Session 4

Yasmeen Shamiya

University of Western Ontario

Fabrication of 3D-printed nanocomposite hydrogels using polymeric nanofibers for wound healing applications

Nanoparticles have gained increasing attention in the last decade owing to their many beneficial properties in regard to drug delivery. Some of their main benefits include (1) their small size and large surface area which provide higher drug loading, (2) their ability to interact with biomolecules, and (3) their persistence in the circulatory system for longer periods of time before being cleared. However, most nanoparticles lose the loaded cargo through the diffusional process as well as by interacting with blood components. Therefore, maintaining the loaded drug inside the drug delivery vehicle is of critical importance and creates the need for delivery vehicles with slow drug release in the blood, but fast drug release when stimulated by environmental factors. Hydrogel scaffolds have emerged as an important tool in biomedical applications due to their biocompatibility, hydrophilicity, extracellular matrix-like structure, and ability to deliver bioactive molecules. However, hydrogels are mechanically weak and have poor hydrophobic drug loading capacities. Here, we have developed self-assembling ascorbyl palmitate (AP) nanofibers capable of enzyme-responsive cleavage in inflammatory conditions. The nanosized features and large surface area of these AP nanofibers maximize the interaction with specific enzymes for disassembly and drug release of a model drug (erythromycin) in the presence of inflammation. Further, we have 3D-printed AP nanofibers in a naturally derived gelatin-based polymeric hydrogel for mechanically resilient scaffolds. We show that the designed nanocomposite hydrogel is (i) cytocompatible with mesenchymal stem cells, (ii) mechanically tough, and (iii) is capable of loading hydrophobic drugs and is suitable for inflammation-responsive drug-delivery based applications while retaining bioactivity of the drug.

Sergio Luna Nino

University of Toronto

Anaerobic digestion can remove small particles in biosludge and improve dewatering

The efficiency of biosludge dewatering has significant economic and environmental implications for both industrial and municipal wastewater treatment facilities. During anaerobic treatment, biosludge dewatering can either improve or deteriorate; this is a highly variable outcome and the underlying mechanisms are not well understood. However, anaerobic digestion can improve the dewatering of biosludge that was pre-treated; studying the factors associated with this benefit to dewatering can uncover the mechanisms that lead to improving biosludge dewatering in anaerobic environments. Biosludge was pre-treated via ultrasonication (20 kHz) in a two-factor design-of-experiment (DOE) approach, varying amplitude (Low: 20 A, Mid: 60 A, High: 100 A) and treatment time (Low: 5 min, Mid: 7.5 min, High: 10 min) to give five experimental groups and a non-pre-treated control. All groups were placed in triplicate 500mL batch mesophilic anaerobic digesters at 6 g COD/g VSS S/I ratio for 66 days. Improved dewatering was observed after 66-day AD, with a reduction in the normalized capillary suction time (N-CST (s-g/L)) for the five experimental groups and non-pretreated control. Crown Press results showed an increase in solids capture efficiency with a lower Crown Press Filtrate TS (g/L) after AD, while the Crown-Press Cake %DS remained within error. The improvement in dewatering coincided with a rightward shift of the respective particle size distribution, which suggests a removal of small particles in the supracolloidal range was a contributing factor in improving biosludge dewatering. This effect was seen in sonicated groups and the control, suggesting the mechanism of improvement and recovery are shared.

Ana Arežina

McMaster University

High-Throughput Multi-Well Screening of Flat Sheet Membrane Adsorbers for Membrane Chromatography

Current bioseparation processes use membrane chromatography for the separation and purification of biomolecules. Unfortunately, this optimization can be costly and time-consuming with its extensive evaluation of process conditions (e.g. salt concentration, pH). High-throughput screening (HTS) tools (i.e. 96-well filter plate) – which use minimal sample and solution amounts – are instead used for this optimization, but are often limited to a few commercial membrane types. Here, a multi-well device with the ability to conduct up to 32 experiments for biomolecule screening to any flat sheet membrane adsorber was developed. This device was also designed with the potential to be stacked, allowing for HTS of biomolecules with the consideration of multiple variables (e.g. salt concentration, pH) In this study, the static binding capacity (SBC) of typical biomolecules (e.g. BSA) on membrane adsorbers (e.g. Sartobind Q, Natrix Q) was analyzed at particular salt concentrations and contact times. The results showed that Natrix Q – a membrane type not sold in a commercial filter plate – had the highest BSA SBC (0 M NaCl, 24 h). Additionally, confocal imaging of a Natrix Q cross-section was conducted after 24 h in contact with green fluorescent protein in the device, and this visually confirmed the protein dispersion throughout the membrane. Finally, the bind-wash-elute chromatography process was replicated in this multi-well device with Natrix Q. Overall, this multi-well device allows for extensive HTS with any flat sheet membrane adsorber, enhancing the potential for superior bioseparation performance in future chromatography processes.

Erica Di Pede

University of Toronto

Engineering High Value Bioactive Substrates from Low-Value Waste: Enzymatically derived manno-oligosaccharides(s) from Coconuts Support the growth of Probiotics and exhibit an antimicrobial effect on Pathogens

Objectives:
1) Use an enzymatic hydrolysis process to develop manno-oligosaccharides of varied degrees of polymerization and characterize structure and chemical composition.
2) Assess the synbiotic potential between two structurally different novel manno-oligosaccharides and key Lactobacillus sp.
3) Evaluate the ability of coconut-meal derived oligosaccharides to inhibit select pathogens responsible for food borne illness and urinary tract infections.

Methods:
4L batch reactors were used to hydrolyze the raw coconut meal to produce manno-oligosaccharides. A range of enzyme doses and hydrolysis times was explored to determine impact on oligosaccharide production. Ultrafiltration and nanofiltration were employed in order to remove high molecular weight components and enzyme resistant fractions of coconut meal. The chemical and structural composition of the manno-oligosaccharide (MOS) derivates was determined using High Performance Liquid Chromatography with an oligomer column. Growth studies were conducted in a microplate reader in dextrose free MRS media, with a range of prebiotic concentrations to assess the prebiotic dose response for L. reuteri, L. rhamnosus and L. plantarum strains. The minimum inhibitory concentration (MIC) was used for engineered MOS and the commercial prebiotic Fructo-oligosaccharides (FOS), against UroPathogenic Escherichia coli (UPEC), S. enteritidis and S. typhimurium. Growth studies were also conducted with FOS, Xylo-oligosaccharides (XOS) and Inulin, to assess comparative performance versus popular commercial prebiotics.
 
Results:
By manipulation of hydrolysis time and enzyme dose, two structurally distinct oligomer mixtures were produced, purified and successfully characterized to be used for subsequent biological in-vitro applications. The coconut-derived MOS’ effectively promoted the proliferation of Lactobacillus rhamnosus GG (ATCC 53103) , Lactobacillus reuteri (WT strain - DSM 20016) and Lactobacillus plantarum (WT strain - nrrl b - 4496). Probiotic growth was comparable on MOS and FOS, and significantly lower on XOS and inulin. This data points to the importance of prebiotic structure and composition for the development of synbiotics. Growth of UroPathogenic Escherichia coli (UPEC), Salmonella Enteritidis and Salmonella Typhimurium was inhibited by MOS in a dose-response manner. The greatest inhibition of pathogen growth was observed when the oligomer mixture with the lowest degree of polymerization was used as the antimicrobial agent.

Conclusions:
This research has identified a potentially impactful and bioactive oligosaccharide produced from low value coconut residues. These bioactive components have potential for future in vivo applications based on their physiological and antimicrobial effects