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 radio protective peptides by Dr. Aaron Goodarzi (a professor at the Charbonneau Institute of Cancer Research). Dr. Goodarzi mentioned the importance of these peptides by emphasizing the damaging effects of ionizing radiation and he stated that the largest sources of ionizing radiation on earth are in 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 where most cancer research in southern Alberta is conducted 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 number of cancer cases caused by medical uses of ionizing radiation is also very low. The engineered controls in medical facilities are also both cost effective and work well. From these conclusions, we began researching other sources of ionizing radiation and found that another area of high ionizing radiation exposure was in 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. Although NASA was 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 space travel.
Conceptualizing a Biological and Physical Delivery System
When conceptualizing a project design, we realized through our research there were multiple design requirements that were specific for use in space. The 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 BBI 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 BBI, meeting our biological requirements. Drawing inspiration from previous NASA inventions such as the biocapsule, we designed a patch system that was portable, durable and would facilitate the diffusion of BBI through a microneedle array directly into the body.
Chassis and Wet Lab Experimentation
Our choice of chassis to deliver the BBi peptide 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, we narrowed the choices down to B. subtilis and D. radiodurans. B. subtilis provided easy integration and sporulation, while D. radiodurans had high desiccation survivability and naturally competent. B. subtilis was chosen for its biocompatibility with humans, chromosomal integration capabilities, robust spores that resist high levels of radiation, and the ability of the spores to survive in long term storage under suboptimal conditions, which met our outlined requirements. To understand what effect changing the environmental conditions had on the production of BBI, 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, a microneedle system array patch was developed. 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 media, and a size and rate controlling membrane that let peptides through while holding bacteria back. The patch designed 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 keep the ease of application, we kept the general design 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 cells within the materials, we can optimize the material for cell growth and production. The requirements outlined were also utilized to find materials such as a rigid backing layer to prevent breakage and an 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 modified our design based on the available materials.
In addition to researching materials, we looked at small scale production to see if our device could be manufactured cost effectively. We met Dr. Uttandaraman (U.T.) Sundararaj, a professor in polymer manufacturing and learned methods used in industry for common polymers. One suggested method was thermoforming which required the use of a two part mold and heating our materials to conform to the molds. By injecting the media between the molds and heating sealing them, we can quickly produce many patches for applications. We also talked to Dr. Amir Sanati Nezhad, a professor on micro and nano technology 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 the bacteria could sustain itself for 16 hours with the initial quantity of media in the patch. This, however, did not meet our requirement for sustainability, as nearly 2 patches per astronaut per day would need to be taken into space.
To extend the lifetime of the patch, we modified the design of the patch to incorporate poppable pockets filled with media. When growth of the cells begins to decline, the user can pop a pocket that will release new media to provide fresh nutrients. To determine how much longer we could extend the lifetime of our patch, we redesigned the growth curves with the help of Dr. Sui-Lam Wong to compare growth with different types of media. By testing the different media and inducing further growth by adding one milliliter 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 a patch, we needed a new method for direct delivery of BBI into the body. Therefore in the genetic constructs for the production and secretion of BBI, a transdermal tag was added to facilitate the diffusion of BBi 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 can be integrated into society given the current political and economic landscape. In our research of biotechnology-related regulations in Canada as well as internationally, we found that there are many gaps in current regulations concerning engineered organisms’ use 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 - to develop a policy brief addressing some of the regulatory gaps we observed in current Health Canada regulations.
