Problem

This is a new opportunity for myopia treatment, but also the need to protect human eye health.

We initially observed that there was a very high rate of myopia among young people in East Asian countries, especially China, so our team went to collect more data. On 8 October 2019, on the eve of World Eye Care Day, the World Health Organization (WHO) released its first World Report on Vision. The report shows that the overall prevalence of myopia has reached 53.4% in high-income countries in the Asia-Pacific region, followed by 51.6% in East Asia. The prevalence of myopia among young people in urban areas is 67% in China and 97% in South Korea. This is a heartbreaking statistic. Eyes are the window of the soul and the precious treasure of human beings. Our team wants to do something about it and make contributions to human health.

We learned about the prominent role of antihypoxic drug salidroside in alleviating scleral hypoxia. As a group of students who love biology, we expected to use synthetic biology methods to solve this problem. As a human practice team, we strive to turn our initial inspiration into reality step by step. We divided our human practice work on salidroside microbial contact lenses into three parts: upstream, midstream and downstream. Upstream is the part of theory and feasibility analysis, including interviews with ophthalmologists and research on target groups. Midstream is the part of the project implementation and technical realization, including interviews with five scholars, which greatly promoted the feedback of the project. Downstream is the part of promotion and user side optimization, including learning product promotion from Youkang Biotechnology Company, and policy consultation with the government.

1 Upstream | Theory and feasibility analysis

It is a process of finding problems, and it is also the starting point of our connection with the world.

1.1 Ophthalmology knowledge consultation

We first went to the Eye Center of the Second Affiliated Hospital of Zhejiang University School of Medicine to consult the factors that cause people to suffer from myopia. Deputy chief physician Nvxia Tong enthusiastically shared with us the possible causes of young people suffering from myopia, of which bad eye habits are the primary factor. Therefore, we think that our contact lens designed to monitor the intraocular pressure in real time to release salidroside will have a prominent role in this.

We also asked about the effects of soft contact lenses on the eyeball. Dr. Tong pointed out that the soft contact lens designed by our team can provide good vision correction under certain conditions, and also has a potential positive effect on the treatment of many eye diseases. In addition, the way the drug is administered through the contact lens will obviously stay longer than the eye drops, and the effect will be more obvious. This also identifies one of the strengths of our project. However, she also mentioned several potential risks to eye health: Wearing soft contact lenses for long periods of time may increase the risk of eye diseases such as corneal infections and dry eyes. In addition, if not properly used and cleaned, it may also lead to corneal inflammation and discomfort.

Based on experience, Dr. Tong gives several suggestions, she reminds us to consider daily disposable biological lenses, and avoid sanitary infections as much as possible by changing contact lenses regularly. In addition, she advised users of the biologic lenses to have regular eye exams and have a regular contact with their doctors to deal with any discomfort or problems.

Fig.1 Interview with Dr. Tong of the Eye Center of the Second Affiliated Hospital of Zhejiang University School of Medicine

1.2 Target group survey

In order to learn more about the needs and concerns of the target user group, our team first conducted a social survey on the topic of "myopia and related device use and experience". The survey content covered the eye use and eye protection habits of the myopia group, myopia conventional treatment options, as well as people's acceptability and concerns about our microbial contact lenses.

Group sample:

There were 209 respondents in this survey, covering 21 provinces/municipalities in China. Among them, respondents aged 18-25 accounted for the largest proportion, accounting for 86.12%. Among all the respondents, the number of people suffering from myopia is as high as 191 (accounting for 91.39%), which shows that the prevalence of myopia in this survey sample is high, and the audience of our project will be very broad. It is worth noting that the average age of all people suffering from myopia is 12.81 years old. It can be seen that the age of myopia among the current public is mostly in teenagers. To this end, we have contacted some communities and schools to carry out eye protection education and science activities on the prevention of myopia among teenagers and children.

Fig.2 Geographical distribution of samples

Fig.3 Age distribution of the sample

Eye use and eye protection habits:

Data shows that the vast majority (86.91%) of people with myopia wear glasses, followed by orthokeratology lenses and contact lenses (3.66% and 2.62% respectively). Glasses are still the mainstream choice for myopia patients today. Most of the respondents said that they would not take relevant measures to protect their eyesight in their daily life. When asked "How much do you attach importance to eye health?", the average score given by all respondents was 5.59 (out of 10 points). It can be seen that the public attaches little importance to eye health , so people need a product that can easily prevent or even treat myopia in daily life, which is consistent with the original intention of our team to choose this project.

