Optimizing Yield for Multilamellar Vesicle (MLV) Platform for Cosmetic Delivery
Advisor: Dr. Sharma
Internship Location: ASDRP/reThink64, 46309 Warm Springs Blvd, Fremont, CA 94539
Onsite Mentor: Dr. Joey Pazzi
Traditional drug delivery methods such as pills and injections can be effective, but they often cause systemic side effects and require strict dosing schedules. Multilamellar vesicles (MLVs) are specialized structures composed of multiple lipid bilayers surrounding aqueous compartments. This project will focus on using MLVs for both drug delivery and cosmetic applications. Recent studies have showcased their use in cosmetics because MLVs can break down into smaller structures as they penetrate deeper, which improves product outcome thus performance. MLVs have also been emphasized in drug delivery because their concentric layers can encapsulate a larger total drug volume than unilamellar vesicles and can support a more stable, controlled release, as layers are expected to break down sequentially rather than all at once. This also allows for more protection of the compound in the most central vesicle. In this study, I will develop a simple method to generate MLVs using DOPC lipid films dried on paper supports and then hydrated with different aqueous buffers. Vesicle formation will then be confirmed using fluorescence microscopy with RhoPE-labeled lipids. I will then compare MLV yield across hydration conditions and test whether saline buffers such as NaCl and CaCl₂ produce higher quantities of MLVs than other solutions, and quantify this using ImageJ. Finally, I will use these results to identify hydration conditions that improve reproducibility and yield, supporting a scalable framework for producing MLVs for both topical cosmeceutical formulations and drug delivery research.
Project Blog
Week 0: Blog:
Hi, I’m Samahith, and this is my introductory Senior Project blog.
If you’ve ever had to get a shot at the doctor’s office or taken a lot of pills, you know the soreness and unpleasant feeling it leaves you with along with tremendous anxiety. My project works on a strategy to help you skip injections & pills and yet get the medication inside your body. This medicine delivery can be done by applying a patch or a cream and yet get the same benefits. This seems like a dream and an amazing scenario, but requires in-depth application of physics, chemistry, and iterative experimentation. This is exactly what I’m investigating for the next ten weeks. My project is all about optimizing Multilamellar Vesicles (MLVs) production, which is the drug delivery mechanism that will be used in these patches and creams.
In layman’s terms, MLVs are microscopic onions with around 20 layers made entirely of lipids (fats). These MLVs are incredible at trapping drugs or cosmetic ingredients inside their layers and carrying them deep into the skin, while also being able to slowly release the payload by spreading the payload throughout the layers. They are safer and more stable than their single-layered counterparts, as once the first layer is popped, the payload is lost. MLVs have one massive problem: they are incredibly inconsistent.
Currently, the standard way to make them is called thin-film hydration, which gives you a different result every single time. Sometimes you get a lot of vesicles without uniformity, sometimes you get barely any, but with perfect uniformity.
I’ve spent a lot of time in the lab at ASDRP working with lipids, Lipid Nanoparticles (LNPs), and Giant Unilamellar Vesicles (GUVs). I’ve seen how inconsistent and difficult making these vesicles can be. I realized that if we want to revolutionize drug delivery, we need a way to manufacture these vesicles reliably and efficiently.
For my Senior Project, I’ll be testing how different salt solutions interact with the lipid headgroups to see if we can increase uniformity and yield. My goal is to optimize the protocol for high-yield MLVs that can be used in pharmaceuticals and cosmeceuticals to make treatment cheaper, scalable, and anxiety-free.
I am incredibly lucky to be working under the guidance of Dr. Joseph Pazzi at ASDRP and Dr. Rachna Sharma at BASIS, and I’m ready to get into the lab and start hydrating some lipids.
– Samahith
Week 1: Blog: Setting the Reagents and Experiments
Hello Everyone! Welcome to the first official week of my Senior Project. While the end goal is to help transform drug delivery, the reality of Week 1 is a bit more grounded, centered on preparation, and focused on reading a lot of research papers. In research, you can’t just jump into experimentation. I have to ensure my materials are pure, ready to use, and stable. I have to ensure my calculations are precise and accurate, and in this specific case, I have to ensure my environment is sterile. Think of this week like the French culinary term "mise en place," which means having all your ingredients measured, cut, and ready before you start cooking.
