(357z) Self-Assembled Recombinant Protein Nanomaterials for Treatment of Sars-Cov-2

Research Interests:

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 micelles. Although the immediate concern is to combat SARS-CoV-2 infection, the strategy is modular, in that domains of proteins embedded in the nanostructurescan be swapped with other bioactive domains of different specificity as required as new infectious agents, such as mutant strains, emerge.

Research Skills:

Molecular Biology, Protein Engineering, Protein characterization, Self-Assembly, Bioconjugation, Colloidal Nanoparticle formulation and characterization, Basic microfluidic techniques, Cell and Tissue culture, In vivo model and data interpretation, Biomedical Imaging (especially Ultrasound), Advanced Microscopy techniques.

Leadership and Mentoring Experience.

As part of a team and team leader in the bioengineering industry R&D, I will draw from my previous experiences as a grad student and a postdoc. I have been in charge of the design and day-to-day running of multiple simultaneous projects involving several lab members and collaborators across science, engineering, and medicine throughout my postdoc and a portion of my Ph.D. I have spearheaded and written proposals which were funded by multiple federal and state agencies. In terms of outreach, I have been part of an initiative in developing online modules for Philadelphia high school teachers to use with their students. I have co-organized hands-on demonstrations on behalf of my lab for outreach when middle/high school students from across Philadelphia take part in “exhibits, demonstrations, tours, and talks highlighting nanotechnology related research and its importance in our lives”. I have been a Teaching Assistant in core courses, during which I had the opportunity to prepare exams, design course structure, conduct office hours, recitations, and give several independent lecturesas part of my training. I have experience giving guest talks in tutorials and as invited speakers in conferences. I have had the rare opportunity of supervising as many as several undergraduate researchers over the course of my PhD and postdoc, including guiding some through their undergraduate senior theses. Additionally, I have trained numerous new graduate students and postdocs in lab techniques.

Additional Research Expertise and Experience.

My experience in graduate school and as a postdoc has been highly interdisciplinary, with focus on protein design, nanoparticle functionalization, 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. I have also designed microbubbles containing therapeutic gases to treat traumatic brain injuries in large animal models, as demonstrated by MRI scans and histopathology of a porcine brain. 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.

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.

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. $50,000.
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. $50,000.

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.