The Beginning of Our Kill Switch Design
Upon deciding to develop biosafety measures, we first considered using antibiotics to clean out the gene-engineered bacteria.
However, after exchanging ideas with Prof. Tsai, he suggested to us that we should not simply use antibiotics to eradicate our engineering bacteria, but should use other measures, such as designing specific nutritional requirements for the bacteria.
Furthermore, Mr. Katsuhito from Tokyo University also reminded us that the gut microbiota is a very delicate system, contributing greatly to the health of not just the intestine, but the whole human body. Therefore, using antibiotics to clean our the gene-engineered bacteria might lead to unwanted result on the human body.
Hence we proposed another design to clean out the LBP in the user’s body, a kill switch design aiming to kill the gene-engineered bacteria when environmental temperatures drop below 25 degrees celsius, which is when it is outside the human body, or when the user takes in L-Arabinose in the purpose of eradicating the bacteria.
From Thrombosis Detection to Deep Vein Thrombosis (DVT) Detection
Another aspect of our project is the thrombosis detection kit we wanted to create. After doing literature research, we decided to use the fibrin degradation product, D-Dimer, as the biomarker for our detection kit. Considering the fact that it could be found in saliva, D-Dimer would make a decent choice for a home thrombosis detection kit.
But after we consulted advice from Dr. Shih-Lin Chang from Taipei Veterans General Hospital, we were reminded that while using D-Dimer as our biomarker may be a feasible measure for thrombosis detection, D-Dimer is in fact more sensitive to deep vein thrombosis (DVT), but not so much for other kinds of thrombosis. He suggested that we narrow down the claimed target of our thrombosis detection kit from “thrombosis” to just “deep vein thrombosis”, and so have we heeded his advice.
The Inspiration to Do a Community Education
Faced with serious population aging problem, Taiwan is in urgent need of a thorough, detailed, and systematic long-term elderly care plan, including the the recruitment of personal care assistants and the establishment of full-day elderly care insitutions, especially for bedridden patients who encountered loss of mobility, relied on nasogastric tubes for food nutritions, or diagnosed with dementia therefore required 24hr care due to safety concerns.
The above-mentioned problems were critically pointed out during our interview with Mr. Yu, the chief of Yi-Ching-Yuan Long-Term Care Center, and he also told us that cardiovascular diseases ought to be early prevented so that the negative impact can be minimized. Moreover, we learned the significance of deep vein thrombosis (DVT) through our interview with Dr. Chang, and that it is actually a fatal disease with severe complications such as pulmonary embolism (PE) but hasn’t received enough attention yet.
Hence, we were greatly motivated to raise the awareness of thrombosis, and conducted a health education for the elderly community by giving out healthcare leaflets focusing on DVT.
Also, we learned the deeper meaning underlying our project - which we did not fully recognize at first - that by producing a home test and aid kit featuring the Nattokinase LBP, we can greatly reduce the thrombosis risk and improve the quality of their life in the later years through early prevention and close monitor of the cardiovascular health of the elderly community.
A Huge Change in Our Oral Delivery Design - 4 Contributing Events
the interview with Prof. Devarajan
the meeting with Biorich Bio Technology
the meeting with MegaPro
the public survey conducted in National Yang Ming Chiao Tung University (NYCU)
The main goal of our oral delivery system is to protect our E. coli
Nissle 1917 (EcN) safely into the small intestine. Before entering the small intestine, orally consumed products must face the acidic environment of the stomach, digestive enzymes, host immune system, bile salts, and so on. The harsh environment of the GI tract would be the main obstacle we have to overcome.
We first invented an idea of encapsulating our engineering bacteria with alginate beads coated with PEGylated chitosan; however, after discussing with experts and asking public preference, our probiotic oral delivery system has undergone significant modifications.
From Powder to Capsule
The original idea of our oral delivery system (click me!)
We first came up with an idea of encapsulating EcN into alginate beads coated with PEGylated chitosan by emulsion-solvent evaporation (ESE) process. Then by lyophilizing the product, shelf storage could be ensured.
Microencapsulation enhances probiotic survival rate in the acidic gut, increases its attachment with intestine mucosa, and maintains cell availability . Accordingly, we thought that microencapsulation might be a perfect way for our delivery system.
When thinking about encapsulation biomaterials, alginate coated with chitosan layer came across our mind: Alginate has been medically used extensively due to its biocompatibility, low toxicity, and relatively low cost . It is stable in acidic environments, and it could pass through the stomach without being degraded . As a result, it is a desirable biomaterial for enteric delivery. Chitosan has been considered as a promising drug coating recently due to its non-toxicity, enhanced bioavailability, mucoadhesive property, permeability, and prolonged target residence time . It is also a common material coated outside of alginate microspheres of controlled drug delivery systems to enhance its stability and cell viability. Alginate coated with chitosan layer is the most widely used method for probiotic fourth generation encapsulation [2, 3]. Studies have shown that alginate-chitosan encapsulation of probiotics help maintain high stability in low pH environments, and ensure cell viability [3, 4, 5].
Nonetheless, alginate-chitosan oral delivery system targets cells to the colon while we hope that Natto It Out targets the small intestine. In order to solve this problem, we included PEG (polyethylene glycol), since studies have shown that high surface density of low molecular weight PEG or high molecular weight PEG perform high mucus penetrating effects . This encapsulation method involves coating alginate microspheres with PEGylated chitosan, and it would be in powder form. This encapsulation method has been used for tissue engineering and oral drug delivery, for example, encapsulation of islet cells for bioartificial pancreas, encapsulation of liver cells for direct liver transplant, encapsulation of NPs-PEG-FA/6-shogaol in treatment of colitis [7, 8].
