Team:UofC Calgary/Integrated Practices
Integrated Practices
Developing a Project Idea
While researching potential topics for the season, the team was introduced to soybean-derived radioprotective peptides by Dr. Aaron Goodarzi, a professor at the Charbonneau Institute of Cancer Research. Dr. Goodarzi shared with us the importance of these peptides, specifically the Bowman Birk Protease Inhibitor (BBI), by emphasizing the damaging effects of ionizing radiation, explaining that the largest sources of human exposure to ionizing radiation on earth result from radiotherapy and imaging.
In order to learn more about the significance of this issue in the medical field, we interviewed many professionals who either utilize ionizing radiation or are exposed to it in their daily lives.This included meeting with researchers, radiation oncologists, medical physicists, and a radiation safety officer. Additionally, we received a tour of the Tom Baker Cancer Centre at Foothills Hospital in Calgary. This is where most cancer research in southern Alberta is conducted, and from our tour we began to understand the process of receiving ionizing radiation in radiotherapy. These consultations revealed that ionizing radiation poses only a small risk in medicine for patients and staff, and that the rate of cancer cases caused by medical uses of ionizing radiation is very low. The engineered controls in medical facilities are also both cost-effective and efficient protection methods. From these conclusions, we began researching other sources of ionizing radiation. We found another area of exposure to high ionizing radiation: space! From NASA’s report for the Mars Mission for 2035, an astronaut will face 1200 mSv of ionizing radiation on a three-year mission – the equivalent of 120 full body CT scans. This limits the amount of time an astronaut can spend in space in their lifetime. Although NASA is currently researching methods of shielding astronauts from most of the radiation, many were not cost- or space-effective. We decided to shift the focus of our project from radiation therapy to long-term space travel.
Conceptualizing a Biological and Physical Delivery System
When conceptualizing a project design, we realized through our research that there were multiple design requirements specific for use in space. Our system had to be lightweight to reduce the launch cost, sustainable to reduce the amount of waste produced, and able to be stored for the entire duration of a mission if need be. Biologically, the system had to continuously deliver our modified radioprotective peptide, mBBI, into the bloodstream at a high enough concentration to confer radioprotection even under changing environmental conditions. With these specifications, we designed a biological system that would use bacteria to continuously produce mBBI, meeting our biological requirements. Drawing inspiration from previous NASA inventions such as the biocapsule, we designed our first patch system. It was portable, durable, and would facilitate the diffusion of mBBI through a microneedle array directly into the body.
Chassis and Wet Lab Experimentation
Our bacterial chassis of choice, which would deliver mBBI to the user, also needed to fulfill several specific requirements. One of the largest concerns for the chassis was whether it could be stored for long durations. After discussions with previous UCalgary iGEM team members and research into many bacteria, we narrowed the choices down to Bacillus subtilis and Deinococcus radiodurans. B. subtilis provided easy integration and sporulation, while D. radiodurans had high ionizing radiation survivability. B. subtilis was chosen because it met all of our requirements: it is known for its biocompatibility with humans, chromosomal integration capabilities, robust spores that resist high levels of radiation, and the ability of these spores to survive long-term storage under extreme conditions. To understand what effect changing the environmental conditions had on the production of mBBI, a growth assay was performed to simulate the temperature conditions of B. subtilis in our patch. Bacterial growth in 10 mL of Luria-Bertani (LB) broth over the course of 24 hours at varying temperatures of 35°C (average skin temperature), 22°C (average room temperature on board the International Space Station) and 4°C (negative control) were tested.
Physical Delivery
For the initial physical delivery system, we decided on a microneedle array for the method of delivery. In the initial design, the patch contained a backing layer that protected the contents from external factors and contamination, a drug reservoir containing bacteria and growth media, and a size- and rate-controlling membrane that let peptides through while holding bacteria back. The patch design with use of microneedles would allow direct delivery of the peptide, ease of application by the user, and easy storage due to its small size and weight. After discussions with Dr. Colin Dalton, a professor researching microneedle arrays, we learned that microneedles are used to disperse the force of injections over a larger area and are not intended for use for long durations as we had initially planned. Keeping the microneedles in the skin long-term can lead to infection and skin irritation, and may result in microneedles breaking and becoming imbedded in the skin. Wanting to ensure ease of application and maintain long-term wearability, we kept the general design of our patch but removed the microneedle array. In its place, we integrated an adhesive layer that would stick to the skin for a duration of seven days.
Looking Forward: Manufacturing
To simulate the patch in real conditions, we furthered our design and researched materials for testing. By better understanding the behavior of our bacterial cells within the material, we could optimize the material for cell growth and production. The requirements for our bacterial chassis also influenced the materials we used such as the rigid backing layer to prevent breakage and cellular leakage, and the adhesive layer that provides flexibility for movement. To determine what types of materials were suitable for our purposes, we contacted representatives from 3M and Dow Corning, two companies that specialize transdermal drug delivery. Through our discussions, we gained a better understanding of the materials currently used for transdermal drug delivery and how we could modify them for our use, and we changed our design based on the best available materials.
In addition to researching materials, we looked into small-scale production to see if our device could be manufactured at an effective cost. We met Dr. Uttandaraman (U.T.) Sundararaj, a professor in polymer manufacturing, and learned about methods used in industry for using common polymers. One suggested method was thermoforming, which required the use of a two-part mold and heating our materials to conform to the mold's shape. By injecting the media between the molds and heating sealing them, we could quickly produce many patches for applications. We also talked to Dr. Amir Sanati Nezhad, a professor on micro and nanotechnology, to better understand the manufacturing of technologies similar to ours. From these interviews, we developed a small-scale mold to manufacture patches for the use in our mouse study.
Feedback
The results of the initial growth curves showed that B. subtilis could sustain itself for 16 hours with the initial quantity of media in the patch (10 mL). This, however, did not meet our requirement for sustainability, as nearly 2 patches per astronaut per day would need to be transported into space.
To extend the lifetime of the patch, we modified its design to incorporate poppable pockets filled with extra media. When growth of the cells begins to decline, the user can pop a pocket that will release new media to provide additional nutrients. To determine how much longer we could extend the lifetime of our patch, we redesigned the growth curve assays with the help of Dr. Sui-Lam Wong to compare bacterial growth in different types of media. By testing the different media and inducing further growth by adding one mL of the given media, we optimized the growth of the cells in the patch environment. Given the change in the delivery design from a microneedle array to an adhesive patch, we needed a new method for direct delivery of mBBI into the body. Therefore, a transdermal tag (TD1) was added to the N-terminus of mBBI to facilitate its diffusion through intact skin.
Addressing Issues Outside of the Lab
Whilst we made modifications to our designs in the lab, we also researched how our device could be integrated into society given the current political and economic landscape. In our research of biotechnology-related regulations in Canada and at the international level, we found that there are many gaps in current regulations concerning the use of engineered organisms in drug applications. This made it very difficult for us to find applicable guidelines for our product had we wanted to bring our device to the market.
In order to remedy this problem, we worked with Dr. Agnes Klein, the Director of the Centre of Evaluation for Radiopharmaceuticals and Biotherapeutics at Health Canada, to develop a policy brief addressing some of the regulatory gaps we observed in current Canadian regulations.
You can find our policy brief here.
