Katarzyna M. Sawicka

Protective antigen composite nanofibers as a transdermal anthrax vaccine

Anthrax, a disease caused by the gram positive bacteria Bacillus anthracis, has become an increasing threat to public health in the last several years, due to its use as an agent of biological warfare. The currently utilized human anthrax vaccine, which confers immunity through the host antibody recognition of protective antigen (PA), requires a three dose regimen and annual booster shots after the initial vaccination to maintain its efficacy. The long term goal of this project is to produce an anthrax vaccine that is capable of delivering protective antigen through human skin. The novel method for transdermal vaccine delivery that we propose utilizes the high surface area to volume ratio offered by protein-containing nanofiber membranes, prepared by the electrospinning technique. Research has already been undertaken to study the effect the main virulent agent of anthrax, lethal toxin (LT), has on a human monocytic cell line, Monomac 6 cells (MM6). Lethal toxin is said to comprise of a Zn2+-dependent metalloprotease known as lethal factor (LF), and a binding protein known as protective antigen. The successful encapsulation of the protective antigen within the nanofibrous membrane was analyzed with the use of an in vitro MM6 assay. The assay was designed to ensure the functionality of PA through the harsh environment of the electrospinning process. Quantitative analysis of IL-6 cytokine production by lipopolysaccharide (LPS) stimulated MM6 cells in the presence of LF and PA provided proof that PA retained its biological activity through the process of electrospinning. This finding provides an innovative platform for the development of a transdermal anthrax vaccine.

Molybdenum and Tungsten Oxide Nanowires Prepared by Electrospinning

Tungsten oxide (WO 3 ) and molybdenum oxide (MoO 3 ) nanowires were synthesized through utilizing sol gel and electrospinning methods. Mixtures of metal oxide sol gel and polyvinylpyrrolidone (PVP) in ethanol solution were electrospun and resulted in metal oxide composite nanofiber mats. Precise annealing process removed all organic material, and pure metal oxide single crystal nanowires remained. Both the as-spun nanocomposite mats and the heat-treated nanofiborous materials were characterized using Scanning and Transmission Electron Microscopes. The average diameter of the nanofibers was concluded to be proportional to the flow rate used and inversely proportional to the metal oxide concentration in the solution.

Encapsulation within nanofibers confers stability to the protective antigen protein

The current vaccination paradigm for the prevention of anthrax is insufficient to deal with a potential, widespread epidemic. To solve this issue, we propose a self-administrable vaccine patch capable of delivering the antigen of interest into the skin. This patch is comprised of solid-state nanofibers containing encapsulated protective antigen (PA), a binding protein secreted by Bacillus anthracis. Polyvinylpyrrolidone (PVP) nanofibers, produced by the electrospinning technique, are utilized as our transdermal delivery vector because the high surface area to volume ratio that they afford maximizes contact with the skin; in turn increasing local concentration gradients of reversibly packaged PA when the hygroscopic PVP is solubilized via transepidermal water loss. Previous studies have confirmed the retention of PA immunoreactivity and functionality after the voltage-intensive electrospinning process. The study described here aimed to compare the retention of PA functionality within the nanofibers to PA in solution over a several month period; since it is theorized that encapsulation within nanofibers may confer protein stability. The functionality of encapsulated PA was retained whereas PA in solution was inactive after a 32 week incubation at 4°C; suggesting that encapsulation within nanofibers confers stability to the PA protein.

Engineering nanofibers for a novel intradermal vaccination method for whooping cough

Current delivery systems for vaccines present major logistical problems when large populations need to be vaccinated within a short time period, such as in the case of a pandemic. An efficient and effective method for pandemic vaccination is the driving force of the research performed by our group. The proposed alternative vaccination technique consists of a self-administrable patch made of electrospun nanofibers, where antigens and adjuvants are imbedded during the electrospinning process. This allows large quantities of immunogens to be encapsulated within the nanofibrous mat. This abstract describes the use of the electrospinning technique for the development of a new intradermal delivery system for biologically functional pertussis toxin (PT) to effectively vaccinate against whooping cough. The functional PT was immobilized within highly hygroscopic polyvinyl pyrrolidone (PVP) nanofibers. To establish the efficiency of the technique to immobilize biologically functional PT, prepared patches were incubated with Chinese Hamster Ovary (CHO, K1) cells. The cellular assay measures the degree of clumping of the natively attached CHO cells upon exposure to biologically functional PT. The simultaneous incubation of diluted stock and electrospun PT allowed for a direct comparison of functional PT concentration. This study has reproducibly demonstrated that approximately 80% of the total PT added to the polymer solution was successfully incorporated into the electrospun mat, and maintained biological functionality.

ArF excimer laser debrides burns without destruction of viable tissue: A pilot study

INTRODUCTION Recent evidence indicates that early removal of eschar by tangential debridement can promote healing. Laser debridement can be used for debridement of areas that prove challenging for debridement using tangential excision. In particular, irradiation with an ArF excimer laser ablates desiccated eschar and is self-terminating, preserving hydrated or viable tissue. METHODS Thermal burns were created on the flanks of two outbred, female Yorkshire pigs using aluminum bars heated to 70°C and applied for different lengths of time. Three days after injury, burns were debrided using an ArF excimer laser (193nm). Tissue was harvested immediately after debridement and 7days after debridement (10days after burn). RESULTS Data from a pilot study demonstrates that ArF excimer laser irradiation removes burn eschar and promotes healing at 10days after burn. ArF excimer laser debridement is self-terminating and preserves underlying and adjacent perfused tissue. Potentially, this modality would be ideal for the complex curvilinear structures of the body.

