(2el) Interfacial Design of Nanoparticles and Microbubbles for Treatment of Viral Infection and Brain Injury

Research Interests:

1. Recombinant Protein Amphiphiles for Inactivation of SARS-CoV-2:The current pandemic has laid bare the necessity of multiple therapeutic options, including, but not limited to, vaccines for efficient combating of a highly transmissible infectious disease. Especially useful would be multifunctional therapeutics that would block multiple pathways of viral infection. My chief research interest lies in leveraging the platform of nanostructures like micelles, vesicles, and droplets for assigning multifunctionality to a therapeutic platform to inactivate a viral agent. A powerful strategy to block viral infection is to use inhibitors such as ACE2-derived or de novo designed short chain peptides and mini-proteins that bind to the S1 protein with high affinity. In my work, I have developed micelles purely from a functional recombinant protein called oleosin that will bind and inactivate the Spike S1 protein. Unlike therapeutic antibodies, recombinant proteins can be formulated relatively easily, and unlike free peptides and mini-proteins in many cases, recombinant proteins are uniform, have greater circulation half-life and can be equipped with multiple functions with precision through genetic engineering. Oleosin acts as a stabilizer for fat bodies in plant seeds; it is a rare protein that can be engineered to behave as a free-chain triblock surfactant, capable of assembling into micelles and vesicles. Micelles were formed by spontaneous self-assembly of oleosin that was genetically modified with anti-S1 mini-protein and peptide sequences. Because oleosin is a protein, we can precisely incorporate into it different functional moieties at the gene level, eliminating complex post-formulation functionalization chemistry and washing steps involved in other synthetic nanostructures. Peptides don’t usually self-assemble; however, the spontaneous self-assembly of peptide-modified oleosin has enabled the inclusion of multiple peptide sequences on the same self-assembled structure that has not only blocked SARS-CoV-2 pseudovirus entry into 293T cells, but also blocked infection more efficiently compared with single functionality peptides/micelles. A therapeutic dose response showed reduction in RVP infection at concentrations as low as 5 nM functional protein in micellesAlthough the immediate concern is to combat SARS-CoV-2 infection, the strategy is modular, in I will that embed a variety of domains of proteins in the nanostructureswith other bioactive domains of different specificity as required as new infectious agents and mutant strains emerge, for not just SARS-CoV-2, but for other pathogens and toxins.
2. Ultrasound Responsive Therapeutic Gas Microbubbles: Local, non-systemic, image-guided therapy for brain ailments like traumatic brain injury or stroke is often challenging. Medical gases like xenon, oxygen, nitric oxide, are considered an attractive treatment option. An efficient way to make such treatment affordable, non-systemic, and image-guided is through the use of 1-10 µm gas-filled particles, known as microbubbles (MBs) stabilized by phospholipids, polymers, or proteins. In recent years, MBs have shown promise as carriers of water-soluble therapeutic gases, beyond their more common purpose as imaging contrast agents under clinical ultrasound. Stabilization of highly water-soluble gases inside a MB core is challenging, however, due to a high chemical potential gradient with the bloodstream.I have developed microbubbles (MBs) encapsulating pure xenon gas as a first in the field. Noble gases xenon (Xe) and argon (Ar), known to have cytoprotective effects, for treatment of hypoxic ischemic injuries in the brain with simultaneous local contrast imaging. Through optimization of the phospholipid compositions on the bubble membrane, pure Xe MBs were demonstrated to be stable and echogenic in vivo.The MBs were then shown to act positively in the neuroprotection of porcine brains subjected to traumatic brain injury via a focal contusion model. Magnetic resonance imaging (MRI) scans and histopathology of the pig brain revealed significantly reduced perilesional edema lower perivascular inflammation in the xenon bubble-treated group versus the control bubble group. This first-of-its-kind large animal study thus shows the feasibility of neuroprotection using Xe MBs, as well as providing a highly promising theranostic agent containing xenon and other therapeutic gases for local, non-systemic, gas delivery for ultrasound image-guided treatment of different kinds of injury in different organs.

Successful Grant Proposals:

1. NIH R21. Co-Author. PI: Daniel A. Hammer. “Functionalized lipid inactosomes to bind and clear SARS-CoV-2.”
2. NIH R03. 2021-2023. Authored and submitted entirety of proposalincluding pertinent data. PI: Daeyeon Lee. “Ultrasound image-guided treatment of ischemia-reperfusion injury using argon microbubbles.”
3. Pennsylvania Formula Fund 2021-2024. Authored and submitted entirety of proposalincluding pertinent data. “Image-guided delivery of xenon by microfluidically produced recombinant protein microbubbles.”PI: Daeyeon Lee.
4. UPenn Horing Grant for Covid-19 research, 2020-2021. Co-Author. PI: Daniel A. Hammer and Daeyeon Lee.
5. Transdisciplinary Awards Program in Translational Medicine and Therapeutics, 2020-2021. Co-Author. Grant awarded by the Perelman School of Medicine, University of Pennsylvania. PI: Misun Hwang.

