Overview
Thanks for the inspiration from the exchanges at LZU-China and BUCT! As an old saying goes, the obvious is only obvious when it happens to someone else. We left out some details when designing the project, but other teams provided suggestions and ideas for improvement.
Who are the proposed end users?
Stakeholders
Our project is aimed at all those who care about and value kidney health. After further reflection, we believe that the following are the groups of people who could be the end-users of our program:
- Patients suffering from chronic kidney disease: this group will benefit from our probiotic medicines as they help reduce the burden on the kidneys and promote waste metabolism, which is expected to improve their kidney health.
- High-risk group: people with a family history of kidney disease or other kidney problems, especially those with high-risk factors, can also benefit from our program.
- Health-conscious individuals: Individuals who are concerned about their health and seek ways to take care of their kidneys, including those who are willing to prevent kidney problems, may also be potential end-users.
- Healthcare Professionals: Doctors, nurses, and healthcare professionals may use our probiotic medication in their patients' treatment to provide better kidney healthcare solutions.
Our goal is to provide these users with an effective, convenient, and innovative solution to help them improve their kidney health and enhance their quality of life.
How to use our project?
How do engineered bacteria work?
BUCT wants to create a probiotic that can colonize the intestines for a long period of time to assist the kidneys in their purification function. Our product would continuously absorb metabolic waste from the bloodstream in the intestines and expel it through the intestines while producing nutrients to improve the body's function.
After visiting the Nutrifresh factory in Tianjin, BUCT was inspired by the fact that the form of the Nutrifresh product was very similar to our original concept.
When kidney function is impaired, the metabolic wastes creatinine and urea seep into the gastrointestinal tract, further damaging the body until it breaks down.
But you can try taking our probiotics to tackle these two bad guys, creatinine and urea!
By taking our probiotic bacteria liquid orally, it will cut through the layers of obstacles.
Probiotics will make their home in the gut and take up permanent residence.
Probiotics absorb creatinine and urea, purify the intestinal environment, and produce the beneficial substance sarcosine to energize the body. At the same time, they also produce green and non-polluting polyglutamic acid (γ-PGA), which crosses the intestinal tract and is excreted from the body.
Turn on the toilet water to flush out this metabolic waste.
We have designed a dual biosecurity system of ammonia suicide and oxygen suicide, so you don't have to worry about your toilet being contaminated with probiotics.
Difficulties
The above is the application of the product we envisioned, but it is obvious that it can't be done in a year's time, and it is very difficult to realize it. However, we want our probiotic to benefit kidney patients. To realize our idea, BUCT is trying to solve the difficulties step by step.
Problem 1: How do we get our bacteria through the layers of barriers to reach the gut and keep them alive in sufficient numbers and activity?
The physical and chemical stresses faced by probiotic cells during their entry into the gut are a major obstacle to their survival. The question of how to ensure microbiota abundance and activity in the gut in order to facilitate the enhancement of probiotic drug function has always been a vexing one. For this purpose, we have reviewed a lot of literature and learned about microencapsulated membrane technology. But unfortunately, our lab was not equipped to realize this technology. Later, we went to Jumbo Pharmaceuticals and asked for the relevant knowledge, but we did not get the answer (༼•̀ɷ•́༽) .
However, luckily, we met LZU-China, who realized the capsule embedding experiment, which inspired us. We can adopt their technology to break through this barrier.
LZU-China told us that microencapsulation can be an effective strategy for the industrialization of probiotic drugs.
Sodium alginate has long been used in microencapsulation technology because of its ability to form robust network structures. Alginate/Poly-L-Lysine/Alginate (APA) has been widely used in microencapsulation technology because of its good biocompatibility and biodegradability.
However, the cost of APA is quite high ($300/g) and not suitable for large-scale industrial production. In recent years, researchers have found that replacing polylysine with chitosan can greatly reduce the cost. In addition, ACA is relatively simple to prepare, further simplifying the production process.
The interaction between alginate and chitosan is realized by the attraction between positive and negative charges, which enables the successful encapsulation of probiotic cells.
Literature and experimental results from Lanzhou University showed that E. coli could effectively survive in ACA, so we decided to productize the engineered bacteria using the same microencapsulation technique.
With the help of LZU-China, we did repeated experiments. We have successfully crafted microcapsules, also referred to as ACA, as depicted in the illustrative image below.