Optimization of the electrospinning process parameters for a pandemic vaccine patch

A skin patch composed of an electrospun nanofibrous membrane of a highly hygroscopic polymer, encasing a reversibly packaged antigen and adjuvant cocktail, promises a practical alternative to the current vaccine strategy. The proposed system would utilize the high density of antigen presenting (APC) cells found within the layers of human skin to elicit a vast adaptive immune response. The amount of contact between patches and skin has been said to directly affect the efficiency of load delivery. Incorporation of the immunogens often requires use of aqueous solvents associated with the beads-on-the-string morphology formation, which hinders the high surface area to volume ratio afforded by the electrospun membranes. The long-term goal of this research is to optimize the solution and process parameters to maximize the surface interaction with the skin for improved delivery of immunogens. In this study we attempt to utilize a novel method of morphology control through employment of the electrospinning process pausation. The scanning electron microscopy (SEM) examination indicated that stopping the electrospinning process at different intervals and for various durations impacts the generated morphology. The findings indicate that the surface area to volume ratio for the three-dimensional nonwoven membrane can be maximized through utility of process pausing.

Pertussis composite nanofibrous membranes as an acellular transdermal whooping cough vaccine

Whooping Cough has globally resurfaced due to the suboptimal quality of the traditional vaccines, cyclic variations in its pattern and discovery of new strains of the causative agent, Bordetella pertussis. Our studies provide a proof of principle for the development of a novel, solid state vaccine to counter the disease by successfully immobilizing Pertussis Toxin (PT), which is 200 times larger than molecules traditionally delivered through the skin, in electrospun nanofibrous membranes of the polymer, polyvinylpyrrolidone (PVP). The transdermal delivery of the functional protein was verified using an in vitro assay utilizing Chinese Hamster Ovary (CHO) cells. The semiquantitative assay allowed us to estimate the extent of clumping of the adherent cells in the presence of functional PT from the basal media and homogenized constructs of EFT-200 human skin organotypic models (MatTek). The functionality of PT was compared to that of standard solutions of known concentrations as low as 6.25 ng/ml to quantify the findings. The successful delivery of biologically active PT through a model of uncompromised full thickness human skin indicates that the nanocomposite coating is a promising candidate for a novel transdermal vaccine, and may be employed in future strategies that would supplant traditional vaccination methods.

Confirmation of protective antigen functionality in an electrospun, nanofibrous membrane

The use of Bacillus anthracis as a bioterrorist agent, in 2001, resulted in the death of 5 individuals. The pathogenicity of anthrax is due to the proteolytic effects of one of its components: the enzyme lethal factor (LF). LFs substrate is located within the cytosol of cells and its cleavage results in a silencing of the interleukin-6 (IL-6) cytokine signaling pathway. The entry of LF into the cytosol is facilitated by a binding protein: protective antigen (PA). Proposed here is a nanofibrous membrane, containing encapsulated PA, that can function as a transdermal anthrax vaccine. This membrane is produced using the voltage-intensive process of electrospinning. To confirm retention of protein biological activity throughout the process, Mono Mac 6 cells, a human monocytic cell line, were treated with electrospun PA after being activated with lipopolysaccharide (LPS) and dosed with LF. A down regulation of IL-6 production, determined through an ELISA, was used as an indicator for PA function. Functionality of electrospun PA was confirmed and the amount of functioning PA deposited on a 4 mm × 4 mm silicon wafer substrate was quantified using the dose-dependent response between PA present and IL-6 down regulated.

Composite nanofibrous membrane of immunoreactive H5-hemagglutinin as a transdermal bird flu vaccine

H5N1 has been identified as the most pathogenic strain of avian flu, and therefore has been the most studied in prospective vaccine strategies. The grim reality of material and personnel shortage during a pandemic renders all of the currently proposed technologies inadequate for rapid mass vaccination. The long term goal of this project is to develop a transdermal patch containing a nanoencapsulated H5N1 antigen to deliver the immunogen into the antigen presenting cell (APC)-rich epidermal layer of the human skin. This study examines the capacity of the electrospinning method to create a nanocomposite nonwoven mat that effectively encapsulates a peptide derived from the H5-hemagglutinin (HA) and preserves its immunoreactivity throughout the process. The assay we have employed utilizes immunoblotting with a slot blot apparatus; this method has allowed us to confirm immunoreactivity after exposure of HA peptide to the conditions encountered in producing electrospun nanocomposite mats and to quantitate levels of the immunogen on silicon wafer targets we propose to employ for transdermal delivery. Its ease of use and cost effectiveness offers the potential for incorporation of numerous epitopes of a selected antigen in a rapid and efficient manner to create large supplies of effective bird flu vaccines.

Encapsulation of immunoreactive protective antigen within a nanofibrous membrane for use as a transdermal anthrax vaccine

Bacillus anthracis, the bacterium which causes anthrax, has been a source of major concern since the deadly series of contaminated postal letters back in 2001. The three main proteins secreted by the bacterium are Protective Antigen (PA), Lethal Factor (LF), and Edema Factor (EF), and are the cause of its virulent effect. With the impeding threat of bioterrorist attacks, studies have been undertaken to develop an effective anthrax vaccine. The currently proposed technologies suffer from immunity conferred only in a small percentage of subjects, but most importantly the unrealistic method of delivery during a pandemic situation. Our group has proposed to construct a transdermal patch composed of a nonwoven nanofibrous membrane of PA as a self administrable anthrax vaccine prepared by the cost efficient technique of electrospinning. The fabricated water-soluble membrane with a high surface area-to-volume and -mass ratios is expected to allow antigen penetration into the human skin layers, where antigen presenting cells will enable cutaneous immunity. This study utilized a novel immunoblotting assay to investigate the effect of electrospinning process on the proteins immunoreactivity. The developed method allowed for accurate quantification of the immobilized PA deposited on 4mm × 4mm silicon wafers proposed as delivery vehicles.