Veronesi Lab

Life Health Sciences Internship

Fa21-Sp22

Veronesi Lab Overview

Intranasal delivery of cancer drugs has the potential to bypass the blood brain barrier (BBB) by adsorption through the trigeminal nerves (red) and olfactory neurons (blue). (Adapted from Veronesi et. al., 2020).

Fighting for a Cure

This academic year, I am working with Dr. Michael Veronesi through the Life Health Sciences Institute (LHSI) at IUPUI to develop nanoparticle treatments for glioblastomas (GBMs). Glioblastomas are aggressive brain tumors that resist available chemotherapy, surgery, and radiotherapy treatments. Cell culture studies have demonstrated that some drugs may be effective against GBM, but the drugs must be able to reach the tumor in order to have therapeutic effects. Nanoparticles (NPs) may be able to bypass the blood brain barrier and help facilitate transport of drug to the brain, especially if they are delivered through nose-to-brain pathways. The research objective of the lab is to develop a therapeutic NP treatment which can be delivered to rats intranasally and harnessed for in vivo imaging.

Final Project

Many Minds Make Light Work

Developing intranasally delivered, theranostic nanoparticles to treat glioblastomas in rat models combines expertise in chemistry, biology, medicine, pharmacology, and engineering. The flowchart below demonstrates how disciplinary specialties are combined in the quest to innovate translatable cancer therapeutics. As a biomedical engineering major, I primarily contribute to aerosolizer development. After gaining more experience in the lab, I am now also seeking to contribute to the intranasal delivery and in vivo aspects of the work.

Learning and Skills

My Project

Glioblastoma multiforme (GBM) is a highly malignant, difficult-to-treat brain tumor with a median survival time of only 15-20 months. GBM treatments are especially hard to deliver because most drugs cannot cross the blood brain barrier. Intranasal delivery (IND) is a promising drug delivery strategy which may allow drugs to access the brain directly through nose-to-brain pathways. In this project, a small animal IND device (“aerosolizer”) was characterized and optimized for the delivery of therapeutic nanoparticles/drugs intended to treat brain diseases. The size of aerosolized droplets was measured under various conditions relating to aerosolizer design, actuation pressure, distance from the target site, and drug properties. This data can be used to inform aerosolization conditions when delivering therapeutic nanoparticles into the nasal cavities of small animals during drug testing. By optimizing the aerosolizer for small animals, drugs can be assessed for nose-to-brain delivery in the treatment of neurological diseases prior to testing in humans.

The rat nasal cavity has many similarities to the human nasal cavity (Adapted from Veronesi et. al., 2020).

Top: Side view of aerosolizer. Bottom: Schematic flow of air (purple) and drug (blue) through the top view of the aerosolizer. (Adapted from Veronesi et. al., 2020).

My Contributions

My contributions have impacted the larger lab work of developing nanoparticle treatments for GBM. By characterizing aerosolizer performance under different conditions, I have established data which can inform aerosolization conditions when using the device in animals. As I transition to in vivo studies, I hope to also demonstrate how aerosolized particles are distributed and taken up in the rat nasal cavity. If our drug-loaded nanoparticles are shown to be effective against GBM, this data will elucidate whether nose-to-brain pathways may have played a significant role in the therapeutic effect. In contrast, if the nanoparticles are ineffective, the intranasal delivery data may shed light on which step of drug transport to the brain is ineffective. Most importantly, we take a step closer towards improving the current treatments options for GBM.

Annotated picture of the aerosolizer as it fires drug solution onto a microscope slide for analysis.

Progress and Accomplishments

Fall

In the fall, I developed a microscope slide preparation method which uses an optimized mixture of paraffin oil and white petrolatum to allow visualization of intact droplets under an inverted microscope. This technique provides a more accurate particle size than the previous methods, which used filter paper and an iPhone camera.
By applying MATLAB skills from my BME classes and utilizing online documentation to teach myself ImageJ, I developed a processing protocol which uses ImageJ and MATLAB to return information about the average particle radii in microscope images. With this method, I analyzed original and iterative aerosolizer designs, the latter of which I created in Fusion 360 by relying on my previous experience with CAD software.
Outside of working on the aerosolizer itself, I transferred the controller box circuit from a cardboard box to a more durable, 3D-printed box. My exposure to circuit prototyping in BME 224 proved very helpful to this endeavor.