First, we discussed with Prof. Devarajan. Although “alginate coated with PEGylated chitosan” might be a feasible idea, Prof. Devarajan thought that this encapsulation idea would be too complicated for our project, "I think that your idea might be feasible; however, this encapsulation method is too complicated for your project, simply loading your probiotics into capsules could still protect EcN against gastric fluid and reach your oral delivery goals.”
Then, we asked for second opinion by interviewing MegaPro. They also recommended that we simplify our encapsulation idea. From the perspective of experimental verification, our project has already involved quite a lot of innovative designs, including gene-edited E. coli
Nissle 1917, detection system, and optogenetic system. The more novel designs were put in, the harder it would be to confirm the overall result. That is, it would be harder to confirm which part of the design contributes to unexpected experimental results.
As a result, we decided to change our oral delivery method to simply load the lyophilized EcN into enteric coated capsules.
Verifying the Public’s Preference for Capsules
To verify the public's preference toward capsules, we performed a public survey, and met up with Biorich Bio Technology Co., Ltd. and MegaPro Biomedical Co., Ltd. During this process, we were able to not only stand from the perspectives of consumers, but also companies.
As shown below, Chart 1
indicated that most of the respondents preferred capsules to powder and tablets. It is mentioned that venous thrombosis risk increases rapidly at age 40 and peaks at age 60-70 . We also wanted to know the oral delivery preference of the over 40s, the target audience of Natto It Out product. Chart 2
represented the preference of oral delivery for Natto It Out product of people above 40 that filled out our survey. According to the pie chart, people above 40 also preferred capsules most. Capsules align with the consumer’s preference. During our interview with Manager Wang of Biorich Bio Technology, Mr. Wang also pointed out that in terms of efficiency, capsule forms are better than powder forms, and that most of the consumers prefer capsule form to powder form probiotics.
Therefore, after listening to opinions and advice from experts and the public, we decided to utilize enteric coated HPMC capsules as our oral delivery system.
Chart 1. Public survey result of people’s preference of our Natto It Out delivery form. [n=163; capsule: 92/163 (56%); powder: 11/163 (7%); tablet: 23/163 (14%); No certain preference: 37/163 (23%)].
Chart 2. Public survey result of the preference of the over 40s for the form of the drug delivery system. [n=48; capsule: 23/48 (48%); powder: 3/48 (6%); tablet: 7/48 (15%); no certain preference: 15/48 (31%)].
From the original idea to the final plan, our oral drug delivery system has come a long way. It had gradually taken shape through our continual discussion with experts, modified from trying to encapsulate our product in alginate coated with PEGylated chitosan, to encapsulate them into hard HPMC enteric coated capsules after listening to experts and the public.
 Mawad, A., Helmy, Y.A., Shalkami, AG. et al. E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro. Appl Microbiol Biotechnol 102, 10675–10690 (2018).
 Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Prog Polym Sci. 2012 Jan;37(1):106-126. doi: 10.1016/j.progpolymsci.2011.06.003. PMID: 22125349; PMCID: PMC3223967.
 Salas-Jara, M. J., Ilabaca, A., Vega, M., & García, A. (2016). Biofilm Forming Lactobacillus: New Challenges for the Development of Probiotics. Microorganisms, 4(3), 35. https://doi.org/10.3390/microorganisms4030035
 Chavarri M, Maranon I, Ares R, Ibanez FC, Marzo F, Villaran Mdel C. 2010. Microencapsulation of a probiotic and prebiotic in alginate-chitosan capsules improves
survival in simulated gastro-intestinal conditions. Int J Food Microbiol
 Shori, AB. Microencapsulation Improved Probiotics Survival During Gastric Transit.
Trends in Food Science & Technology 24(1), 1-5 (2017). https://doi.org/10.1016/j.tifs.2007.01.004
 Wang YY, Lai SK, Suk JS, Pace A, Cone R, Hanes J. Addressing the PEG mucoadhesivity paradox to engineer nanoparticles that "slip" through the human mucus barrier. Angew Chem Int Ed Engl. 2008;47(50):9726-9. doi: 10.1002/anie.200803526. PMID: 18979480; PMCID: PMC2666733.
 Haque, T., Chen, H., Ouyang, W., Metz, T., Lawuyi, B., & Prakash, S. (2005). Effect of Integrating Polyethylene Glycol to Alginate-Poly-L-Lysine and Alginate Chitosan Microcapsules for Oral Delivery of Live Cells and Cell Transplant for Therapy.
 Zhang M, Xu C, Liu D, Han MK, Wang L, Merlin D. Oral Delivery of Nanoparticles Loaded With Ginger Active Compound, 6-Shogaol, Attenuates Ulcerative Colitis and Promotes Wound Healing in a Murine Model of Ulcerative Colitis. J Crohns Colitis. 2018 Jan 24;12(2):217-229. doi: 10.1093/ecco-jcc/jjx115. PMID: 28961808; PMCID: PMC5881712.
 Cushman M. (2007). Epidemiology and risk factors for venous thrombosis. Seminars in hematology, 44(2), 62–69. https://doi.org/10.1053/j.seminhematol.2007.02.004