold surface and allowed to dry. A photo-crosslinked poly(vinyl alcohol) layer containing PyOx was then formed on the poly- l-lysine/poly(4-styrenesulfonate)-complex layer. The polyion complex layer was effective for eliminating electrochemical interferents such as l-ascorbic acid, uric acid, l-cysteine and acetaminophen, whereas the hydrogen peroxide produced through the PyOx-catalyzed reaction permeated easily through the layer. This resulted in a high sensitivity (detection limit, 50 nM) and a low interference level (e.g. the ratio of response for l-ascorbic acid to that for the same concentration of pyruvic acid, 0.18). The electrode could be used for determining phosphoric acid (detection limit, 0.2 μM), since PyOx consumes phospholic acid as the co-substrate during the course of pyruvate oxidation. Further, an amperometric urea-sensing electrode (detection limit, 0.5 μM) was prepared by coupling the phosphate-sensing system with urea amidolyase which catalyzes an ATP-consuming urea hydrolyzation.

Synthesis and characterization of novel degradable photocrosslinked poly(ether-anhydride) networks

New degradable poly(ether-anhydride) networks were synthesized by UV photopolymerization. Dicarboxylated poly(ethylene glycol) (PEG) or poly(tetramethylene glycol) (PTMG) was reacted with an excess of methacrylic anhydride to form dimethacrylated macromers containing anhydride linkages. The percent of conversion for the macromer formation was more than 80% at 60 °C after 24 h. 1H NMR and IR spectroscopies show the presence of anhydride linkages in the macromer. In vitro degradation studies were carried out at 37 °C in PBS with crosslinked polymer networks formed by UV irradiation. All PEG-based polymers degraded within 2 days, while PTMG-based polymers degraded by 50% of the initial weight after 14 days. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 1277–1282, 2000

Synthesis and characterization of novel degradable photocrosslinked poly(ether-anhydride) networks

New degradable poly(ether-anhydride) networks were synthesized by UV photopolymerization. Dicarboxylated poly(ethylene glycol) (PEG) or poly(tetramethylene glycol) (PTMG) was reacted with an excess of methacrylic anhydride to form dimethacrylated macromers containing anhydride linkages. The percent of conversion for the macromer formation was more than 80% at 60 °C after 24 h. 1H NMR and IR spectroscopies show the presence of anhydride linkages in the macromer. In vitro degradation studies were carried out at 37 °C in PBS with crosslinked polymer networks formed by UV irradiation. All PEG-based polymers degraded within 2 days, while PTMG-based polymers degraded by 50% of the initial weight after 14 days. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 1277–1282, 2000