Spring

I created a MATLAB script which parses slow-motion video footage frame-by-frame to determine the velocity of fluid ejected from the aerosolizer. After researching the basics of fluid dynamics, I calculated the Reynold's number from the velocity and demonstrated turbulence for each aerosolizer design.
This spring, I improved my literature review and scientific writing skills by submitting a thesis proposal to continue my research. In addition, I created a project summary PowerPoint presentation through which I had the chance to practice using visuals to communicate scientific methodology and results.
My primary focus this semester has been on transitioning to animal work, which has included: receiving training on how to handle and care for rats, learning the surgical procedure for GBM implantation, and planning a new experiment to characterize nasal mucosa uptake following intranasal delivery.
In working on experiment development, I have been researching fluorophores and testing their compatibility with various decalcifying solutions. In addition, I have been working with the Indiana Center for Biological Microscopy to determine an appropriate method for tissue sectioning and visualization.

Favorite Moments

Throughout my work over the past two semesters, I have found the in vivo work to be the most humbling and gratifying. Performing GBM implantations is a high-risk, high-responsibility endeavor which requires excellent surgical skill, careful attention to detail, and diligent post-operative monitoring. The learning curve is tremendous and has required me to extend patience and grace to myself and others. In the process, I have discovered that I love the synergy of the operating room when team members are communicating and performing their roles well. The end result is a successful outcome for the rats and a tremendous sense of satisfaction for the surgical team. I find it rewarding to be a part of this cooperative dynamic. This sense of fulfillment and enjoyment of surgery pushes my vocational interests toward medicine.

Personal Growth and Skills Development

Becoming a Better Scientist and Professional

Communication: This year has pushed me to improve my oral presentation skills through weekly lab meeting updates, and to expand my scientific reading and writing skills through literature searches and thesis proposal writing. In addition, I have learned to adapt emails and conversations to a wide variety of audiences including other lab members, students, IU faculty, animal or microscopy personnel, technical support staff at scientific supply companies, and ePortfolio visitors.

Independence and work progress: Working in a dynamic research environment has challenged me to perfect the art of planning, prioritizing, and setting goals. When I first came to the lab, I had a difficult time translating abstract research objectives into concrete weekly tasks. Through experience and observing other lab members, I learned that I could better utilize my time by creating action plans during group meetings so that my priorities would be aligned with those of the lab.

Confronting challenges and failure: Throughout the course of my internship, I have had many encounters with failure: finding the optimal slide mixture through trial and error, choosing quantum dots that turned out to be incompatible with decalcifying solutions, and losing a rat after surgery. From these experiences, I have discovered that after using all tools and resources at my disposal to plan ahead, I must be willing to risk failure when trying something new.

I want to build the habit of viewing failure as a step on the journey to success. While I still have work to do in this area, I look forward to refining my mindset during my upcoming summer internship. As my first experience in industry, I expect to experience many opportunities for growth through both my successes and my failures.

Looking to the Future...

As a future physician or engineer, I am learning skills through my internship that will help me in my vocational aspirations. For instance, developing an in vitro testing method builds upon the problem-solving skills and initiative that I will need to face unsolved design challenges as an engineer. Though animal experiments, I am learning about sterile technique and imaging topics which are relevant to the medical field. In addition, I am learning skills about how to read and write scientific literature that will benefit my ability to communicate with others in my field, keep my knowledge current, and contribute to the disciplinary body of work.

