Extended release of bevacizumab by thermosensitive biodegradable and biocompatible hydrogel

The antibody bevacizumab (Avastin) has been used clinically to treat intraocular neovascular diseases based on its antivascular endothelial growth factor (VEGF) character. The anti-VEGF strategy for retinal neovascular diseases is limited by the short half-life of bevacizumab and thus requires frequent injections. This Article reports the sustained release of bevacizumab from a biocompatible material that is composed of a triblock copolymer of poly(2-ethyl-2-oxazoline)-b-poly(ε-caprolactone)-b-poly(2-ethyl-2-oxazoline) (PEOz-PCL-PEOz). The amphiphilic PEOz-PCL-PEOz triblock copolymer was synthesized in three steps. First, the PEOz was polymerized by methyl p-toluenesulfonate and 2-ethyl-2-oxazoline (EOz), and the living end was terminated by potassium hydroxide methanolic solution. Subsequently, the hydroxyl-PEOz was used as a macroinitiator for the ring-opening polymerization of ε-caprolactone using a Tin(II) octoate catalyst to synthesize the telechelic hydroxylated PEOz-PCL. Finally, the PEOz-PCL-PEOz triblock copolymer was obtained using the 1,6-hexamethylene diisocyanateas a coupling reagent. The PEOz-PCL-PEOz was chemically and molecularly characterized by GPC, (1)H NMR, and FTIR, and its aqueous solution (ECE hydrogel) showed a reversible sol (room temperature)-gel (physiological temperature) phase transition, which serves as an easy antibody-packing system with extended release. The biodegradability of ECE hydrogel was assessed by the porosity formation at different periods by scanning electron microscopy. The ECE hydrogel had no in vitro cytotoxicity on the human retinal pigment epithelial cell line by flow cytometry. The histomorphology and electrophysiology of the rabbit neuroretina were preserved after 2 months of intravitreal injection. In conclusion, the ECE hydrogel has a temperature-sensitive sol-gel phase transition and is effective in vitro. Its intraocular biocompatibility demonstrated its great potential to be widely used in biomedical applications for extended drug release.     

Injectable in situ physically and chemically crosslinkable gellan hydrogel

An injectable, in situ physically and chemically crosslinkable gellan hydrogel is synthesized via gellan thiolation. The thiolation does not alter the gellan's unique 3-D conformation, but leads to a lower phase transition temperature under physiological conditions and stable chemical crosslinking. The synthesis and hydrogels are characterized by (1)H NMR, FT-IR, CD, or rheology measurements. The injectability and the tissue culture cell viability is also tested. The thiolated gellan hydrogel exhibits merits, such as ease for injection, quick gelation, lower gelling temperature, stable structure, and nontoxicity, which make it promising in biomedicine and bioengineering as an injectable hydrogel.     

Functionalisation of chemically sensitive surfaces for biosensors and microreactors fabrication

In this paper, several surface functionalisation techniques for application in biosensors and microreactors, namely silanisation of glass and silicon and polymer modification were described. The modified surface of silicon samples was investigated by atomic force microscopy (AFM) and Fourier transformed infrared spectroscopy (FTIR) techniques. The found with AFM distinction between the surfaces of three silicon samples, bare, and the ones modified with aminopropyltriethoxysilane/glutaraldehyde (APTS/GA) and enzyme, consisted in a change of roughness of the surfaces, namely for the non-modified and modified ones were 3, 8 and more than 40 nm, respectively. The IR results for the third sample, modified with enzyme, proved the presence of the peptide coupling, visible as Amide-I and Amide-II bounds, specific for proteins. In addition, the performances of two types of microreactors batch and lamella type were compared. In the case of the batch type microreactors, the most effective immobilisation technique was those using polymeric beads made of reduced polyacrylonitrile (PAN) and glass beads modified with APTS/GA, where maximal pH difference (ΔpH_max) was about 3.3 and 2.5 for over 30 days, respectively. The worse results were obtained for the batch type microreactor loaded with beads of polyacrylamide type copolymer EUPERGIT^® where the output signal was decreased to 1.2 pH unit after 10 days of the microreactor operation. The performances of both lamella type microreactors modified with APTS/GA and glycidoxypropyltriethoxysilane (GOPS) were rather poor, i.e. the maximal output signal was about 0.3 pH unit after 10 and five days of their operation, respectively.     

