Team:UofC Calgary/HP/Gold
HP - Gold
Device
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Colin Dalton
Adjunct Professor, Facility Manager of Advanced Micro/Nano Integration Facility (AMIF)
We initially contacted Dr. Dalton to get his opinion on our first draft design of the patch. The main concern he had was with our proposed use of microneedles. Incorporating microneedles into the design can cause the rate of diffusion of the peptide out of the patch to decrease, meaning additional mechanisms such as pumps would need be used. More importantly, microneedles are meant for short term delivery and the continuous usage could cause irritation and inflammation. Microneedles are fragile, and there is a risk that they will break off and become imbedded in the skin if pressure is applied to the patch or the patch moves.
We decided to NOT pursue our microneedle design because it defeats our goal to have a transdermal drug delivery system for long term use. We decided instead to design a transdermal reservoir-adhesive patch to deliver peptides into the body. Our conversation with Dr. Dalton became one of the major turning points in this project.
- Amir Sanati-Nezhad
Professor, Schulich School of Engineering
Department of Electrical and Computer Engineering
Dr. Sanati-Nezhad invited us into his lab where microfluidic systems or “labs-on-a-chip” are being studied. We discussed the difficulty of creating devices incorporating living cells, because the cultures must be supplied with food and waste must be removed. This requires a method to transfer materials in and out of the system. We looked into this problem, and decided that we would design the patch to have pockets containing media that could be supplied to give the cells fresh media and dilute waste to extend the lifetime of the patch.
These concepts directly apply to our project as we are dealing with living organisms and must ensure they are are not deprived of nutrients, as this will result in lowered peptide production. He stressed the importance of drafting up a prototype of our design in AutoCAD for better understanding of our design.
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Uttandaraman (U.T.) Sundararaj
Professor, Schulich School of Engineering
Department of Mechanical and Manufacturing Engineering
Dr. UT brought up three key considerations for our patch: safety, choice of materials, and manufacturing. He suggested that gel media might be a better choice than liquid media in case of puncturing or storage issues, however, this would lead to diffusion being compromised. Overall, he said that the flux of materials should be the same in equilibrium. We decided on a liquid media because it is better for cell survival, and if the cells are not producing peptides, it doesn’t matter how stable or durable the patch is.
He also approved our materials and recommended us to consider the solubility parameters of the materials to ensure compatibility of materials involved in the system ie. nothing will dissolve into each other when heated.
Lastly, he recommended us to use the process of thermoforming to manufacture mouse patches. The general idea is to make a mold made of wood or metal and form the materials into it with the use of heat. The molds are shaped as reservoirs or sinks. Once the material has been formed to the mold, media is added into the sinks. Heat will be applied once again with the covering material to seal everything together. We incorporated this suggestion into our mouse study.
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Robert Mayall
Biotarget
BBI is 3 kDa big
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Dr. Ebba Kurz
Oncologist, University of Calgary
Dr. Kurz suggested several cell lines we could use to perform our clonogenics assay: MCF -7 (Breast), U205 (Osteosarcoma) and HCT116. All of these tissue cell lines lie flat, which made them ideal for running Clonogenics and the H2AX Assays. We chose to work with the HCT116, as we had already started to work with them for the clonogenics assay.
HCT116 cells are irradiated in space, but not in cancer treatments. Because we had decided to switch the focus of our project from cancer to space travel, these cells fit well for our purposes.
The other cell lines that were mentioned by Dr. Kurz were taken into consideration for the H2AX and FAC assays. The FAC assay will be performed in the near future.
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Aaron Goodarzi
Assistant Professor, University of Calgary
Dr. Goodarzi introduced us to BBi and its ability to enhance DNA repair mechanisms, and acted as an advisor/mentor as we performed our clonogenics and H2AX assays. He helped us to troubleshoot our experiments, and based on his advice, we decided to switch from the HCT116 cell line to 1B3 for the H2AX assay. Dr. Goodarzi suggested, based on the results of our first round of assays, that HCT116 would not be the most suitable cell line because it is a cancer cell line and so cuts out downstream signalling, causing BBi to exert only weak effects.