As a professional, I have become more confident in my ability to think critically, take initiative, and implement feedback from others. As an engineer, these skills would prove helpful in troubleshooting problems, collaborating with others on design ideas, and foreseeing challenges that might arise with a particular design or process. Likewise, as a physician these skills would help me diagnose patients, continually improve my practices and bedside manner, and to actively seek out ways in which I can improve patient care.

The Workplace

Successes and Challenges

Microscopy: One of my greatest successes as an intern has been the development of a method for viewing aerosolized droplets under the microscope. Previous students used an imaging method of petroleum jelly on filter paper. Switching to an inverted microscope allowed the visualization of even smaller droplets, yet required me to first develop a new oil-based mixture because petrolatum jelly was too opaque to allow light through the microscope slide. In addition to perfecting the ratio of paraffin oil to white petrolatum, I also had to optimize microwaving time to obtain the thinnest layer possible. A mixture that was too runny or too thick would be displaced by the aerosolize plume and degrade the image quality.

Organization: The other contribution of which I am especially proud is the reorganization of the aerosolizer files. When I arrived at the lab, CAD files had been haphazardly deposited into a folder and the physical aerosolizers were not labeled. After careful correlation of the physical aerosolizer properties with dimensions of the various designs, I was able to painstakingly match each file and aerosolizer while creating a better labeling system to prevent such confusion in the future.

Meeting Efficiency: My greatest challenge when I first came to the lab was learning how to most effectively communicate progress and needs during lab meetings. I didn't anticipate the difficulty of presenting experimental results in a concise and relevant manner. I also didn't realize how much a purposeful meeting agenda could improve the efficiency and clarity of communication, rather than coming with a verbal explanation of updates and questions. After noticing how other lab members presented their work, I have started the practice of making an explicit list of progress updates, questions, and action items prior to each meeting. I have found this habit very helpful and plan to continue refining it.

Animal Work: My greatest challenge thus far in the semester has been transitioning to animal work. Getting the required trainings, ordering animals, and planning experiments has turned out to involve a high degree of logistical planning, communication, and foresight. Through experience, I am learning how to navigate the various institutions and procedures for working with animals. While this process has not been perfect, I am trying to maximize every learning experience by relying on more experienced lab members for help and creating a written record of my experiences.

I faced a challenge upon ordering rats for the first time and discovering that I had specified the wrong gender and age. I was able to overcome this challenge by implementing the order change protocol and communicating with my PI and the vet staff regarding animal updates.
Recently, I learned aspects of sterile technique and successfully assisted with pre-surgery preparation and post-surgery care for twenty rats undergoing GBM intercranial implantation surgery.

Finding Mentors

Through working in the lab, I am learning about the kind of behaviors and attitudes that I wish to have as a professional from the strengths exhibited by the investigators and other students with whom I collaborate. For instance, Dr. Butch is confident in her knowledge of cell culture and uses this expertise to take decisive next steps and communicate matter-of-factly about progress with the in vitro experiments. At the same time, Dr. Butch is honest and clear about what she doesn't know. Whether in weekly lab meetings or in my future career, I want to communicate in an equally concise and effective manner while striking a balance between confidence in what I do know and openness about what I don't.

The dynamic between Dr. Das and Dr. Veronesi informs how I might contribute in a multidisciplinary team setting. Dr. Veronesi excels at keeping the larger lab goals in mind and consistently directing the team's energy towards an evolving, ambitious vision. In contrast, Dr. Das is especially attentive to details and often draws on his vast knowledge of pharmaceuticals and research methods to offer suggestions on specific protocols and analysis techniques. These two perspectives balance one another and together contribute to the overall productivity of the lab. Knowing this, I want to seek out coworkers with big-picture mindsets to complement my own detail-oriented approach in the workplace.

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Representative firing of the aerosolizer onto a microscope slide for analysis.

I also want to learn from Luke, an older student intern who improves lab cohesiveness by keeping everyone in the loop about lab-related meetings and resources. In addition, Luke makes an intentional effort to connect with younger student interns such as myself so that he can answer our questions, share hard-learned lessons, and train us on skills and technologies related to the lab. In my current and future workplaces, I want to exhibit this same intention about connecting with people and passing along knowledge.