Rapidly serum-degradable hydrogel templating fabrication of spherical tissues and curved tubular structures

Development of the techniques for fabricating three‐dimensional tissues still poses significant challenges for tissue engineering. We used hydrogels obtained from phenol‐substituted amylopectin (AP‐Ph) as templates for preparing multicellular spherical tissues (MSTs) and endothelialized curved tubular structures in type I collagen gel. AP‐Ph hydrogel microparticles of diameter 200 µm and fibers of diameter 500 µm disappeared within hours of soaking in a serum‐containing medium. HeLa cells and human endothelial cells were enclosed in the microparticles and hydrogel fibers, respectively, and then embedded in Ca‐alginate microcapsules or the collagen gel. The enclosed cells were released in cavities formed by hydrogel degradation in the serum‐containing medium. The released HeLa cells in the spherical cavities grew and formed MSTs, eventually filling the cavities. The spherical tissues were easily harvested by liquefying the Ca‐alginate hydrogel microcapsule membrane by chelation using sodium citrate. The released endothelial cells grew on the tubular cavity surfaces and formed tubular structures. An endothelial cell network was formed by cell migration into the collagen gel. These results demonstrate the potential of serum‐degradable AP‐Ph hydrogels in constructing three‐dimensional tissues. Biotechnol. Bioeng. 2012; 109: 2911–2919. © 2012 Wiley Periodicals, Inc.     

Facile fabrication of gelatin‐based biopolymeric optical waveguides

The rapid development in optical detection techniques for sensing applications has led to an increased need for biocompatible, biodegradable, and disposable optical components. We present a controllable fabrication technique for an entirely biopolymeric planar optical waveguide via simple spin‐coating. The refractive index difference, thermal responsive properties, and inherent biocompatibility of gelatin and agarose were exploited in the fabrication of thin, stacked films that efficiently guide light in a core layer with higher index of refraction. These planar waveguides were fabricated using a simple spin‐coating technique, which resulted in controllable layer thicknesses and smooth layer interfaces. This technique, therefore, offers a path for routine engineering of biopolymer structures with contrasting refractive indices. The thermal stability of the gelatin core layer was improved using two crosslinkers; glutaraldehyde or microbial Transglutaminase. Light guiding in the core layer of the waveguide was demonstrated using a simple He–Ne laser setup. Guiding efficiency was further illustrated by directly embedding fluorescent markers within the core layer and detecting their spectral signature. Combined with the biopolymers' inherent biocompatibility and biodegradability, our simple strategy to fabricate disposable optical components holds the potential for the development of applications in biological sensing and implantable biomedical devices. Biotechnol. Bioeng. 2009;103: 725–732. © 2009 Wiley Periodicals, Inc.     

Facile fabrication of distance-tunable Au-nanorod chips for single-nanoparticle plasmonic biosensors

In this work, we present a simple and effective method to fabricate distance-controllable, Au nanorod (AuNR) chips thorough electrostatic assembly. Cetyltrimethylammonium bromide (CTAB)-capped AuNRs were immobilized on a hydroxyl-functionalized glass substrate by immersion of the glass into AuNR-suspension. The electrostatic surfacial assembly of AuNRs offers significant advantages over conventional thiol-induced chemistry, i.e., direct control of self-assembly of AuNRs, easy fabrication in ambient environment and most importantly, broad range of tunable inter-particle distance, ranging from 0.25 to 10 μm. The mechanism of time-dependant deposition process of AuNRs was described via competitive bindings of AuNRs and free CTAB molecules in AuNR-suspension. In addition, the electrostatically anchored AuNRs on a glass substrate provide sufficient stability under harsh experimental conditions with flow of basic/acidic solutions and organic solvents with different polarity. The feasibility of the AuNR-chips fabricated by the proposed method for single-nanoparticle plasmonic biosensors was demonstrated by the plasmonic measurement of aptamer-thrombin binding event. The corresponding limit of detection of thrombin molecule was found to be ～278 pM based on the signal to noise ratio of 4.     

