Gwen Lawrie

Phosphorylation of alginate: Synthesis, characterization, and evaluation of in vitro mineralization capacity

Phosphorylation of alginate was achieved using a heterogeneous urea/phosphate reaction. The degree and stereoselectivity of phosphorylation as well as the effects on the physical properties of the polysaccharide were investigated by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies, inductively coupled plasma optical-emission spectroscopy (ICP-OES), and size exclusion chromatography (SEC). Multidimensional NMR studies of the phosporylated alginate revealed that phosphorylation of the M residues occurred predominantly at the C3 (equatorial) carbon of the polysaccharide ring. In addition, a more comprehensive assignment of the (1)H NMR spectrum of alginate, compared with those previously reported in the literature, is provided here. Hydrogel materials were formed from ionically cross-linked blends of phosphorylated alginate and alginate. These blended hydrogels showed an enhanced resistance to degradation by chelating agents compared with cross-linked alginate hydrogels and a reduction in their mineralization potential.

A novel strategy for preparing mechanically robust ionically cross-linked alginate hydrogels

The properties of alginate films modified using two cross-linker ions (Ca(2+) and Ba(2+)), comparing two separate cross-linking techniques (the traditional immersion (IM) method and a new strategy in a pressure-assisted diffusion (PD) method), are evaluated. This was achieved through measuring metal ion content, water uptake and film stability in an ionic solution ([Ca(2+)] = 2 mM). Characterization of the internal structure and mechanical properties of hydrated films were established by cryogenic scanning electron microscopy and tensile testing, respectively. It was found that gels formed by the PD technique possessed greater stability and did not exhibit any delamination after 21 day immersion as compared to gels formed by the IM technique. The Ba(2+) cross-linked gels possessed significantly higher cross-linking density as reflected in lower water content, a more dense internal structure and higher Youngs modulus compared to Ca(2+) cross-linked gels. For the Ca(2+) cross-linked gels, a large improvement in the mechanical properties was observed in gels produced by the PD technique and this was attributed to thicker pore walls observed within the hydrogel structure. In contrast, for the Ba(2+) cross-linked gels, the PD technique resulted in gels that had lower tensile strength and strain energy density and this was attributed to phase separation and larger macropores in this gel.

Encapsulation of a glycosaminoglycan in hydroxyapatite/alginate capsules

The development of suitable vehicles for the delivery of growth-inducing factors to fracture sites is a challenging area of bone repair. Bone-specific glycosaminoglycan molecules are of particular interest because of their high stability and proven effect on bone growth. Calcium alginate capsules are popular as delivery vehicles because of their low immunogenic response; they offer a versatile route that enables the controlled release of heparin (a member of the glycosaminoglycan family). In this study, hydroxyapatite (HA)/alginate composite capsules are explored as novel drug delivery vehicles for heparin, using both medium- and low-viscosity alginates. The composition, structure, and stability of the capsules are fully characterized and correlated to the release of heparin in vitro. Heparin is found to associate both with the alginate matrix through polymeric flocculation and also with the HA crystals in the composite beads. The mechanism by which heparin is released is dictated by the stability of the capsule in a particular release media and by the composition of the capsule. The use of medium-viscosity alginate is advantageous with respect to both drug loading and prolonging the release. The inclusion of HA increases the encapsulation efficiency, but because of its destabilizing effect to the alginate hydrogel matrix, it also increases the rate of heparin release. The bioactivity of heparin is fully retained throughout the assembly and release processes.

Synthesis and characterization of alginate/poly-L-ornithine/alginate microcapsules for local immunosuppression

Alginate/poly-L-ornithine/alginate (APA) coherent microencapsulation, which provides an immunoselective and highly biocompatible membrane, creates a viable option for cellular or tissue transplantation. This study explored the potential of incorporating immunosuppressive drugs onto the capsule surface to provide local immunosuppression in addition to immunoisolation. A thorough investigation has been conducted to optimize and characterize alginate biotinylation via carbodiimide chemistry by a 4′-hydroxyazobenzene-2-carboxylic acid (HABA) based assay and by ATR-FTIR, H-NMR and XPS. To minimize the formation of by-product, a theoretical 40% activation of the carboxylic group on the alginate was employed to manufacture an optimal modification of ∼10% biotinylated alginate. Confocal fluorescence microscopy was used to assess the conjugation of streptavidin and assembly of antibodies on the microcapsules. Local immunosuppressive capacity was assimilated on the APA microcapsules by binding of anti-tumour necrosis factor-alpha (TNF-α) antibodies via streptavidin-biotin conjugation, shown from the clear reduction of TNF-α in in-vitro medium.

