Caryn L. Heldt

Virus adsorption of water-stable quaternized chitosan nanofibers

Abstract The burden of unsafe drinking water is responsible for millions of deaths each year. To relieve this burden, we are in search of an inexpensive material that can adsorb pathogens from drinking water. In this pursuit, we have studied the natural carbohydrate, chitosan. To impart virus removal features, chitosan has been functionalized with a quaternary amine to form quaternized chitosan N-[(2-hydroxyl-3-trimethylammonium) propyl] chitosan (HTCC). HTCC can be electrospun into nanofibers with the non-ionogenic polyvinyl alcohol (PVA), creating a high surface area mat. High surface area is a major requirement for effective adsorption processes. HTCC is antiviral and antimicrobial, making it a good material for water purification. However, HTCC dissolves in water. We have explored the parameters to crosslink the nanofibers with glutaraldehyde. We have imparted water stability so there is a maximum of 30% swelling of the fibers after 6h in water. The water stable fibers retain their ability to adsorb virus, as shown for an enveloped and nonenveloped virus. HTCC now has the potential to be incorporated into a microfiltration membrane that can remove viruses. This could create an inexpensive, low pressure filtration membrane for drinking water purification.

Biocompatibility of soft-templated mesoporous carbons.

Soft-templated mesoporous carbon is morphologically a non-nano type of carbon. It is a relatively newer variety of biomaterial, which has already demonstrated its successful role in drug delivery applications. To investigate the toxicity and biocompatibility, we introduced three types of mesoporous carbons with varying synthesis conditions and pore textural properties. We compared the Brunauer-Emmett-Teller (BET) surface area and pore width and performed cytotoxicity experiments with HeLa cells, cell viability studies with fibroblast cells and hemocomapatibility studies. Cytotoxicity tests reveal that two of the carbons are not cytotoxic, with cell survival over 90%. The mesoporous carbon with the highest surface area showed slight toxicity (∼ 70% cell survival) at the highest carbon concentration of 500 μg/mL. Fibroblast cell viability assays suggested high and constant viability of over 98% after 3 days with no apparent relation with materials property and good visible cell-carbon compatibility. No hemolysis (<1%) was confirmed for all the carbon materials. Protein adsorption experiments with bovine serum albumin (BSA) and fibrinogen revealed a lower protein binding capacity of 0.2-0.6 mg/m(2) and 2-4 mg/m(2) for BSA and fibrinogen, respectively, with lower binding associated with an increase in surface area. The results of this study confirm the biocompatibility of soft-templated mesoporous carbons.

Reduction of porcine parvovirus infectivity in the presence of protecting osmolytes

Osmolytes are natural compounds found in the cells of many organisms that stabilize intracellular proteins against environmental stresses. Protecting osmolytes can promote protein folding, whereas denaturing osmolytes have the opposite effect. A variety of osmolytes were tested for their antiviral activity against porcine parvovirus (PPV). PPV is a non-enveloped, icosahedral, single-strand DNA virus. We have discovered two protecting osmolytes, trimethylamine N-oxide (TMAO) and glycine that reduce the infectivity of PPV by four logs (99.99%). We hypothesize that both osmolytes stabilize viral capsid proteins and prevent them from assembling into viable virus particles. The advantage of the antiviral compounds found is that they can be applied post-infection, which increases their potential to serve as a therapeutic drug.

Separation of porcine parvovirus from bovine serum albumin using PEG–salt aqueous two-phase system

Vaccine production faces a challenge in adopting conventional downstream processing steps that can efficiently purify large viral particles. Some major issues that plague vaccine purification are purity, potency, and quality. The industry currently considers 30% as an acceptable virus recovery for a vaccine purification process, including all downstream processes, whereas antibody recovery from CHO cell culture is generally around 80-85%. A platform technology with an improved virus recovery would revolutionize vaccine production. In a quest to fulfill this goal, we have been exploring aqueous two-phase systems (ATPSs) as an optional mechanism to purify virus. ATPS has been unable to gain wide implementation mainly due to loss of virus infectivity, co-purification of proteins, and difficulty of polymer recycling. Non-enveloped viruses are chemically resistant enough to withstand the high polymer and salt concentrations that are required for effective ATPS separations. We used infectious porcine parvovirus (PPV), a non-enveloped, DNA virus as a model virus to test and develop an ATPS separation method. We successfully tackled two of the three main disadvantages of ATPS previously stated; we achieved a high infectious yield of 64% in a PEG-citrate ATPS process while separating out the main contaminate protein, bovine serum albumin (BSA). The most dominant forces in the separation were biomolecule charge, virus surface hydrophobicity, and the ATPS surface tension. Highly hydrophobic viruses are likely to benefit from the discovered ATPS for high-purity vaccine production and ease of implementation.

Porcine parvovirus flocculation and removal in the presence of osmolytes

Viruses can be modified into viral vaccines or gene therapy vectors in order to treat acquired or genetic diseases. To satisfy the current market demand, an improvement in current vaccine manufacturing is needed. Chromatography and nanofiltration are not suitable for all types of viruses. In this study, we propose to use virus flocculation with osmolytes, followed by microfiltration, as a potential virus purification process. We hypothesize that osmolytes strongly bind to water, thus leading to the formation of a hydration layer around the virus particles and stimulation of aggregation. We have discovered that osmolytes, including sugars, sugar alcohols and amino acids, preferentially flocculate porcine parvovirus (PPV), and demonstrate a >80% removal with a 0.2 μm filter while leaving model proteins in solution. This large pore size filter increases the flux and decreases the transmembrane pressure of typical virus filters. The best flocculants were tested for their ability to aggregate PPV at different concentrations, shear stress, pH and ionic strength. We were able to remove 96% of PPV in 3.0M glycine at a pH of 5. Glycine is also an excipient, and therefore may not require removal later in the process. Virus flocculation using osmolytes, followed by microfiltration could be used as an integrated process for virus purification.