What I did this week:
Material Procurement: I successfully bought and prepared the core components for my experiments: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids and Rhodamine B, the fluorescent dye that will allow me to visualize the vesicles. I also prepared the aqueous ionic buffer solutions I need. I prepared 0.1 M solutions of Sodium Chloride (NaCl), Calcium Chloride (CaCl₂), Potassium Chloride (KCl), and dPBS. I also tested for contamination.
Lipid Stock Solution: After I prepared the primary lipid stock solution. This involved dissolving the DOPC and trace amounts of dye into a 2:1 Chloroform/Methanol mixture to create a 10 mg/mL concentration.
Sterilization: Purity is essential in biology and chemistry. I spent a significant portion of the week autoclaving pipette tips and glass slides to ensure absolutely no contaminants interfere with the delicate self-assembly process.
Safety Review: Since I am handling chloroform and other volatile organic solvents, my advisor made me review the Safety Data Sheets (SDS) and established the proper fume hood protocols and other safety procedures to remain compliant with lab safety standards.
I have also started developing the Standard Operating Procedure (SOP) for the fluorescence microscope and Dynamic Light Scattering. While I haven't finalized the protocol yet, I am currently testing different exposure times and gain settings to determine the optimal configuration. Locking these settings in now is important so there are no potential confounding variables or changes in arbitrary units. It basically ensures that any future differences I observe in vesicle yield or uniformity are due to the solvents, not inconsistent imaging or testing. Next week, I will continue this Protocol Optimization and then run the first trial runs. Thanks for reading!
– Samahith
Week 2: Trial, Error, and Standardization
If Week 1 was the "mise en place," Week 2 was the test cook. This week, I moved from prepping materials to actually running the protocol. In science, you can’t just run an experiment once and hope for the best. You have to prove that your method is repeatable before you even touch your test variables. On top of that, my results also have to be repeatable.
Locking in the SOP:
The biggest milestone this week was finalizing the Standard Operating Procedure (SOP) for the thin-film hydration method. While the concept is simple, the actual execution is quite delicate. It involves drying the lipids, adding water, and shaking it up to perfection.
I spent the early part of the week conducting trial runs using just DI water and dPBS (Dulbecco’s Phosphate-Buffered Saline) to work out the kinks and resolve any issues in my procedure. One immediate challenge was the vacuum desiccation step. I noticed that if the vacuum pressure was applied too quickly, the solvent bubbled aggressively rather than evaporating smoothly, creating an uneven lipid film and a non-uniform spread lipid layer. By adjusting the pressure ramp-up time, I managed to create a uniformly smooth film on the tracing paper to essentially ensure that there is consistent hydration later on.
Microscopy:
I also finished optimizing the fluorescence microscope setup that I had started working on last week. It is tempting to adjust the brightness for every picture to make it look "good," but that ruins the data and doesn’t allow for consistency amongst the samples. To ensure scientific validity, I locked in the exposure time and gain settings. I tested this on my trial dPBS batch and confirmed that I can clearly distinguish the Rhodamine-labeled vesicles from the background noise without over-saturating the image.
I have successfully created MLVs in my control groups (Water and dPBS), which is a huge relief since it means the chemistry is functional. I executed a final "dry run" of my image processing workflow. I took the images from the dPBS trial, stitched them into a tile scan, and ran them through my ImageJ macro to ensure the counting algorithm picks up the vesicles correctly. Since this is now verified, I’ll start working on my experimental variables. Next week, I will start the actual data collection with Sodium Chloride and Calcium Chloride.
Thanks for following along!
– Samahith
Week 3: Testing Different Ions (Sodium vs. Calcium)
Week 3: Testing Different Ions (Sodium vs. Calcium)
This week, I started Phase 2 of my methodology. I tested the first set of experimental variables (0.1M Sodium Chloride and 0.1M Calcium Chloride) to create MLVs.