Expectations vs. Reality

Upon joining the lab, I thought I would be trained on experimental protocols involving rats and then expected to perform experiments and report on the results. When I came to the lab, I discovered instead that my engineering expertise was needed on the aerosolizer aspect of the project, which led to an unexpected change of focus. I have since discovered that frequent pivoting in response to new information and objectives is a regular and integral part of the research setting. Through the day-to-day routine of the lab, I have discovered that research often entails a "hurry up and wait" mindset. Much background research can be poured into a decision about a single aspect of the experimental method, and experiments may entail alternating periods of urgent work and waiting. In this dynamic environment, I have grown in my adaptability and resilience and discovered a deeper level of what it means to have "attention to detail."

The innovation and persistence required to create a new microscopy method proved to be far more challenging than I ever imagined, but also more rewarding as I stretched the limits of my own creativity and learned the value of incorporating the multidisciplinary ideas of other lab team members. Likewise, transitioning to animal work has been full of obstacles and unknowns, but I have learned how to own my mistakes and be a more proactive communicator because of it. Throughout both of these experiences, I have developed an appreciation for the ideas and advice of my fellow lab members, who each bring their unique educational, vocational, and cultural background and perspectives. I will carry these lessons in how to be a better researcher, teammate, and person into the remaining months of the internship and beyond.

Posterior (above) versus medial (below) sections of the rat nasal cavity. Tissues were sectioned after decalcification and stained with H&E.

Workplace Culture

The culture in the Veronesi lab is defined by a common passion for investigating unknown questions and driven by faculty who are deeply invested in academia. The organizational structure involves close communication and daily interactions within subgroups (nanoparticle, in vitro, and in vivo), and somewhat looser communication between subgroups. To coordinate the various research activities, value is placed on exhibiting timely communication and a collaborative attitude.

One of my favorite aspects of the Veronesi lab is the comradery between members. As students and faculty go about their individual tasks in the lab, the work environment is a balance of focus and friendliness in which lab members take a genuine interest in what others are working on. From this experience and my time as an interfaith intern, I've learned that I value a tight-knit culture in which employees care about one another as people. If I could change something about my site culture, I would create a meeting structure to facilitate the transition between subgroup and whole lab communication. In the fall semester, we met weekly as an entire lab group which would sometimes result in long and inefficient discussions about small details pertaining to one subgroup. This semester, we are meeting as subgroups to cut down on meeting times and increase the relevance of meeting content. While this has greatly simplified meeting time logistics and improved efficiency, it has come at the cost of decreased sharing of ideas and progress between subgroups. I wish there was a way to meet as a whole group less frequently, perhaps once a month or a few times a semester, to learn about progress in other areas of the lab, clarify goals and deadlines pertaining to the whole group, and increase lab rapport.

Learning through Difference

Throughout my internship, I have learned about the unique backgrounds of each of my fellow lab members. Within the group, people come from the United States, the Middle East, and South Asia; celebrate religion traditions ranging from Hinduism to Eastern Orthodox Christianity to Islam; originate from all sizes of families; and have unique career histories and/or career aspirations which span teaching, medicine, industry, and research. Whenever I get the opportunity to speak with a lab member about his or her background, I find myself awed by their accomplishments and aspirations. Hearing their stories inspires me to think about how I can be a better coworker by wishing someone a happy holiday specific to their tradition, learning about the histories and cultural values of other geographical regions, and being an open-minded and respectful listener at all times.

Because I get to know each lab member on a personal level, it is easy to appreciate the diversity and commonalities of our respective stories. When it comes to larger groups of people, however, this can be more difficult. As I continue my professional journey, I want to grow my experiences with diverse populations so that I am better equipped to serve a wide variety of communities. As a future physician or biomedical engineer, understanding these needs is an important part of equitable medical device design and healthcare delivery.

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