Non-enveloped virus reduction with quaternized chitosan nanofibers containing graphene

Membranes are an accepted technology for water purification. Membrane filtration can remove pathogens, including bacteria and viruses, by size. For small viruses that can have a diameter <25nm, removal by size leads to large membrane areas, high transmembrane pressures, low water flux, and frequent changing of membranes. In this work, we discovered that electrospun nanofibers made of chitosan and functionalized with a quaternary amine (HTCC) have the ability to adsorb a model non-enveloped virus, porcine parvovirus (PPV). To improve the virus removal of HTCC, we added graphene. Graphene both enhanced the ability to form nanofibers with HTCC and improved the virus removal. The hydrophobicity of graphene and the high charge of the HTCC create a system that can bind 95% of PPV. The HTCC/graphene nanofibers could be incorporated into microfiltration membranes and remove virus by adsorption. This would create a low pressure system that is more likely to benefit areas in need of fresh water.

Experimental and computational surface hydrophobicity analysis of a non-enveloped virus and proteins

The physical characteristics of viruses needs to be understood in order to manipulate the interaction of viruses with host cells, as well as to create specific molecular recognition techniques to detect, purify, and remove viruses. Viruses are generally believed to be positively charged at physiological pH, but there are few other defining characteristics. Here, we have experimentally and computationally demonstrated that a non-enveloped virus is more hydrophobic than a panel of model proteins. Reverse-phase and hydrophobic interaction chromatography and ANS fluorescence determined the experimental hydrophobic strength of each entity. Computational surface hydrophobicity was calculated by the solvent exposed surface area of the protein weighted by the hydrophobicity of each amino acid. The results obtained indicate a strong correlation between the computational surface hydrophobicity and experimentally determined hydrophobicity using reverse-phase chromatography and ANS fluorescence. The surface hydrophobicity did not compare strongly to the weighted average of the amino acid sequence hydrophobicity. This demonstrates that our simple method of calculating the surface hydrophobicity gives general hydrophobicity information about proteins and viruses with crystal structures. In the process, this method demonstrated that porcine parvovirus (PPV) is more hydrophobic than the model proteins used in this study. This adds an additional dimension to currently known virus characteristics and can improve our manipulation of viruses for gene therapy targeting, surface adsorption and general understanding of virus interactions.

Adsorption of a non-enveloped mammalian virus to functionalized nanofibers.

In the pursuit of finding superior methods to remove pathogens from drinking water, this study examines the adsorption of a non-enveloped, mammalian virus to highly charged nanofibers. N-[(2-Hydroxyl-3-trimethylammonium) propyl] chitosan (HTCC) nanofibers were synthesized by the addition of a quaternary amine to chitosan. HTCC was blended with polyvinyl alcohol (PVA) to produce nanofibers by electrospinning. The nanofibers were stabilized against water by crosslinking with glutaraldehyde. When studied in the range of 100-200nm in diameter, larger fibers were able to adsorb about 90% more virus than smaller fibers. The kinetics of the adsorption was modeled with pseudo-first order kinetics and equilibrium was achieved in as little as 10min. Equilibrium adsorption was modeled with the Freundlich isotherm with a Freundlich constant of 1.4. When the Freundlich constant deviates from 1, this demonstrates that there is heterogeneity at the adsorption surface. The heterogeneity likely occurs at the nanofiber surface since a polymeric blend of two polymers was used to electrospin the nanofibers. The model mammalian virus, porcine parvovirus (PPV), has a fairly homogeneous, icosahedral protein capsid available for adsorption. The fast adsorption kinetics and high capacity of the nanofibers make HTCC/PVA a potential filter material for the removal of pathogens from drinking water.

Enveloped virus flocculation and removal in osmolyte solutions.

Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a >80% removal with a 0.2 μm microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, this method has the potential to be a future platform purification process for vaccines.

A study on the cytotoxicity of carbon-based materials

With an aim to understand the origin and key contributing factors towards carbon-induced cytotoxicity, we have studied five different carbon samples with diverse surface area, pore width, shape and size, conductivity and surface functionality. All the carbon materials were characterized with surface area and pore size distribution, X-ray photoelectron spectroscopy (XPS) and electron microscopic imaging. We performed cytotoxicity study in Caco-2 cells by colorimetric assay, oxidative stress analysis by reactive oxygen species (ROS) detection, cellular metabolic activity measurement by adenosine triphosphate (ATP) depletion and visualization of cellular internalization by TEM imaging. The carbon materials demonstrated a varying degree of cytotoxicity in contact with Caco-2 cells. The lowest cell survival rate was observed for nanographene, which possessed the minimal size amongst all the carbon samples under this study. None of the carbons induced oxidative stress to the cells as indicated by the ROS generation results. Cellular metabolic activity study revealed that the carbon materials caused ATP depletion in cells and nanographene caused the highest depletion. Visual observation by TEM imaging indicated the cellular internalization of nanographene. This study confirmed that the size is the key cause of carbon-induced cytotoxicity and it is probably caused by the ATP depletion within the cell.