The main reason for selecting these specific salts is to see how different ionic charges affect the DOPC lipid headgroups and in turn the formation of MLVs. Calcium is a divalent cation (2+ charge), which means it binds strongly to the negatively charged lipids and can potentially cause the membrane to become too rigid. Sodium is a monovalent cation (1+ charge), so it interacts weakly in comparison, which should theoretically allow the lipids to have greater flexibility needed to form proper MLVs, without forming clumps, tubes, or other residues.
Over the past few days, I completed the entire thin film hydration protocol six times for these two buffers. I dried the lipid films, hydrated them with their solvents, and vortexed them for exactly five minutes to ensure consistency across all my samples.
When I took the completed samples to the darkroom for fluorescence microscopy, the difference between the two groups was immediately obvious. Even without running the images through the ImageJ counting software, I could clearly see that the NaCl samples were much more uniform. The overall yield of multilamellar vesicles in the Sodium buffer was also visibly higher, whereas the Calcium Chloride samples looked noticeably sparser and often clumped together. I was elated to see my results align perfectly with the biophysical theory right away.
For tomorrow, I plan to finish the imaging process for the final calcium chloride replicates. Once everything is captured, I will organize the tile scans so that they are fully prepped for the quantitative data analysis phase. Seeing such a noticeable difference, even before data analysis, already gives me a lot of confidence moving forward. I am eagerly looking forward to getting the actual numbers to confirm this later in my project.
-Samahith
Week 4: Update on Potassium Chloride and dPBS
Welcome back to my Senior Project blog! As a quick refresher, my research focuses on optimizing the production of Multilamellar Vesicles (MLVs). You can think of MLVs as microscopic, layered fat bubbles that can carry and deliver therapeutic drugs and cosmetics. Because these vesicles are notoriously difficult to manufacture consistently, my project investigates how different salt solutions affect their assembly. Last week, I observed that a Sodium Chloride solution, which carries a single positive charge, created highly uniform vesicles compared to salt solutions with a double positive charge.
Building on that, my fourth week in the lab is focused on testing a new set of solutions. This time it's Potassium Chloride (KCl) and dPBS (Saline).
The reasoning for choosing Potassium Chloride is tied to the physical interactions I explored last week. Potassium is an ion with a single positive charge, just like Sodium. Due to this similarity, it should interact gently with the negatively charged outer shell of the lipid molecules. This gentle interaction preserves the flexibility to the lipid layers, which is absolutely necessary for them to assemble into uniform delivery bubbles. Testing Potassium allows me to verify if the high yield I saw with Sodium last week was truly due to its single charge rather than just a specific quirk of Sodium itself.
Furthermore, introducing dPBS into the experiment serves a very practical purpose. The dPBS solution is a specially balanced saltwater mixture that closely mimics the natural fluids found inside the human body. Since the ultimate goal of these vesicles is drug and cosmetic delivery within human biological systems, testing a liquid that copies a natural cellular environment provides an important baseline for how these structures might actually form in real-world applications.
To maintain the rigorous consistency required for this project, I completed the hydration protocol six times over the last few days, running three replicates for KCl and three for dPBS. Even before the full data analysis, the visual results under the microscope were very encouraging. I observed a high degree of uniformity among the KCl samples. This closely mirrored the successful outcomes of the Sodium trials and supported my hypothesis.
Going into next week, my primary objective is to finish getting the final microscope images for the dPBS MLVs. Hoping to share some exciting results next week.
- Samahith
Week 5: Creating Control Groups for my Project
Welcome back to my blog! If you are following my blog, you know that my research focuses on improving the quality of Multilamellar Vesicles. These are microscopic, multi-layered fat bubbles used to deliver drugs and cosmetics directly through the skin, which will eventually allow us to replace painful injections. The main challenge is that they are difficult to produce in a reasonable quantity consistently. Over the past few weeks, I have been testing how different saltwater solutions interact with the lipids to increase the total number of vesicles formed and the uniformity.
This week is the final week of the data collection phase. After testing various positively charged ions like Sodium and Calcium, this week I assembled my vesicles with water and a 0.1 M DMSO (Dimethyl sulfoxide) solution.
Testing with water served as my control group. Since water lacks the charged ions found in my previous solvents, it provides a clear baseline. It allows me to see exactly how the lipids assemble without any chemical interference on their negatively charged outer shells.
