(3ec) Stabilization of Therapeutic and Water-Soluble Gas Microbubbles By Phospholipids and Recombinant Proteins for Ultrasound Mediated Theranostic Applications

Research Interests: Microbubbles (MBs) are 1-10 µm gas-filled particles, stabilized by phospholipids, polymers, or proteins, and used primarily for contrast imaging, along with more recent applications in drug/gene delivery, sonoporation, photoacoustics, among others. Traditionally, MBs contain an insoluble fluorocarbon core gas to prevent dissolution of the bubble in aqueous media. In recent years however, MBs have shown promise as carriers of water-soluble therapeutic gases, such as oxygen, and more recently, xenon. Stabilization of highly water-soluble gases inside a MB core is challenging due to a high chemical potential gradient with the bloodstream.

First, we have developed a new class of therapeutic microbubbles containing 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. Delivering Xe locally to the injury site has been a challenge due to non-specificity of inhalation protocols. Encapsulating pure Xe inside stable sub-10 µm bubbles had thus far not been achieved due to the high aqueous solubility of Xe as opposed to that of traditionally used PFCs. Optimization of the shell composition of these bubbles led to significant and persistent ultrasound contrast both in phantoms and in vivo, making them excellent potential candidates for image-guided therapeutic gas delivery, with ongoing studies being planned for treatment in a large animal porcine brain injury model.

Second, in addition to therapeutic gases, stabilizing water-soluble gases in MBs in general can be a hurdle. Nitrogen, for instance, would prove a much cheaper and more widely available gas than fluorocarbons; however, traditionally used phospholipids can keep nitrogen stable in MBs only for a few hours. We have discovered a new material, a recombinantly produced variant of the protein oleosin, that is extremely effective in stabilizing nitrogen in the MB core. Because of a lack of a robust secondary structure, oleosin molecules can stabilize bubbles without the need of denaturation, unlike albumin UCAs. Furthermore, using recombinant methods to form the UCA shell material allows for bottom-up functionalization of the shell instead of post-formulation chemical modification and necessary washing steps. Most importantly, however, we observe that oleosin MBs with a nitrogen core show exceptional stability for weeks along with a robust acoustic signal. Variants of this molecule are thus promising materials for formulation of functional bubbles, and potentially droplets and vesicles, with different therapeutic core gases.

Successful Proposals: “Transdisciplinary Awards Program in Translational Medicine and Therapeutics, 2020-2021” grant awarded by the Perelman School of Medicine, University of Pennsylvania, for work on xenon microbubbles. PI: Prof. Misun Hwang. Role: Co-author.

UPenn internal seed grant based on (Horing Fund) for Covid-19 research, 2020-2021. Proposal title: Functionalized lipid inactosomes to bind and clear SARS-CoV-2. PI: Prof. Daniel A. Hammer and Prof. Daeyeon Lee. Role: Co-author..

Postdoctoral Projects:

“Ultrasound responsive xenon microbubbles for imaging and neuroprotective therapy.”

Under supervision Daeyeon Lee (Chemical and Biomolecular Engineering), Chandra M. Sehgal (Radiology) at the University of Pennsylvania, and Misun Hwang (Radiology) at the Children’s Hospital of Philadelphia.

“Amphiphilic recombinant protein engineering for design of liposomes and droplets for treatment of the SARS-CoV-2 virus”. Under supervision Daniel A. Hammer and Daeyeon Lee, Chemical and Biomolecular Engineering, University of Pennsylvania.

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. That experience enabled one of my two current projects to design functional amphiphiles to self-assemble into vesicles to treat the Sars-CoV-2 virus. The formulation of colloidal structures has also provided me with experience in microfluidics and soft lithography so as to create bubbles and vesicles of uniform size and composition. Lastly, the other half of my postdoc has yielded the formulation of echogenic xenon microbubbles, which has led to an ongoing collaboration with clinicians at the Children’s Hospital of Philadelphia. All of this has provided me with a varied knowhow in the fields of micro and nanomaterial fabrication, imaging techniques, ultrasound therapy, animal model design for tumors or injuries, microfluidic procedures, and recombinant protein synthesis, 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. Additionally, I have experience mentoring new graduate students as well as supervising 7 undergraduate researchers over the course of my PhD and postdoc, including mentoring 2 of them on their undergraduate senior theses. As a faculty member, I would like to design elective courses encompassing (1) colloidal and interfacial phenomenon in biology, (2) Biomedical Imaging, (3) Nanobio interactions, in addition to teaching core chemical, bio, and materials engineering courses.

Future Direction: As faculty, I would like to establish two key sides to my research: (1) Uniform microbubbles and nanodroplets with different free or dissolved gas cores, and (2) Droplets, bubbles, and vesicles functionalized with recombinant proteins and their ultrasound-mediated biointeractions. For area (1), I will use my knowledge about droplet and bubble fabrication and microfluidics to design particles with xenon, argon, oxygen, and nitric oxide cores for image-guided therapeutic gas delivery for treatment of in cancer and hypoxic ishemic injury related conditions. Inspiration for area (2) comes from a vast amount of relevant literature suggesting that altering functional groups can greatly affect protein interactions, complement activation, cell interactions, immune response, and targeting. Recombinant proteins can be functionalized from the bottom-up by genetic engineering of the plasmid instead of complex chemical functionalization and post-formulation washing. I will unite my knowledge about droplet/vesicle functionalization with aptamers and peptides from my PhD with that of recombinant protein design during my postdoc for developing particles to tune their interactions with physiological targets for stimulus (pH, pressure) sensitive imaging and protein delivery. My hope as a faculty will be to collaborate with clinicians to both study the fundamental dynamics of biocolloids under physiological conditions and to apply those results in the localized therapy of tumor and injury models.

Selected Publications:

• 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.
• Chen, R. Chattaraj, K.W. Pulsipher, M.B. Karmacharya, D.A. Hammer, D. Lee, and C.M. Sehgal. “Photoacoustic and Ultrasound Dual-Mode Imaging via Functionalization of Recombinant Protein-Stabilized Microbubbles with Methylene Blue.”ACS Applied Bio Materials, 2019, 2(9), 4020-4026.
• Chattaraj and A.P. Goodwin. “Design and application of stimulus-responsive droplets and bubbles stabilized by phospholipid monolayers.” Current Opinion in Colloid & Interface Science, 2018
• 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, 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.