Fig.4 The survey of the types of glasses worn

Comparison with conventional myopia treatment methods:

We found that there are currently three mainstream methods for treating myopia: eye drops to relieve eye fatigue, corrective functional glasses, and oral medications. All methods known to society are based on "drug treatment" or "medical device treatment" alone. Our team's design is "combination of drug and device treatment", which is also our innovation point. Among the respondents who have had refractive surgery or used orthokeratology lenses, 1/6 are not satisfied with the treatment effect. The biological lenses we designed can be used as auxiliary treatment to make this type of surgery more effective. The final effect is better. The reason why we choose soft contact lenses as the carrier is because the direct contact between the "carrier" and the eyeball can more accurately and quickly sense the corneal hypoxia, thereby releasing salidroside more efficiently and the drug action time is longer and long-lasting, thus achieving the effect of relieving corneal hypoxia.

Fig.5 The survey of different kinds of myopia treatment

Concerns:

Regarding the use of soft contact lenses, 66.03% of the respondents said they had no relevant experience, indicating that its popularity among myopia groups is not high. Our team also conducted social education and publicity on soft contact lenses to address this issue. Another concern we have is the comfort level of soft contact lenses. Among the respondents who have worn them, the majority (73.24%) believe that the comfort of wearing contact lenses is uncertain and accidental. Therefore, we have also fully considered relevant factors during the lens design process, such as choicing appropriate lens water content, using silicone hydrogel lenses as carriers.

Fig.6 The survey of the comfort of wearing contact lenses

The main concerns of interviewees about the therapeutic contact lenses designed by our team are "microbial safety", "price" and "care and preservation". In response to concerns about the "safety of microorganisms", on the one hand, we have carried out some popular science education activities on synthetic biology to enhance people's understanding and attention to synthetic biology products; on the other hand, we have consulted with Youkang Biology Company to learn more about them Experience in promoting microbial products to customer base. We hope to use relevant technologies when putting them into production in the future and do our best to reduce patients' worries.

Fig.7 The survey of concerns about Biolens

2 Midstream | Project implementation and technical realization

Here, we gained a lot of good feedback for the wet lab experiment.

2.1 AND gate of blue light and temperature

In our original design, we intended to use blue-sensitive components and vitamin C as detection ingredients, allowing them to track the duration of blue light exposure to the eye throughout the day and serve as a warning. Dr. Haorao Yu, researcher of College of Chemical and Biological Engineering, Zhejiang University remined us that blue light also existed in natural light, and normal exposure to blue light will not cause damage to the eyes. He suggested that we use blue light as a condition for production. Since we cannot distinguish between blue light in natural light and blue light emitted by electronics, we decided to cancel the module for health detection. We use blue light as the response condition of the light-controlled switch, and form AND gate together with the RNA thermometer to control the recovery of engineering bacteria.

Fig.8 Consult Dr. Haoran Yu

2.2 Enhance the stability of metabolic modification

To further enhance the stability of metabolic modification, we consulted Associate Professor Ye Lidan, an expert in metabolic modification at the College of Chemical and Biological Engineering, Zhejiang University. Dr. Ye believes that if we keep the ARO10 and UGT85A1 genes in the form of plasmids in E. coli, they are likely to be lost during passage. She suggested that we integrate genes into the E. coli genome to increase genetic stability. We were fortunate to have a transposon system guided by CRISPR-RNA from Dr. Ye's lab, including pEffector (BBa_K4761052), pDonor (BBa_K4761108) and pCutamp (BBa_K4761109). In the follow-up experiment, we took the advice of Dr. Ye and successfully completed the gene integration with this powerful gene integration tool. See our Results page for details.

2.3 Inspiration of liposomal vesicles

To find a membrane material suitable for wrapping engineered bacteria in contact lenses, we consulted researcher Zhikan Yao, an expert in membrane science and technology at the College of Chemical and Biological Engineering at Zhejiang University. Dr. Yao pointed out that to prevent bacterial leakage, biosafety should be the focus of membrane materials to consider. In addition, because our membrane material is in direct contact with the eye, biocompatibility is also important. Dr. Yao admitted that he did not know much about membrane materials in biomedicine, but he proposed that bacteria could be embedded in a spherical phospholipid membrane and combined with contact lenses to enhance biosecurity. This gives us a great inspiration! To this end, we decided to add a liposomal vesicle module to the wet lab, embedding engineering bacteria and media in the chamber of the vesicle, so as to prevent the bacteria and their metabolic waste from leaking.

