Contribution

We originally aimed to carry out this procedure as a portion of our protocol, with lipid nanoparticles encapsulating our drug as a delivery method into our cells. However, due to the numerous variables and suggestions given to us by professionals1, our team opted to design a concept model in order to contribute to future teams’ research or if this experiment ever expands into an in vivo production.
1. For more information, visit our Human Practices Page.

1. Dissolve the lipid powders in Ethanol:
2. Add ethanol to dry lipid residue and dissolve completely using moderate heat (40-50 °C) and sonication if necessary. Mix the following phospholipids: 13.4 mM 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), 10 mM 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 20 mM 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP), 5 mM 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2k), and 20 mM cholesterol. Store the stock solutions at -20 °C prior to the experiment. The total lipid concentration should be adjusted to 8mM.

1.Prepare 54 mM NaCl (saline), 25 mM acetate buffer at pH 4.0 using DNase/RNase-free distilled water. (The amounts are in relative to the concentrations of the LNP)
2.Filter the solutions through 0.2 µm sized membrane filters or syringe filters. This is the compression method using a syringe.

1.Dissolve 70 µg of siRNA into 1 mL of 25 mM acetate buffer (pH 4.0).

1. Fill 1 mL glass syringes with lipid and aqueous solutions

One syringe with the AQ buffer with saline + acetate buffer

One Syringe with the acetate buffer + siRNA

2. Connect glass syringes to Microfluidic to PEEK capillaries
3. Set machine flow rate ratio at a gradient from 3: 1 to 9:1 Aqueous phase to lipid phase, set to rapid dilution
4. Introduce the lipid and aqueous solutions separately into the iLiNP device using syringe pumps.
5. Collect LNP suspensions in a microtube from the outlet of the iLiNP device

Purpose: Prepare and purify the LNPs for cell transfections

Dialysis will remove the solvents from the LNPs

Separate the LNPs from non encapsulated molecules, lipids, floating siRNA

1. Dialyze the LNP suspension using a dialysis membrane (12−14 kDa MW cutoffs) at 4 °C overnight against saline or D-PBS for POPC LNPs and siRNA-loaded LNPs, respectively.
2. Store at 4 degrees celsius for up to 1 week
Notes: from procedure- POPC is not dissolved into saline. POPC/ethanol solution is diluted with saline.
Saline is a mixture of salt and water and acts as an osmotically balanced environment that the LNP is comfortable around for transfection.

Purpose: Allows us to know quantify the amount of siRNA that was encapsulated
1. Dilute 2 mg/mL of siRNA with 10 mM HEPES buffer (pH 7.4) to 500 ng/mL siGL4 solution.
2. Prepare the dilution series (0, 12.5, 25, 50, 100, 200 ng/ mL) of siRNA4 solution to make a calibration curve for Triton (+) and Triton (-) samples.
3. Dilute the LNP suspension 100 times with 10 mM HEPES buffer (pH 7.4).
4. Mix the following for Triton (+) solution: 980 µL of 10 mM HEPES (pH 7.4), 20 µL of 10% w/v TritonX-100, and 1.25 µL of RiboGreen for 10 wells of a 96-well microplate
5. Mix the following for Triton (-) solution: 1000 µL of 10 mM HEPES (pH 7.4) and 1.25 µL of RiboGreen for 10 wells of a 96-well microplate.
6. Pipette 100 µL of the dilution series of siRNA4 solution and diluted LNP suspensions into the wells of a black 96-well microplate.
7. Pipette 100 µL of the detection solution (TritonX-100 (+) or Triton (-)) into the wells. 8. Incubate the microplate for 5 min at room temperature.
9. Measure the fluorescence intensity using a microplate reader at a wavelength of 475 nm

Analysis calculation [% siRNA encapsulation efficiency] =
[siRNA concentration of Triton[+] — siRNA concentration of Triton[-]] /siRNA concentration of Triton[+]

1. Day 1–5: Immunization of mice. Anesthetize mice by intraperitoneally injecting 0.1 mL of a 20% ketamine, 10% xylazine cocktail in distilled water per mouse (this assumes a mouse of 25 g, the volume of anesthesia cocktail injected should be adjusted based on weight and observed response to anesthesia).
2. Immunize each mouse with 10 μg of siRNA-LNPs diluted in 125 μL 1x PBS via retro-orbital injection. For negative control, immunize mice with vehicle control (here 1X PBS).
3. Plan experiment for testing physical notable changes and track behavior of rats for self-administration of substances such as cocaine.

Due to the fact that we were a team of high school students with only our passion for synthetic biology and little else, we wanted to contribute our knowledge and insight of this process to those who are also limited in resources and don’t know where to start. The process of building up a strong team dynamic, social media presence, and reaching out to other institutions is a set of skills we learned to build through trial and error.

Figure 1: Circle Model of Leadership

Team Dynamic/Introductions: Below the Green Line

• Getting to know the team: This is a crucial and foundational step regarding developing a healthy team dynamic. This portion can be described as “below” the green line, or establishing core values in the team and building relationships. In this step, we are essentially fabricating the “heart” of the team. By creating a tight knit group, this ensures effective communication in the research process and following steps.
• Team introductions and icebreakers are encouraged when the team is meeting for the first time. Let every member have a chance to speak.
• Foster a collaborative environment and discourage competitiveness amongst the team. A game involving working together is suggested. Suggestions for questions to ask for icebreakers.
• What we would do differently: Hold team bondings sooner into the project, if time allows. The team bonding could be as simple as going out to eat together.