Dr. Hans Vogel
Adjunct Professor, Biochemistry & Molecular Biology
University of Calgary
Dr. Vogel highlighted several possible safety issues relating to our project. These included the possibility of an immunological response to Bowman- Birk Inhibitor (BBI), which was originally derived from Glycine max. (soybeans), or filtering of the BBi peptide by the Kidney due to its small size. Based on this feedback, an extensive literature search was performed to gain more insight into the possible immunological responses induced by BBI, and the underlying mechanisms. A possible solution that could be implemented to minimize the adverse effects of BBI is to introduce BBI to Astronauts at increasing concentration over time to monitor and minimize immunological responses. She suggested we consider yeast as an alternate chassis, but we decided to continue with Bacillus subtilis because of its robustness, ability to form spores, and the availability of protease knockout strains that allow for increased peptide secretion.
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Dr. Susan Lees Miller
Professor, Departments of Biochemistry and Molecular Biology, Oncology and Biological Sciences
In order to gain more insight in the possible mechanisms of DNA repair with BBi, we consulted with Dr. Lees-Miller. Dr. Miller familiarized us with some of the mechanisms behind double stranded breaks in mammalian cell DNA, and shared her hypothesis for the mechanism of action of BBi.
Dr. Miller also provided advice to help us design, perform, and troubleshoot for our clonogenics and H2AX assays. She emphasized the importance of dose rate, and suggested we conduct assays looking into the autophosphorylation site 2056 in DNA PKCs to see if ionizing radiation activates DNA PKC activity. She hoped this may help us learn more about the mechanism of BBi.
Another key issue was determining BBI levels in blood, comparing it to the mathematical models, and using the data to improve our patch design so it would deliver the desired dosage consistently. In order to measure the levels of BBI in the blood, the team agreed to carry out in vivo testing with mice models. Dr. Miller confirmed that we can look at the serum from blood samples and use mass spectrophotometry or NMR/ gas chromatography to detect BBI. She cautioned us that if there were proteases present in the samples, it would change the predicted mass of BBI, so we would need to consider peptide degradation rates .
Chassis
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Dr. Sui-Lam Wong
Microbiology Professor, Specialization with Bacillus subtilis
University of Calgary
One of the key aspects of our project was the determination of an ideal chassis. The choices of potential chassis the team considered were Bacillus subtilis WB800, Deinococcus radiodurans, Escherichia coli, and Saccharomyces cerevisiae. After weighing the pros and cons for each with the help of Dr. Wong, the team reached a consensus that B. subtilis WB800 was the most appropriate choice as the chassis for our project. It is biocompatible, robust, and suitable for long term storage due to its ability to form spores. Several protease knockout strains were available for our use, and the mechanisms for chromosomal integration and peptide secretion are well understood.
Dr. Wong aided us throughout the project, providing advice on working with bacillus subtilis and allowing us to use his protocols.
Human Practices
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Agnes Klein
Director of Centre of Evaluation of Radiopharmaceuticals and Biotherapeutics
Health Canada
Dr. Agnes Klein was instrumental in helping to shape the direction of our policies and practices work. First, she helped us gain insight into the steps and studies necessary before you bring a new drug product to market. Secondly, she suggested we consider writing a policy brief.
We realized through our research that there are no distinct drug regulations for recombinant organisms (as opposed to the genetic engineering guidelines for the food sector) in Canada. We planned to address this shortcoming by writing up a policy brief outlining the importance of regulation for cell based therapeutics and share this document with Health Canada to advocate for improved regulations and policies. We will also publish this policy brief so that we can increase awareness for the emerging field of cell based therapeutics and the regulatory challenges it brings.
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Terry Johnson
Former iGEM Judge and Chemical Engineer
From a Skype conference with Terry Johnson, it was clear that we would need to focus on how we would ensure our patch was safe for human use, as well as the issues of safe disposal and prevention of colonization of foreign planets by engineered cells. Based on Terry Johnson’s feedback, we decided to implement double auxotrophies through the knockout of genes encoding the essential amino acids threonine and tryptophan in B. subtilis WB800. This would ensure that if the bacteria was accidently released from the patch the bacteria will not survive on human skin or in the environment. Creation of these double auxotrophic strains is still in progress. In addition to biological controls, we also incorporated engineering controls in the design of the transdermal patch. The patch was designed to be flexible to reduce strain caused by movement, and designed to distribute applied pressure evenly over the surface to prevent the patch’s seams from opening. The choice of a patch over an implant also increases safety because it is less invasive.
Based on Terry Johnson’s feedback on safe disposal of our transdermal patch, we created a manual for our patch that outlines the safety controls, design features, and procedures for safe disposal. We also decided to incorporate mathematical modelling into our design at his suggestion to illustrate the pharmacokinetics of BBi and the patch to demonstrate its safety in comparison to alternate methods such as injections or implants.