Facile fabrication of thermo-responsive and reduction-sensitive polymeric micelles for anticancer drug delivery

The development of thermo-responsive and reduction-sensitive polymeric micelles based on an amphiphilic block copolymer poly[(PEG-MEMA)-co-(Boc-Cyst-MMAm)]-block-PEG (denoted PEG-P-SS-HP) for the intracellular delivery of anticancer drugs is reported. PTX, as model drug, was loaded into the PEG-P-SS-HP micelles with an encapsulation efficiency >90%, resulting in a high drug loading content (up to 35?wt%). The PTX-loaded PEG-P-SS-HP micelles show slow drug release in PBS and rapid release after incubation with DTT. The PTX-loaded micelles display a better cytotoxic effect than the free drug, whereas empty micelles are found to be non-toxic. The thermo-responsive and reduction-sensitive polymeric micelles described may serve as promising carriers for cytostatic drugs.     

A study on the fabrication of chemically modified bacteriorhodopsin film and its photochemical properties

Chemically modified bacteriorhodopsin (BR) films embedded in gelatin matrix were fabricated. It was found that chemically modified BR/gelatin film remained the original conformation of BR and possessed homogeneity. Then, the photochemical conversion from all-trans O state to 9-cis P state in the chemically modified BR film/gelatin was investigated. For comparison purpose, the same conversion in the wild-type BR (BRwt) film was also presented here. To our know, it is the first report to show that the absorption changes of the P state in the chemically modified BR film were larger than in the BRwt film upon illumination with red light at the low actinic power density.     

Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition

Glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase have been adsorbed to colloidal gold sols with good retention of enzymatic activity. Adsorption of xanthine oxidase on colloidal gold did not result in a change in enzymatic activity as determined by active site titration with the stoichiometric inhibitor pterin aldehyde and by measurement of the apparent Michaelis constant (K'(M)). Gold sols with adsorbed glucose oxidase, horseradish peroxidase, and xanthine oxidase have also been electrodeposited onto conducting matrices (platinum gauze and/or glassy carbon) to make enzyme electrodes. These electrodes retained enzymatic activity and, more importantly, gave an electrochemical response to the enzyme substrate in the presence of an appropriate electron transfer mediator. Our results demonstrate the utility of colloidal gold as a biocompatible enzyme imobilization matrix suitable for the fabrication of enzyme electrodes. (c) 1992 John Wiley & Sons, Inc.     

A thixotropic hydrogel from chemically cross-linked guar gum: synthesis, characterization and rheological behaviour

Polysaccharide guar gum (GG) was cross-linked in an alkaline solution with polyethylene glycol diglycidyl ether (PEGDGE) to create a new hydrogel. The GG hydrogel was examined by FT-IR spectroscopy, AFM analysis and SEM analysis. The water uptake of the GG hydrogel was measured at different pHs, and rheological studies were performed to verify the thixotropic nature of the material. Rheological studies revealed the pseudoplastic behaviour of the GG hydrogel and its thixotropic nature. AFM analysis on a sample which was subjected to shear stress showed the presence of nanoparticles in the hydrogel. When the sample was left to settle, the gel surface returned to its original homogenous morphology. The thixotropic and injectable nature of the GG hydrogel suggest its possible use in biomedical applications.     

Facile fabrication of eco-friendly nano-mosquitocides: Biophysical characterization and effectiveness on neglected tropical mosquito vectors

Mosquito (Diptera: Culicidae) vectors are solely responsible for transmitting important diseases such as malaria, dengue, chikungunya, Japanese encephalitis, lymphatic filariasis and Zika virus. Eco-friendly control tools of Culicidae vectors are a priority. In this study, we proposed a facile fabrication process of poly-disperse and stable silver nanoparticles (Ag NPs) using a cheap leaf extract of Ichnocarpus frutescens (Apocyanaceae). Bio-reduced Ag NPs were characterized by UV–vis spectrophotometry, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The acute toxicity of I. frutescens leaf extract and green-synthesized Ag NPs was evaluated against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Compared to the leaf aqueous extract, Ag NPs showed higher toxicity against A. subpictus, A. albopictus, and C. tritaeniorhynchus with LC₅₀ values of 14.22, 15.84 and 17.26 μg/mL, respectively. Ag NPs were found safer to non-target mosquito predators Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC₅₀ values ranging from 636.61 to 2098.61 μg/mL. Overall, this research firstly shed light on the mosquitocidal potential of I. frutescens, a potential bio-resource for rapid, cheap and effective synthesis of poly-disperse and highly stable silver nanocrystals.     