In our replicated experiments, it was clearly observed that the optical density (OD) values of E. coli encapsulated by microcapsules increased dramatically compared to those of E. coli without such protective microcapsules. This effect was consistently observed in both a 4% bile salt solution and simulated gastric fluid (SGF). These results confirm the great protective efficacy of the microcapsules against bacteria.
Here are the experimental methods that LZU-China passed on to us. Many thanks to LZU-China!
Experiment
Physiological saline (0.9%), 1.5% sodium alginate, 4% calcium chloride solution, 0.3% chitosan solution, and E. coli.
All solutions for the experiments were prepared in saline.
- Overnight activation of EcN and EcN-lac (2 copies of bacteria) After incubation for the logarithmic growth period, determine the OD value of the strain and record it.
- Centrifuge the two 5 ml bacterial solutions at 6000 rpm for 10 minutes at 4 °C.
- Mix the centrifuged EcN with 50 ml of sodium alginate solution (two) at a concentration of 1.5%, and wait until the air bubbles are eliminated.
- Prepare 200 ml of 0.3% chitosan solution with 1% acetic acid solution, dissolve it fully at 60 °C, and then mix it with 20 ml of 4% calcium chloride solution.
- Aspirate the solution with a syringe (inner diameter 0.5 mm) at 20 cm from the liquid surface and stir while stirring under a magnetic stirrer in the calcium chloride-chitosan mixture (drop acceleration: 40 drops/min).
- Wash the gumballs twice with physiological saline. After that, the gumballs were filtered out to produce sodium alginate-chitosan microcapsules, also called ACA.
- The microcapsules are immersed in a 4% sterilized calcium chloride solution at 4°C for one week. When used, preheat at 37°C for 5 minutes.
In vitro verification
- Prepare 10 ml of 4% bile salt solution, 10 ml of PBS, and 10 ml of SGF solution.
- After measuring the free EcN ODvalue, take 500μl and suspend it with 5ml EcN(C/A)2 in 5ml PBS (control), 4% bile salt solution, and SGF, respectively, and incubate for 2 h at 37 °C with shaking.
- After 2 h, the bacterial solution was aspirated, centrifuged at 6000 rpm for 10 min at 4°C, washed with phosphate buffer, and the OD value measured.
- For EcN and EcN-lac pellets, after mechanical crushing, liquefy with 1.42% sodium citrate. Centrifuge at 1500 rpm/min for 10 minutes, remove microcapsule fragments, collect the bacteriophage, and measure OD.
Take 100 ul EcN-lac, and 1 ml EcN-lac spheres in the test tube, add 5 ml of LB medium, respectively, incubate at 37 °C for 24 hours, and then observe the fluorescence phenomenon
Problem 2: How do we monitor the growth of probiotics in the gut and their ability to purify metabolic waste?
After interviewing doctors, BUCT learned that animal testing is the best test. But it was difficult for us to get that far. Therefore, we wanted to use modeling to make predictions. To get a correct prediction, we need to have the correct equation.
Finding the correct modeling result is our next step.
Security issue
Possible problems:
In terms of safety, based on the information obtained from interviews with different people, we realized that we also need to consider the following safety issues:
1. Stability of probiotics: Probiotics may be affected by environmental factors such as temperature and humidity during preparation, storage, and transportation. This may affect the activity of the probiotics.
Solution: We will take measures to ensure the stability of probiotics, including the use of appropriate packaging and storage conditions to minimize the impact of the external environment on product quality.
2. Antibiotic resistance: Probiotics may develop resistance to antibiotics during long-term use, which may result in reduced therapeutic efficacy.
Solution: We will ensure that probiotics are used appropriately and avoid unnecessary long-term use to minimize the risk of antibiotic resistance. In addition, we will monitor the sensitivity of probiotics regularly.
3. Abnormal reaction: Some people may have an uncomfortable reaction to probiotics, such as an allergic reaction, gastrointestinal discomfort, or other adverse reactions.
Solution: We will conduct rigorous allergy testing on potential users and provide detailed instructions for use to help minimize the risk of uncomfortable reactions. In addition, we will establish a feedback system to monitor and respond quickly to user discomfort.
4. Compliance with Regulatory Standards: Probiotics are medications that require compliance with relevant regulations and regulatory standards. Failure to comply with these standards may result in legal and safety issues.
Solution: We will actively cooperate and comply with all regulatory and supervisory requirements to ensure that our products meet relevant safety and quality standards.
These safety considerations will help ensure that our programs are developed and used in a way that minimizes potential risks to protect the health and rights of end users. At the same time, we will continue to work with experts in specialized fields to obtain the latest information and advice on the safety of probiotics.