On the other hand, DMSO is a vital liquid to test. In the medical and cosmetic fields, DMSO is widely used for its ability to easily penetrate the human skin. Since my end goal is topical delivery, understanding how a penetration enhancer like DMSO affects the actual formation and yield of the vesicles is highly relevant to my vesicle assembly. Furthermore, because many compounds use DMSO as their primary solvent, it is important to understand its role in vesicle formation. We need to figure out if DMSO can safely coexist with the lipid bilayers, or if it's actually disrupting and breaking down the vesicle structure during assembly.
To keep my data perfectly consistent, I followed the exact same hydration and five minute vortex (rigorous mixing) protocol for these new liquids. I have already finished the three replicates for the water control group and observed the results under the microscope. As expected, there was essentially no yield, since there were no ions to help with vesicle formation.
Tomorrow I will conclude this week's work with my final task, which is to capture the fluorescence microscopy images for the DMSO replicates. Once I finish taking those pictures, my core experimental phase will be finished. I hope to analyze my data next week. Looking forward to sharing some exciting results.
Thank you!
Samahith
Week 6: Revisiting Inconsistencies and Literature Review
Welcome back to my blog! If you are following my project, you know that my research focuses on improving the quality and yield of Multilamellar Vesicles. These are microscopic, multilayered fat bubbles used to deliver drugs and cosmetics directly through the skin, which will eventually allow us to replace painful injections. Over the past five weeks, I have been testing how different salt solutions interact with lipids to increase vesicle formation and uniformity.
This week marks the official end of my data collection phase, but science is never perfect on the first try. While reviewing my images, I noticed a large inconsistency in my dPBS (stable solvent that mimics the human body’s natural conditions) replicates regarding yield and formation. All my samples had varying amounts of MLVs of different sizes. I dedicated this week solely to work out any problems and finalize my results.
I did not want to cherry-pick my final results. I decided to redo some of the experiments. I re-ran the thin film hydration protocol for a new dPBS batch. I am happy to say that the new samples are all consistent. This means the outlier that I previously got was just a minor execution error during the lipid drying phase and not a failure of the solvent or my protocol.
Since my lab work is finally wrapping up, I also spent a good chunk of this week doing literature reviews for my final research paper. I went back to a research paper by Melcrova and her team about how Calcium cations interact with phospholipid bilayers. This really helped me understand the exact chemistry behind why my Calcium samples from Week 3 were clumping up so much. I also read a foundational paper by M.J. Hope on vesicle generation techniques, which was very informative.
Next week, I will finally run my images through the ImageJ software to finalize the quantitative data for my experiments. Looking forward to sharing some exciting results. Thank you!
Samahith
Week 7: Image Processing and Tile Scanning
Week 7: Image Processing and Tile Scanning
Welcome back to my Senior Project blog! As a quick reminder, my research focuses on optimizing the production of Multilamellar Vesicles. These are microscopic bubbles that can deliver drugs and cosmetics through the skin and potentially replace painful injections. Over the past six weeks, I completed all my wet lab work, which consisted of testing the effect of different salt solutions on the formation and yield of these vesicles.
This week marks the next phase of my project. I am officially moving away from the lab to my computer to begin Phase 3 of my methodology. Now that I have captured hundreds of fluorescence microscopy images, I need to digitally process them to quantify my data.
My primary task this week was creating tile scans. When looking through a fluorescence microscope, you only see a tiny fraction of the sample at one time. To get a complete picture of the overall vesicle yield, I used a software program called ImageJ to stitch nine separate images together into one giant map. This gives me a comprehensive view of the entire lipid film and ensures I am not biased towards the most populated spots to analyze.
After I made some of the tile scans, I had to prep them for actual counting. Computers are not great at analyzing glowing colored circles on a dark background, so I converted every stitched image into an 8-bit grayscale format. I then applied a standardized brightness and contrast filter to the images. This step removes all the background noise and turns the vesicles into quantifiable shapes, making them incredibly easy for the software to identify accurately.