PhD Dissertation: “Engineering the Phospholipid Monolayer on Fluorocarbon, Hydrocarbon, and Liquid Crystal Nanodroplets for Applications in Biosensing.”

Under supervision of Andrew P. Goodwin, Chemical and Biological Engineering, University of Colorado Boulder.

Research Experience: My experience in graduate school and as a postdoc has been highly interdisciplinary, with focus on biocolloid design and ultrasound imaging. For instance, my PhD involved designing the interface of nanodroplets to tune their mechanical properties or their functionalities, ultimately leading to them being used for rapid sensing of biomarkers like VEGF. The crux of my work revolved around engineering the phospholipid monolayers on perfluorocarbon nanodroplets to alter their acoustic response on interactions with analytes. Additionally, a longstanding collaboration with a postdoc led me to co-design mesoporous silica nanoparticles which were ultrasound theranostics of tumor tissue. As a postdoc, I learned another way of functionalizing the colloidal interface using synthetically designed recombinant protein amphiphiles as shell materials for microbubbles and nanodroplets. My years of transdisciplinary experience has provided me with a varied knowhow in the fields of recombinant protein synthesis, micro and nanomaterial fabrication, imaging techniques, ultrasound therapy, animal model design for tumors or injuries, microfluidic procedures, all of which I look to unite to pursue different projects as a faculty member.

Teaching Interests: My roles in graduate and postdoc positions has enabled me to acquire significant teaching and mentoring experience. I have worked as a Teaching Assistant at the University of Colorado, Boulder in core courses such as Heat Transfer and Introduction to Materials Science, while conducting a number of independent lectures during this time. I have also taught a seminar on Biomedical Ultrasound as a part of a tutorial series at the University of Pennsylvania. I have acquired a strong foundation to serve as a mentor to students in my lab and successfully teach courses both at the undergraduate and graduate levels (I plan to design and offer elective courses, such as in Ultrasound Imaging & Contrast Agents and Recombinant Proteins and Medicine).Additionally, I have experience mentoring new graduate students as well as supervising 9 undergraduate researchers over the course of my PhD and postdoc, including mentoring 2 of them on their undergraduate senior theses.

Future Direction: As faculty, my plan for the next 5 years is to develop next-generation ultrasound-responsive, recombinant proteinmaterials for image-guided therapy, for cardiac injury treatment, inactivation of viruses and toxins, and deep tissue cancer treatment. I look forward to discussing more details with recruiters.

Selected Publications:

• Chattaraj, C. Kim, D. Lee, and D.A. Hammer. “Recombinant Protein Micelles for Inactivation of SARS-CoV-2.”In Revision (ACS Nano)
• Hwang, R. Chattaraj, A. Sridharan, S. Shin, A.N. Viaene, S. Haddad, D. Khrichenko, C.M. Sehgal, D. Lee, and T. Kilbaugh. “Can Ultrasound-Guided Xenon Delivery Provide Neuroprotection in Traumatic Brain Injury?” Neurotrauma Reports,2022, 3(1), 97-104.
• Chattaraj, D.A. Hammer, D. Lee, and C.M. Sehgal. “Multivariable dependence of acoustic contrast of fluorocarbon and xenon microbubbles under flow.”Ultrasound in Medicine and Biology, 2021.
• Chattaraj, Misun Hwang, Serge D. Zemerov, Ivan J. Dmochowski, D.A. Hammer, D. Lee, and C.M. Sehgal. “Ultrasound Responsive Noble Gas Microbubbles for Applications in Image Guided Gas Delivery”Advanced Healthcare Materials, 2020.
• Chattaraj, G. M. Goldscheitter, A. Yildirim, and A. P. Goodwin. "Phase behavior of mixed lipid monolayers on perfluorocarbon nanoemulsions and its effect on acoustic contrast." RSC Advances,2016, 6,111318-25.
• Yildirim, R. Chattaraj, N. T. Blum, and A. P. Goodwin. “Understanding Acoustic Cavitation Initiation by Porous Nanoparticles: Toward Nanoscale Agents for Ultrasound Imaging and Therapy.” Chemistry of Materials, 2016,28, 5962-72.
• Chattaraj, P. Mohan, C. R. Livingston, J. D. Besmer, K. Kumar, and A. P. Goodwin. “Mutually-Reactive, Fluorogenic Reporter Molecules for In-Solution Biosensing via Droplet Association.” ACS Applied Materials & Interfaces,2016, 8, 802-808.
• Chattaraj, P. Mohan, J. D. Besmer, and A. P. Goodwin. “Selective Vaporization of Superheated Nanodroplets for Rapid, Sensitive Acoustic Biosensing.” Advanced Healthcare Materials. 2015, 4, 1790-1795.