Fig.9 With Dr. Zhikan Yao

2.4 Refine the details of liposome vesicle experiments

Our team didn't know much about how to build liposomal vesicles, and we could only find some relevant literature, but there were few similar cases of stable encapsulation of bacteria in giant liposomes. Therefore, we consulted Zhuxian Zhou, associate professor of the College of Chemical and Biological Engineering at Zhejiang University, who is an expert in the field of nanobiomaterials, targeted drug delivery, and controlled drug release. We originally thought that when preparing the hydrogel film for contact lenses, the bacterial solution would be mixed in it, so that the bacterial solution would be embedded in the hydrogel chamber. But Dr. Zhou reminded us that we should pay attention to the problem of metabolic waste. Dr. Zhou suggested that we use a phospholipid membrane embedded with salidroside transporter protein as a shell, encased in the bacterial solution, and encapsulated between the two membranes of the contact lens. In this way, the shipment of salidroside can be strictly controlled, while the bacterial solution and its metabolic waste will not be leaked.

Dr. Zhou warmly invited us to conduct experiments in his biomedical polymer laboratory. In terms of experimental details, Zhou believes that the size of our liposomes needs to be in the micron level (so as to encapsulate engineered bacteria), but at the same time, the increase in the size of the liposomes will also increase its instability. In addition, he reminded us that the traditional method of preparing liposomes - film water coating method, if applied to our experiment, may need to consider the effect of organic reagent extraction, ultrasound and other steps on the activity of our bacteria. They are a very delicate species!

Fig.10 Team members had a cordial interview with Dr. Zhou

2.5 Vitamin E introduction

Researcher Jianzhang Lian is an expert in building artificial cell factories and also the primary PI of our iGEM team. He told us that the eye is a complex system, the production of salidroside can not be simply released directly into the eye, the complex structure of the cornea of the eye will prevent salidroside from entering. In response to Dr. Lian Jianchang's suggestion, we found a solution to add vitamin E by consulting the relevant literature on eye administration of hydrophilic drugs. Its aromatic ring can form hydrophobic interaction with the aromatic ring of salidroside, and enhance the distribution of salidroside in the water phase. This makes our experimental design more complete and makes our project more feasible. See our Design page for more details.

Fig.11 Present flowers to our primary PI, Dr. Lian

3 Downstream | Promotion and user side optimization

Future-oriented product promotion.

3.1 Meeting with Youkang Biotechnology Company

We visited Youkang Biotechnology Company, Zhejiang Province, a young biotechnology company founded in 2018, which is also our partner in testing the experimental results. Although this company has not been established for a long time, it has been able to share the university market with many established biotechnology companies in China, and we think they must have super superb social promotion ability. We have learned in the previous questionnaire survey that concerns about microorganisms are the primary concern of the user community for our contact lenses, so we are very much looking forward to gaining some experience in promoting biological products from Youkang, especially those related to biosafety.

There are a few key factors shared by the Biologics receptionist for us. One is to lower the price, and the other is to prefer to trouble themselves to facilitate customers. In addition, for products that may have biosafety concerns, it is also important to display official product quality recognition certificates. This gives us a great inspiration! We also gained a lot of sales skills from the salesmen who had Kangbio, which was a pleasant experience.

Fig.12 Team members are visiting Youkang Biotechnology Company

3.2 Visit Provincial Drug Administration

Our team interviewed Mr. Shenke Fang, a first-level chief clerk of the Medical Device Supervision and Administration Department, Zhejiang Provincial Drug Administration, and consulted on the market prospects of products that combine drugs and medical devices. He told us that therapeutic products that combine drugs and medical devices are not common in the market, and may face challenges in the approval of relevant units. He reminded us to fully prepare the persuasiveness of the efficacy and safety of the product. Only in this way can the public accept the bio-lens we designed.

Mr. Fang's guidance means that we need to fully prepare relevant data and research results to prove the efficacy and safety of the product. In addition, he emphasized the importance of compliance review, including technical assessment, quality control, production process, etc., to ensure the quality and safety of the product. When facing approval challenges, Mr. Fang encouraged us to maintain close communication with the drug regulatory authorities and seek professional advice and guidance.

We are very grateful to Mr. Fang for his guidance and advice. He reminded us of the need for thorough preparation before designing a biologic lens and entering the market.

Fig.13 Due to the particularity of Mr. Fang's job, we just took a photo at the gate of the Zhejiang Provincial Drug Administration