Electrospun chitosan nanofibers for virus removal

Electrospinning uses electrostatic forces to create nanofibers that are far smaller than conventional fiber spinning process. Nanofibers made with chitosan were created and techniques to control fibers diameter and were well developed. However, the adsorption of porcine parvovirus (PPV) was low. PPV is a small, nonenveloped virus that is difficult to remove due to its size, 18-26 nm in diameter, and its chemical stability. To improve virus adsorption, we functionalized the nanofibers with a quaternized amine, forming N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC). This was blended with additives to increase the ability to form HTCC nanofibers. The additives changed the viscosity and conductivity of the electrospinning solution. We have successfully synthesized and functionalized HTCC nanofibers that absorb PPV. HTCC blend with graphene have the ability to remove a minimum of 99% of PPV present in solution.

Virus purification, detection and removal

The biopharmaceutical industry has a growing demand and an increasing need to improve the current virus purification technologies, especially as more and more vaccines are produced from cell-culture derived virus particles. Downstream purification strategies can be expensive and account for 70% of the overall manufacturing costs. The economic pressure and purification processes can be particularly challenging when the virus to be purified is small, as in our model virus, porcine parvovirus (PPV). Our efforts are focused on designing an easy, economical, scalable and efficient system for virus purification, and we focused on aqueous two-phase systems. Industry acceptable standards for virus vaccine recovery can be as low as 30% due to demand of high final titer, virus transduction inhibitors and presence of empty or defective virus capsids as impurities. We have overcome these shortcomings by recovering a high 64% of infectious virus using an aqueous two-phase system. We used high molecular weight polymer and citrate salt to achieve a good yield and eliminated the major contaminant bovine serum albumin. Viruses are also studied for ensuring pure and safe drinking water. Low pressure microfiltrations are continuously being investigated for water filters as they allow high permeate flux and low fouling. Viruses such as PPV are small enough to pass through the microporous membranes. Control of viruses in water is crucial for public health and we have designed an affinity based membrane filter to capture virus. Nanofibers have a high surface to volume ratio providing a highly accessible surface area for virus adsorption. Chitosan an insoluble, biocompatible and biodegradable polymer was used for adsorbing trimer peptide WRW. About 0.2 μmoles of cysteine terminal WRW peptide was conjugated to amine terminal chitosan using maleimide conjugation chemistry. We achieved 90-99% virus removal from water adjusted to a neutral pH. The virus removal from affinity based chitosan was attributed to electrostatic and hydrophobic driven binding effect.

Electrospun Quaternized Chitosan Fibers for Virus Removal from Drinking Water

Membrane filtration has become an accepted technology for the removal of pathogens from drinking water. Viruses, known to contaminate water supplies, are too small to be removed by a size-exclusion mechanism without a large energy penalty. Thus, functionalized electrospun membranes that can adsorb viruses have drawn our interest. We chose a quaternized chitosan derivative (HTCC) which carries a positively-charged quaternary amine, known to bind negatively-charged virus particles, as a functionalized membrane material. The technique of electrospinning was utilized to produce nanofiber mats with large pore diameters to increase water flux and decrease membrane fouling. In this study, stable, functionalized, electrospun HTCC-PVA nanofibers that can remove 3.6 logs (99.97%) of a model virus, porcine parvovirus (PPV), from water by adsorption and filtration have been successfully produced. This technology has the potential to purify drinking water in undeveloped countries and reduce the number of deaths due to lack of sanitation.

STUDIES OF FUNCTIONALIZED NANOPARTICLES FOR SMART SELF-ASSEMBLY AND AS CONTROLLED DRUG DELIVERY

This dissertation is related to the studies of functionalized nanoparticles for self-assembly and as controlled drug delivery system. The whole topic is composed of two parts. In the first part, the research was conducted to design and synthesize a new type of ionic peptide-functionalized copolymer conjugates for self-assembly into nanoparticle fibers and 3D scaffolds with the ability of multi-drug loading and governing the release rate of each drug for tissue engineering. The self-assembly study confirmed that such peptide-functionalized amphiphilic copolymers underwent different self-assembly behavior. The bigger nanoparticles were more easily assembled into nanoparticle fibers and 3D scaffolds with larger pore size, while the smaller nanoparticle underwent faster self-assembly to form more compact 3D scaffolds with smaller porosity but more stable structure. Controlled release studies confirmed the ability of governing simultaneous release of different model drugs with independent release rate from a same scaffold. Cytotoxicity tests showed that all synthesized peptides, copolymers and peptide-copolymer conjugates were biocompatible with SW-620 cell lines and NIH3T3 cell lines. This new type of self-assembled scaffolds combined the advantages of peptide nanofibers and versatile controlled release of polymeric nanoparticles to achieve simultaneous multi-drug loading and controlled release of each drug, uniform distribution and flexibility of hydrogel scaffolds. The investigations in second part were first to design and synthesize organic biocide-loaded nanoparticles for low-leaching wood preservation using a cost-effective one-pot method to synthesize amphiphilic chitosan-g-PMMA nanoparticles loading with ~25-28 wt.% of the fungicide tebuconazole with particle size of ~100 nm diameter by FESEM. FESEM analysis confirmed efficient penetration of nanoparticles throughout the treated wooden stake with dimension of 19 × 19 × 455 mm^3. Leaching studies showed that biocide introduced into sapwood via nanoparticles leached only ~9% compared with the amount leached from tebuconazole solution-treated control, while soil jar tests showed that the nanoparticle-treated wood blocks were effectively protected from biological decay tested against G. trabeum, a brown rot fungus. Copper oxide nanoparticles with and without polymer stabilizers were also investigated to use as inorganic wood preservatives to clarify the factor affecting copper leaching from treated wood. Copper oxide nanoparticles with uniform diameters of ~10 nm and ~50 nm were prepared, and the leachates from southern pine sapwood treated with these nanoparticles were analyzed. It was found by TEM and EDS analysis that significant numbers of nanoparticles leached from the treated wood. The 50 nm nanoparticles leached slightly less than a soluble copper salt control, but 10 nm nanoparticles leached substantially more than the control. The effect of polymer stabilizers on nanoparticle leaching was also investigated. Results showed that polymer stabilizers increased leaching. The trends showed that nanoparticle size was a major factor in copper leaching.