Bossone Research Center, Room 709, located at 32nd and Market Streets. Also on Zoom.
BIOMED Master's Thesis Defense
Title:
Design of “Armored” Lipid-Based Nanoparticles for Prolonged Drug Circulation via Complement Pathway Attenuation
Speaker:
Emily Wolfe, Master's Candidate
School of Biomedical Engineering, Science and Health Systems
Drexel University
Advisors:
Jacob S. Brenner, MD, PhD
Assistant Professor
Perelman School of Medicine
Associate Director, Penn Health-Tech
University of Pennsylvania
Kara L. Spiller, PhD
URBN Professor of Biomedical Innovation
School of Biomedical Engineering, Science and Health Systems
Drexel University
Details:
Nanomedicine shows great promise in drug delivery due to its versatility in drug loading and ability to target the tissue of interest; however, its current applicability is significantly hindered by the innate immune response. The complement system is a set of blood-circulating proteins that differentiate foreign entities from host cells in order to aid in the rapid removal of pathogens.
The alternative pathway, specifically, remains a stringent barrier to efficient therapeutic delivery via nanocarriers. In this cascade, complement is initiated via random hydrolysis of the C3 protein, leading to mass deposition of the C3b portion on the nanoparticle surface. C3b is a recognizable ligand for circulating macrophages, which bind to the subunit through the CR1 receptor and engulf the entity. The other subunit resulting from C3 cleavage is C3a, an anaphylatoxin that contributes to complement-activation-related pseudoallergy (CARPA)--an acute distress syndrome characterized by hypotension, bronchospasms, and capillary leak. Currently-approved nanomedicines induce CARPA in clinical settings, worsening patient outcomes.
It has been previously found that conjugating regulators of complement activation (RCAs) to lipid-based nanoparticles (NPs) not only decreases C3-adduct formation, but also increases targeting efficiency in vivo. However, the proposed NP formulations were not suitable for scaled-up manufacturing due to their short-term instability and poor conjugate retention. Full protein conjugates are not optimal candidates for automated drug formulation systems, given their complexity and size. Therefore, the purpose of this work is to find a new sub-protein RCA candidate that could prove viable in manufacturing processes, while maintaining complement-attenuating functionality when presented on the nanoparticle surface.
Four candidates are presented in this work, all of which provide replacement mechanisms for conjugating Factor H (FH)--the inhibitory protein responsible for C3b surface recognition and inhibition of alternative pathway amplification. The first conjugate, minimal-size FH (mFH) is a FH subunit solely composed of the functional areas required for C3-adduct inhibition. A FH-binding oligonucleotide sequence, or aptamer (FHA), is the second candidate, and has been previously used in affinity purification of the full protein. The third and fourth candidates are linearized and circularized versions of a FH-binding peptide (FHP), which have been previously determined to attenuate C3 hydrolysis.
To be considered industrially feasible, conjugated NPs first need to have an established conjugation efficiency threshold to account for loss of the moiety during fabrication and produce uniformly-decorated particles. The resulting NPs also need to be stable in solution in order to prevent increased rates of clearance in vivo. Here, we confirm manufacturability ratings of each candidate through size-exclusion chromatography, dynamic light scattering, and particle counts.
Functionality post-conjugation is also considered, as fabrication processes may lead to steric hindrance of the candidate’s active site. Each candidate’s viability as a solution for in vitro C3-adduct formation, as well as peptide and aptamer FH-binding capabilities are assessed. The linearized FHP proves to be the leading contender, due to its dramatic depletion of C3 hydrolysis in comparison to the positive control.
Finally, this work investigates the translation of the chosen candidate, linearized FHP, into complex nanocarriers. Nuances surrounding lipid nanoparticle formulation processes, size, drug encapsulation efficiency, and charge introduce new challenges in conjugating the sub-protein moieties listed in this study. The knowledge gained from this work can drive innovations in immune-evading drug delivery mechanisms, potentially improving patient outcomes by reducing necessary dosing regimens and enhancing therapeutic targeting capabilities.