Is the undergraduate research experience (URE) always best?: The power of choice in a bifurcated practical stream for a large introductory biochemistry class

Science undergraduate courses typically cater to a mixed‐learner cohort, with a diversity of motivations and skills. This diversity introduces pressure for designers of the practical laboratory curriculum. Students who are struggling with the course need a series of tasks that begin simply, and transition to more conceptually difficult material. More capable students need opportunities for conceptual extension and creative activity. In this report, we examine an approach we have used to address this problem in the context of a large introductory biochemistry undergraduate class. Rather than attempting to compromise on a single practical series for our 470 students, we devised two parallel but equivalent practical streams and offered students their choice of laboratory experience. One stream (called Laboratory Experience for Acquiring Practical Skills) was designed to allow acquisition of a range of common biochemistry and molecular biology laboratory skills. The other (called Active Learning Laboratory Undergraduate Research Experience) was designed to offer an authentic (but scaffolded) undergraduate research project. We discuss the ramifications and implications of our approach in terms of funding, staffing, and assessment while also examining student motivation, satisfaction, and skills acquisition. We present data supporting the practical and pedagogical value of laboratory exercise streaming to meet the diverse needs of students. We suggest a framework that can be used to pre‐emptively identify and address problems associated with a bifurcated practical series and increase the sustainability of the approach.

Do We Need to Design Course-Based Undergraduate Research Experiences for Authenticity?

The authors conducted a metareview of published conceptions of “authentic” science laboratory education and used their students’ reflections to examine the authenticity of their own laboratory curriculum design. They find that preauthentication of a learning design is not necessary to deliver an authentic experience to students.

Modification and optimization of organosilica microspheres for peptide synthesis and microsphere-based immunoassays

A new generation of optically encoded organosilica microspheres, suitable for both solid phase synthesis and multiplexed microsphere-based assays, has recently been described. One of the challenges of producing this type of dual-purpose solid support is that the particles must maintain their morphology as well as their encoding during exposure to the solvents used for solid phase synthesis. In this article, organosilica microspheres are subjected to ammonia treatment methods for enhancing the condensation of the silica matrix and their subsequent resilience toward organic solvents and peptide synthesis reagents is described. The instability of the untreated supports toward organic synthesis reagents was found to be associated with the swelling and permeability of these microspheres in organic solvents. Post-synthesis ammonia treatment resulted in reduced permeability, as demonstrated by dye uptake studies. The treated microspheres exhibited enhanced stability against organic synthesis conditions and were characterized via a variety of techniques including electron microscopy, (29)Si-nuclear magnetic resonance (NMR) and optical microscopy. The ammonia-treated supports were subjected to an Fmoc peptide synthesis procedure and successfully applied in a model microsphere-based flow cytometric immunoassay.

Alginate-based drug delivery devices

Alginates are ubiquitous in the fields of biomaterials science and drug delivery. These polysaccharides are versatile due to their biocompatibility and structural functionality. This chapter provides a comprehensive overview of the recent advances in the development of new alginate-based drug delivery systems through the exploration of the manipulation of chemical and structural properties. The encapsulation of both small molecular drugs and cells producing active biomolecules is considered. The refinement of the assembly of alginate-based matrices for drug delivery has made significant advances and research is now focusing on tailoring the release profiles of the bioactive species.

Organosilica Particles for DNA Screening Applications

Particle-based libraries for chemical screening are of interest for robust biochemical assays and molecular diagnostic applications. Silica particles are chemically stable and possess two separate chemical modification pathways that facilitate the attachment of fluorescent dyes for encoding purposes and specific oligonucleotides for hybridization assays. Characterization of surface modifications of the particles are presented and quantitation of oligonucleotide probes on the particle surface is described. The application of the particles in biological screening experiments is presented by means of a single base mismatch detection assay.