Preparation and characterization of a novel pachyman-based pharmaceutical aid. II: A pH-sensitive,biodegradable and biocompatible hydrogel for controlled release of protein drugs

In this investigation, the fabrication, physico-chemical and biological characterization of a novel smart hydrogel had been evaluated for its potentials in effective controlling protein delivery. The hydrophilic pachyman-based hydrogel was generated facilely by crosslinking hydrosoluble carboxymethyl pachyman (CMP) with epichlorohydrin (ECH). The ECH concentration possessing maximum (99.7%) encapsulation efficiency and the most appropriate swelling characteristics was found to be 1.25% (w/v). The resultant hydrogel exhibited swelling ratios most favorable for drug release in simulated intestinal media. It could release two model protein drugs (bovine serum albumin and lysozyme) in the controlled manner and with full preservation of the protein stability and enzymatic activity. Importantly, the ECH-CMP hydrogel was confirmed to be biocompatible and biodegradable. From these findings, we were able to conclude that the synthesized pachyman-based hydrogel would be a promising delivery carrier candidate for site-specific delivery of protein drugs.     

Facile fabrication of promising protein tyrosine phosphatase (PTP) inhibitor entities based on 'clicked' serine/threonine-monosaccharide hybrids

Protein tyrosine phosphatases (PTPs) are well-validated therapeutic targets for many human major diseases. The development of their potent inhibitors has therefore become a main focus of both academia and the pharmaceutical industry. We report herein a facile strategy toward the fabrication of new and competent PTP inhibitor entities by simply 'clicking' alkynyl amino acids onto diverse azido sugar templates. Triazolyl glucosyl, galactosyl, and mannosyl serine and threonine derivatives were efficiently synthesized via click reaction, which were then identified as potent CDC25B and PTP1B inhibitors selective over a panel of homologous PTPs tested. Their inhibitory activity and selectivity were found to largely lie on the structurally and configurationally diversified monosaccharide moieties whereon serinyl and threoninyl residues were introduced. In addition, MTT assay revealed the triazole-connected sugar-amino acid hybrids may also inhibit the growth of several human cancer cell lines including A549, Hela, and especially HCT-116. On the basis of such compelling evidence, we consider that this compound series could furnish promising chemical entities serving as new CDC25B and PTP1B inhibitors with potential cellular activity. Furthermore, the 'click' strategy starting from easily accessible and biocompatible amino acids and sugar templates would allow the modular fabrication of a rich library of new PTP inhibitors efficaciously and productively.     

On‐demand three‐dimensional freeform fabrication of multi‐layered hydrogel scaffold with fluidic channels

One of the challenges in tissue engineering is to provide adequate supplies of oxygen and nutrients to cells within the engineered tissue construct. Soft‐lithographic techniques have allowed the generation of hydrogel scaffolds containing a network of fluidic channels, but at the cost of complicated and often time‐consuming manufacturing steps. We report a three‐dimensional (3D) direct printing technique to construct hydrogel scaffolds containing fluidic channels. Cells can also be printed on to and embedded in the scaffold with this technique. Collagen hydrogel precursor was printed and subsequently crosslinked via nebulized sodium bicarbonate solution. A heated gelatin solution, which served as a sacrificial element for the fluidic channels, was printed between the collagen layers. The process was repeated layer‐by‐layer to form a 3D hydrogel block. The printed hydrogel block was heated to 37°C, which allowed the gelatin to be selectively liquefied and drained, generating a hollow channel within the collagen scaffold. The dermal fibroblasts grown in a scaffold containing fluidic channels showed significantly elevated cell viability compared to the ones without any channels. The on‐demand capability to print fluidic channel structures and cells in a 3D hydrogel scaffold offers flexibility in generating perfusable 3D artificial tissue composites. Biotechnol. Bioeng. 2010;105: 1178–1186. © 2009 Wiley Periodicals, Inc.     