We have addressed this key issue in _____________________ page. (INSERT hyperlink) where a mathematical model was designed to determine the initial concentration of BBI needed in the patch and the production rate that we needed to maintain for constant delivery.
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Corinne Doll
Oncologist at Tom Baker Cancer Centre
Calgary
The modularity of our transdermal patch made it difficult for us to decide which application our patch was more suitable for: space or cancer. After the interview with Corinne Doll, it was clear that our transdermal patch was more applicable in space for providing radioprotection to Astronauts. She told us that it would be difficult to localize BBI to healthy cells without also affecting cancerous cells and conferring radiation protection to them, thereby reducing the effectiveness of treatments (we want double strand breaks to persist in the DNA of cancerous cells in order to kills them and reduce the adverse effects of the therapy on non-cancerous cells by enhancing DNA repair mechanisms). As well, she mentioned that each Cancer patient has a unique gene signature. Therefore, it will be difficult to make sure that our patch works for all Cancer patients. We confirmed based on these considerations that we should focus on designing our patch for applications in space travel rather than cancer treatment, and target our design for astronauts.
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Eduardo and Nicholas
During our tour of the Tom Baker Cancer Centre, we learned about current radiation therapies, and how patients and staff are protected from radiation damage. Although radiation damage is a concern in diagnostics and cancer treatments, most patients and staff are not at a significantly higher risk for radiation damage than the general population. We realized that focusing on cancer treatment as the main application of our product may not be useful, because few patients would benefit, and the dangers of radiation therapies were not as significant as we first believed. We decided following this tour to focus on space travel as the primary application of our product, although future medical applications would still be possible due to the modularity of the patch.
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Ruth Wilkins and Lindsay Beaton
Health Canada: Radiation Department, Work with Astronauts
Both Ruth Wilkins and Lindsay Beaton thought our project was a novel and modular approach to radiation protection. Currently, the maximum amount of time astronauts can spend in space is 6 months. Our product would increase this time and improve space missions. They confirmed for our clonogenic assays that small doses over a long period of time is not equivalent to a large dose all at once, and that cosmic radiation is not the same as gamma radiation to help us design our clonogenic and H2XA assays. When performing our clonogenics and H2AX assays, we irradiated the tissue cell lines, HCT116 for clonogenics and 1BR3 for H2AX, with gamma radiation. In the future, it will be useful to irradiate the tissue cells lines with cosmic radiation to gain a better understanding of BBI function in space. In addition, this interview further strengthened our decision to focus on space application of our transdermal patch rather than Cancer.
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Jahan Quaji
Radiation Safety Officer at the Tom Baker Cancer Centre
The interview with Jahan Quaji assisted the team in choosing to pursue space over cancer as the most applicable use of our transdermal patch. She confirmed with us that current methods of radiation protection are usually sufficient to protect patients and staff from any harm, and that cells are given time to recover naturally between doses in radiation therapy, so the risk of significant DNA damage is very low. In space, there is a much higher dosage of radiation and no rest period between periods of exposure, so cells have no chance to recover.
In vivo Testing
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Craig Jenne
Immunologist, Department of Microbiology, Immunology, and Infectious Disease (MIID)
With our interview with Dr. Jenne, he mentioned a few considerations that we need to consider; such as, skin irritation, possible immunological responses related to the transdermal patch materials and BBI, similar to what Dr. Vogel brought up. In order to address these issues, we performed an extensive literature search on the properties of the materials used for the manufacture of the transdermal patch, and confirmed that the materials are safe for administration. Also, he mentioned alternative modes of drug delivery like injections, similar to what Terry Johnson brought up. In order to address this, we looked into the advantages and disadvantages of all the possible modes of drug delivery. Based on this, it was more beneficial to have a transdermal patch due to the pharmacokinetics, and low degree of invasiveness, and lower degradation levels of bioreactor.
Dr. Jenne offered to mentor some team members for ethics approval, creation and implementation of mice protocols, and mice handling for in vivo testing of our transdermal patch in mice models.
Refer to this document for further info on how interviews influenced our project:
https://docs.google.com/spreadsheets/d/1Gcm7inuUyQO-IqK-th1-mKubIl9_sKXgepbOIUt0s2U/edit#gid=0
