Week 1 HW: Final Project Ideas

1. What do you want to make?

I want to biomanufacture targeted Lipid Nanoparticles (LNPs)—microscopic lipid bubbles—carrying temporary messenger RNA (mRNA) and microRNA instructions. These nanoparticles are engineered to specifically target activated scar cells (cardiac fibroblasts) in the heart attack scar bed of adults.

This therapy is designed for post-myocardial infarction (heart attack) patients who are at risk of progressive heart failure. When a heart attack occurs, oxygen starvation kills heart muscle cells (cardiomyocytes). Because adult human hearts cannot naturally regenerate, the body panics and forms a dense, rigid collagen scar to prevent the heart wall from rupturing. However, this stiff scar creates a massive physical and electrical barrier that blocks transplanted stem cells (like iPSC-derived cardiomyocytes) from successfully taking root and surviving.

My project creates a transitional “stall and settle” bridge for cardiac regenerative medicine. Rather than altering the patient’s DNA permanently or surgically cutting out the scar tissue, the biomanufactured nanoparticles deliver temporary mRNA/microRNA “instruction notes.” These instructions briefly turn down collagen production in the scar cells, softening the existing hard matrix right as iPSC-derived cardiomyocytes are introduced. This mimics natural regenerative species (like zebrafish and neonatal mice), keeping the structural integrity of the heart wall intact while stalling time for the new stem cells to safely integrate, receive blood supply, and rebuild functional heart muscle.

My motivation combines systemic health challenges, utilitarian biomedical engineering, and a little bit of personal experience. Globally, cardiovascular disease remains the number one cause of death in many countries. The heart is the primary engine of the body, responsible for pumping oxygen-rich blood to every other organ. When the heart fails, it triggers a domino effect where other organs slowly starve and fail as well. As the global population ages with passing generations, finding effective solutions for post-heart attack remodeling is becoming increasingly urgent. Beyond the statistics, watching a loved one or patient slowly lose their organ function and independence to heart failure is heartbreaking. It robs people of their vitality, mobility, and dignity long before life ends. While volunteering, I met a patient who suffered from recurrent heart attacks while serving as the primary caregiver for his terminally ill father. He expressed intense fear that his weakening heart would fail before he could finish caring for his parent. That conversation showed me that heart disease strips away a person’s capability to care for those they love too. It’s not just elders but also family members who may be the primary provider. Also, designing a biomanufactured solution that gives damaged hearts a realistic, safe path to heal is a problem I find intriguing as it opens more possibilities for other organ regeneration.

2. What living system could make it?

To be completely honest, I don’t have a background in science, so setting up an experiment like this was definitely confusing, and I’m still reading through research to figure it all out. So, I might have a few many things that are wrong. My project builds directly on the stem cell research pioneered by Dr. Chuck Murry. As Dr. Murry demonstrated in his work on regenerating heart muscle, we can take human pluripotent stem cells (iPSCs), differentiate them with high efficiency into cardiac muscle cells (iPSC-cardiomyocytes), and transplant them into injured heart tissue to re-seed the heart wall. However, one of the biggest clinical hurdles stem cell therapies face after a heart attack is the stiff, dense scar tissue left behind. If the scar bed is too rigid, transplanted stem cells struggle to integrate, take root, and achieve the synchronous beating and electromechanical coupling needed to restore function without causing arrhythmias. To solve this, my system focuses on temporarily softening that scar tissue right when the stem cells are introduced. For my biomanufacturing living system, I chose a cell-free enzymatic system (In Vitro Transcription) paired with a microfluidic lipid assembly platform, rather than growing whole living cells in big tanks. By isolating just the essential purified enzymes, we can produce our targeted matrix-softening mRNA and microRNA payloads directly—without having to feed living cell cultures, clean up cellular waste, or worry about viral contamination.

This part I got alot of help from the papers and Gemini The setup works by feeding in (1)Linearized DNA templates encoding matrix-softening signals (like matrix metalloproteinases [MMPs] to gently break down rigid collagen, and microRNA-29 to temporarily suppress new collagen synthesis) (2)Modified NTP building blocks and T7 RNA polymerase to synthesize the RNA strands. (3)Targeting lipids conjugated with peptides that specifically recognize activated cardiac scar cells (myofibroblasts). Inside the cell-free reactor, the enzymes build the RNA, and a microfluidic mixer immediately wraps it into uniform lipid nanoparticles (LNPs). The output is a batch of sterile, targeted LNPs designed to give temporary scar-softening instructions to cardiac myofibroblasts at the transplant site. My main assumption and biological mechanism— cell-free principles in Karim & Jewett (2021) and research on cardiac scar reprogramming from Dr. Conrad Hodgkinson’s lab at Duke and the University of Sydney (2026)—is that synthetic mRNA naturally degrades within 24 to 72 hours. That short lifespan acts as a built-in safety timer: it pauses collagen production and softens the matrix just long enough for Dr. Murry’s transplanted iPSC-cardiomyocytes to settle in and couple with host tissue, before normal scar repair naturally resumes to prevent wall rupture.