Team Dynamic/Introductions: Above the Green Line

• Creating order in the team: This is another foundational step regarding developing an efficient and organized team. This portion can be described as “above” the green line, or establishing structures and patterns within the team. In this step, we are essentially fabricating the “brain” of the team. Our team consists of high schoolers from all over California, making it necessary for us to create a system where misunderstandings are prevented.
• To overcome these challenges we:
1. Created an organizational system. Our team chose to fall into the groups they found interest in: Tech group, Research group, and Business group. The responsibilities of each group and what they encompass were made clear at the beginning of the project.
2. We hold weekly meetings every Saturday at 8:00 pm, establishing a routine of which every member is expected to follow, unless team leaders are informed 24 hours prior that a member would not be able to attend. Setting meetings routinely fosters strong communication, holds each member accountable for the work they put in, and boosts productivity.

Funding and Publicity:

• Funding
• The business team made a Google Sheet of 200+ pharmaceutical and biotechnology companies to email and request grants and donations. We also searched through each company’s websites to see their grant request forms and check their requirements.
• A big issue we came across was lack of credibility. Most companies only gave funding to legal nonprofit organizations, and though our team researched the process of filing for a nonprofit organization, it would’ve taken longer than the time allotted before we had to start buying our lab materials and renting a lab space.
• We tried selling drinks and popsicles at Fourth of July and made some sales, but it was not enough to meet our demands.
• Professor Outreach
• Each of our team members searched for 20+ university professors in departments such as pharmacology and read the papers they wrote to see alignment in their research goals and ours. Combined, we emailed approximately 200+ professors and 50+ companies, getting about 10 responses total.

• Publicity
• We started an Instagram page to introduce our team and what we aim to do, warn people about addiction, inform the public about the field of synthetic biology, share our progress in the lab, and show our peers that lab research as high schoolers is achievable.
• We posted photos highlighting each day at the lab, writing a step-by-step description of what we did and why
• At the lab, we would take videos and pictures of all of our progress to later put together in a post.
• We also filmed some “silly” Reels to make our research project more “approachable” then added in depth explanations in the comments section or the caption. In one of our Reels, we reached over 19,700 accounts and got over 28,000 views. Through constant efforts, our Instagram profile gained over 20K views each month, and we got 100+ followers
• Filming Reels consisted of finding an audio that was trending (which would help our video to reach a bigger audience), planning which members will be featured and what they would be doing in the video, and putting it into action at our in-perosn meetings or at the lab during waiting periods

Research Process

• Choosing a Topic: Topic choosing requires extensive research and is something that cannot be overlooked. The team’s ideas and passions are important when choosing a research topic. Our process was as follows:
• The team leads narrowed some major topics discussed beforehand into a few topics of interest expressed amongst the team members. Our general topic chosen circulated the idea of addiction prevention.
• Choosing a general topic is not enough. Addiction is a vague subject that needs to be pinpointed down to a specific enzyme, gene, or molecule of interest. This is where the research team comes in.
• From the instructions, “research the causes of addiction and find anything you can about it”, the research team gets to work. ChatGPT was used as members of the research team gained background information of how dopamine receptors and the brain work together. Once a member believes a potential lead has been found from information gained by ChatGPT, the member would then search up the specifics on reliable sources. Major sources we used include: NCBI, NIH, Uniprot, Protein Atlas, and Thermofisher
• The potential detailed topics each member of the research team found were organized in a chart on a google doc. Every member was assigned to pick their top three choices of detailed topics to look into. The final detailed topic is selected from there.
• Note: it is ok to change the final detailed topic if enough research backs up the new topic. A red flag to change the final detailed topic is if not enough sources back up the topic. In our project, we were originally planning to delve deeper into FosB regulators, but changed to G9a once we realized the limits to our capabilities
• Researching the Detailed Topic of Interest:
• We first made it a priority to understand what G9a was.
1. Initial Inquiry: Began by asking ChatGPT to gather preliminary information about G9a.
2. Verification: After receiving that information, we cross-referenced it with Google to verify the accuracy of the details.
3. Research Findings: We focused on understanding the role G9a plays, specifically in the context of addiction, using the resources and research we found.
• Once we understood the relationship between G9a and addiction, the next step was to research how to prevent the effects and come up with more questions. (eg. Higher expression levels of G9a lead to signs of addiction. How do we lower expression levels?)
• During this time, we developed our knowledge in biotechnology and the techniques used by professionals, including plasmids, shrna, sirna, antibody resistance, and different cell lines. This helped us develop our procedure later on.
• (eg. In our project, we were researching shRNA and siRNA to determine which would be more effective in your project involving G9a. Ultimately, we chose siRNA because it allowed for a partial knockdown of G9a expression rather than a complete knockdown, which aligned better with our research goals (IMPORTANT). This approach aimed to modulate G9a levels without fully eliminating its activity.)
• Procedure
• Each team member was assigned a specific procedure of a technique, and developed a detailed process of how to incorporate it into our project. In our project, we researched western blotting, gel electrophoresis, RT-qpcr, cell culturing, and even smaller components like primers, fluorescent dye, antibodies, and preparing growth medium. This step varies depending on each project’s needs.
• Essentially, starting from scratch required us to do comprehensive web searches in order to self learn all the information. Therefore, having a strong team dynamic is very important to feel like the learning process is more efficient.