By standardizing this digital process, I am removing any personal bias from the results. Tomorrow, my goal is to finish applying these grayscale filters to all my control group images. Once every single file is perfectly prepped, I will dedicate all of next week to actually counting the vesicles and running the final statistical analysis. I might also try to train a YOLO (image detection model) to count for me. Looking forward to sharing my final results.
Thank you for reading!
Samahith
Week 8: Data Analysis and Final Graphs
Welcome back to my Senior Project blog! As a quick reminder, my research focuses on optimizing the production of Multilamellar Vesicles. These are microscopic bubbles that can deliver drugs and cosmetics through the skin and potentially replace painful injections. Over the past six weeks, I completed all my physical lab work by testing how different salt solutions affect the formation and yield of these vesicles.
This week, I started extracting data from my images. To do this properly, I usually rely on a tool called a tile scan. Because a high-powered microscope captures only a very tiny square of the sample at a time, a tile scan stitches together many small images to create a map of the entire lipid film. I successfully did this for my Sodium and Calcium samples. However, I ran into an issue while stitching the images for my water and dPBS groups. The software could not align the pictures properly because my setup was incorrect. I completely restarted the digital stitching process for these two groups, but this time the software failed again. To continue the project, I decided to change my approach and decided to just use the raw individual images to count the vesicles for all my groups.
Despite that issue, this week was incredible because I finally translated my visual observations into quantitative data. My main goal was to manually count the number of vesicles formed in every single image to determine the overall yield. I compiled all of this raw data into a spreadsheet to calculate the average yield, CV, SEM, and SD for each solvent. I then created figures to easily compare the different solvents.
Going into tomorrow, my goal is to put the finishing touches on these graphs and ensure all my data tables are formatted correctly. With the data analysis phase complete, I will dedicate next week to writing my final research paper. Thank you for following along!
Samahith
Week 9: Drafting my Final Paper
Welcome back to my Senior Project blog! As a quick reminder, my research focuses on optimizing the production of Multilamellar Vesicles. These are microscopic bubbles that can deliver drugs and cosmetics through the skin and potentially replace painful injections. Over the past six weeks, I completed all my physical lab work by testing how different salt solutions affect the formation and yield of these vesicles.
This week marked the beginning of the final phase of my project. I officially finished data analysis and am now focusing entirely on writing my final research paper. The goal was to take all my raw data, graphs, and hypotheses and combine them into a research paper and poster.
I decided to focus heavily on writing and to get my paper done early. I spent the first two days writing the entire formal paper and followed the standard scientific structure.
I started with the introduction with the goal of making audience familiar with the challenges that are encountered during vesicle production currently and holding back vesicle technology. I then detailed my methodology so other researchers could replicate my thin-film hydration process. The most important part was the results and discussion section, where I explained how my graphs definitively prove that single charge ions like Sodium create significantly higher and more uniform yields than double charge ions like Calcium.
Next week, I will send the paper to my internal and external advisors for feedback and begin my final revisions. I will also work on adding greater detail to my methodology and results section. Finally I will format my paper in the ACS format.
Thanks for reading!
Samahith
Week 10: Finishing up my Senior Project
Welcome to the final week of my Senior Project blog! If you have been following along, you know my research focuses on improving the manufacturing process for Multilamellar Vesicles. These are microscopic bubbles that can carry drugs and cosmetics through the skin to replace painful injections. Over the past ten weeks, I tested different salt solutions, collected and analyzed data, and drafted my research paper.
This week was all about wrapping up my project. Since I finished the bulk of the writing last week, I spent the past few days writing the abstract and the conclusion. I made sure my conclusion highlighted the real-world impact of this research. By standardizing this process, this project supports the growing nanomedicine market and helps pave the way for more topical treatments.
I also spent time editing to ensure my theories and hypotheses were explained perfectly. I want to make sure the paper clearly highlights how single-charge ions like Sodium preserve membrane flexibility while double-charge ions like Calcium cause rigidity and clumping.
Going into tomorrow, my goal is to finally send the completed draft to my internal advisor and my on-site mentor. Once I get their feedback over the weekend, I will make any necessary revisions and officially submit the final research paper.
With the paper almost complete, I look to finish up my slide deck for my presentation as well as my poster. Thank you so much to everyone who followed my project!
Samahith