3. How could automation help you make it?

Automation seems to cut through a lot of tedious manual work across two key parts of the project: Before making a working nanoparticle at scale, hundreds of different combinations of RNA sequences (MMPs, miR-29) and lipid formulations have to be tested to find the exact right ratio. I would use a desktop liquid-handling robot (like an Opentrons OT-2) to automate mixing reactions in 96-well plates. The robot gives us sub-microliter precision, speeds up testing, and makes sure our cell-free transcription assays are completely reproducible. Once we have the right recipe, we need to package the RNA into tiny lipid bubbles (LNPs). Pushing fluids through microfluidic channels manually with a syringe is risky because pushing too hard or unevenly creates turbulent flow or damages the chip. Instead, I would use an automated microfluidic platform (like a Precision Nanosystems or KNAUER LNP Skid). This system automatically controls the exact flow rates, pressures, and mixing speeds of the liquid RNA and organic lipid streams as they converge in microscopic channels. Because nanoparticle self-assembly is super sensitive to fluid dynamics, automating the flow rates guarantees uniform particle sizes (~80–100 nm) and high encapsulation efficiency without batch-to-batch variation.

4.What would it take to make it at a meaningful scale?

What I understood after talking with AI and going through readings on this topic, biomanufacturing scale is all about working backward from the patient dose while accounting for real-world biological losses. To make a meaningful impact, I defined my target scale as treating 10,000 post-myocardial infarction patients per year who are receiving stem cell therapy. A standard therapeutic dose for targeted lipid nanoparticles (LNPs) in cardiac applications is roughly 1X10^{13 active particles per patient. However, because most particles in a real human body get cleared by the liver or break down before reaching the heart, I assumed a 20% target delivery efficiency (meaning 80% is lost). That means we actually need to manufacture 5X10}13 LNPs per patient, bringing our total annual requirement for 10,000 patients to 5X10^17 total LNPs per year. Dividing our total required particles by this yield shows that we need to produce 500 Liters of bulk LNP liquid formulation per year. When it comes to manufacturing time, what I learned from the readings is that the automation tools used during early prototyping are completely different from what you use for full-scale production. During the R&D phase in the lab, when we are testing small 10 mL to 100mL batches using a desktop liquid handler (like an Opentrons OT-2) paired with a benchtop microfluidic mixer, setting up a cell-free in vitro transcription reaction and encapsulating it into LNPs takes under 2 hours per trial batch. But once we move to full clinical production for our 500-liter annual supply, we switch to a continuous-flow industrial microfluidic skid operating at a throughput of roughly 500mL/min (30Liters/hour). Running 500 Liters through that system takes approximately 16.7 hours of continuous fluidic operation. Even after factoring in extra time for tangential flow filtration purification and sterile vial packaging, the entire year’s supply for 10,000 patients could be manufactured in under 3 to 5 operational days.

5. What don’t you know yet?

The biggest unknown is figuring out the exact types and ratios of mRNA and microRNA constructs needed to temporarily soften the dense collagen scar without interrupting or damaging the incoming iPSC-cardiomyocyte stem cells. If we break down the scar tissue too aggressively, we risk causing the left ventricular wall to rupture, but if we don’t break it down enough, Dr. Chuck Murry’s transplanted stem cells won’t be able to integrate or form the electromechanical gap junctions (Connexin-43) required to beat synchronously with the host heart. Additionally, while I’m assuming our peptide-guided lipid nanoparticles will target activated cardiac myofibroblasts, I don’t know if the liver will filter most of them out before they even reach the heart. To actually validate this idea in the lab, I would need to measure the mechanical stiffness of human cardiac tissue after treatment, run biodistribution assays to track LNP clearance, and perform electrophysiological mapping on co-cultured scar and stem-cell tissues to ensure the cells couple properly without causing dangerous arrhythmias.

Here are the reading/videos:

Note: I used Gemini to brainstorm my ideas before answering the question. Specifically, the AI helped translate my strategy—creating a transitional “stall and settle” stage using temporary mRNA/microRNA signals to soften post-infarction heart scars while iPSC-derived cardiomyocytes integrate—into technical engineering terms. Gemini assisted in evaluating biomanufacturing platforms by comparing living cell cultures against cell-free enzymatic synthesis (In Vitro Transcription), clarifying the biological composition of cardiac scars (such as Collagen type I and myofibroblast activity), identifying suitable automation platforms (Opentrons liquid handlers for R&D screening and microfluidic mixers for lipid nanoparticle assembly), and structuring back-of-the-envelope scale calculations and bottleneck analyses (such as off-target liver accumulation and cell-free enzyme costs). Additionally, I used the AI to help organize my raw ideas into logical responses for Prompts 1 through 5. I remain completely responsible for all final concepts, calculations, and written content presented in this assignment.