Facile fabrication of magnetic gold electrode for magnetic beads-based electrochemical immunoassay: application to the diagnosis of Japanese encephalitis virus

A novel magnetic beads-based electrochemical immunoassay strategy has been developed for the detection of Japanese encephalitis virus (JEV). The magnetic gold electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. The proteins (e.g., antibodies or immunocomplexes) attached on the surface of magnetic beads were found to induce a significant decline in their electric conductivity. Multiwalled carbon nanotubes were introduced to improve sensitivity of the assay. The envelope (E) protein, a major immunogenic protein of JEV, was utilized to optimize the assay parameters. Under the optimal conditions, the linear response range of E protein was 0.84 to 11,200 ng/mL with a detection limit of 0.56 ng/mL. When applied for detection of JEV, the proposed method generated a linear response range between 2×10(3) and 5×10(5) PFU/mL. The detection limit for JEV was 2.0×10(3) PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. This method was also successfully applied to detect JEV in clinical specimens.     

Synthesis and magnetic properties of biocompatible hybrid hollow spheres

Magnetic hybrid hollow spheres of about 200 nm were prepared by a core-template-free route, that is, adding Fe3O4 nanoparticles stabilized by poly(vinyl alcohol) (PVA) to an aqueous solution of polymer-monomer pairs composed of a cationic polymer, chitosan (CS), and an anionic monomer, acrylic acid (AA), followed by polymerization of acrylic acid and selective cross-linking of chitosan at the end of polymerization. The obtained hybrid spheres were characterized by dynamic light scattering (DLS) in aqueous solution and observed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) in the solid state. Fourier transform infrared spectroscopy (FTIR) and X-ray and electron diffractions revealed that the Fe3O4 nanoparticles were incorporated into the shells of chitosan-poly(acrylic acid) (CS-AA) hollow spheres. Magnetization studies and M?ssbauer spectroscopy suggested that the chains (or islands) of iron oxide nanoparticles were most likely formed in the walls of the hollow spheres. The phantom test of magnetic resonance imaging showed that the synthesized hybrid hollow spheres had a significant magnetic resonance signal enhancement in T2-weighted image.     

Novel fabrication of morphology tailored nanostructures with Gelatin/Chitosan Co-polymeric bio-composited hydrogel system to accelerate bone fracture healing and hard tissue nursing care management

In this study, we are successfully fabricated on a hydrogel consisting of TiO₂ nanoparticles loaded onto a gelatin/chitosan matrix to control the acceleration of bone fracture healing and improved the nursing care applications. Each specimen (chitosan, gelatin and titanium dioxide) were characterized and confirmed by using different techniques, Fourier transforms infrared spectroscopy, X-ray diffraction analysis, Scanning Electron Microscopy with Elemental dispersive X-ray analysis, Thermo-gravimetric and Differential thermal analysis. In addition, the cell cytotoxicity results verified that the TiO₂/gelatin-chitosan hydrogel are nontoxic to osteoblasts. And cell fixation outcome after 5 days of incubation condition revels that the enhanced in vitro cell survival and cell spreading on the prepared TiO₂ incorporated hydrogel with respect to gelatin/chitosan hydrogel. Furthermore, TiO₂/gelatin-chitosan hydrogel nanostructures can modulate the bone fracture healing, indicating a potential application on nursing care.     

Synthesis and preliminary biological assessments of RGD bearing biocompatible telomers

Work reported herein deals with the synthesis and preliminary biological assessments of a new class of biocompatible telomeric carriers bearing peptidic RGDSK sequences as tumor cell targeting and tyrosine moieties labelled with 125I as in vivo probe. The radioactivity levels obtained in several tissues, after the intravenous injections of these telomers in mice bearing grafted B16 syngenic melanoma showed that the addition of a RGD residue to a telomeric structure confers it an increased affinity for the highly vascularized zone surrounding the tumor.     

Synthesis and characterization of biocompatible polymer interlayers on titanium implant materials

A bifunctional copolymer series of (4-vinylbenzyl)phosphonic acid diethylester and N-acryloxysuccinimide was developed as an interlayer with the aim of immobilizing proteins on titanium surfaces. Copolymers with varying compositions were synthesized, and an alternating copolymerization of the two monomers was found. The copolymers form ultrathin films of about 2-8 nm on titanium surfaces in a simple dipping process, as estimated from the attenuation of the titanium X-ray photoelectron spectroscopy (Ti-XPS) signal. The films were characterized by infrared spectroscopy, XPS, and time-of-flight secondary ion mass spectrometry. The results indicate that the immobilization is due to phosphonate groups, and thus the phosphonate content of the copolymers is decisive for the final film thickness. These polymer films were examined for their potential protein binding capacity by using trifluoroethylamine derivatization and subsequent XPS analysis as a reactivity assay.