Researcher(s)
- Kelly Czapor, Biomedical Engineering, University of Delaware
Faculty Mentor(s)
- Christopher Price, Biomedical Engineering, University of Delaware
- Kristi Kiick, Biomedical Engineering, Material Science and Engineering, University of Delaware
Abstract
Nanoparticle-systems are widely used for the targeted and controlled delivery of various therapeutics. We are developing novel peptide-based elastin-collagen nanovesicles(ECnVs) for delivering disease modifying anti-osteoarthritis drugs, like dexamethasone(Dex),to injured/diseased joints. Elastin’s lower critical solution temperature behavior allows for tunable thermoresponsive, on-demand cargo release, while collagen-like peptides(CLPs) form triple helices that stabilize the elastin-like peptides(ELPs) and permit binding/targeting to denatured collagen(found in abundance in injured joints). Together, these characteristics permit enhanced particle retention and in vivo drug-delivery. This work focuses on i) investigating the cellular uptake of ECnVs in both naïve and LPS-Activated RAW264.7 macrophages and ii) Dex’s anti-inflammatory effects in activated macrophages.
To construct ECnVs, ELPs and CLPs having the sequences (VPGWG)6G’ & (GPO)8GG) respectively, were synthesized via solid-phase peptide synthesis, purified(by HPLC), and confirmed(by mass spectroscopy). Peptides were conjugated via click chemistry and the resultant ELP-CLP conjugates (named W6G8) can undergo self-assembly to form ECnVs.
Macrophages are typically spherical, but take on a more spindle-like, branched shape when activated(by LPS). Cell morphology was assessed using epifluorescent and confocal microscopy after staining with CellMask Blue. RAW264.7 cells treated with 1:1000 LPS media exhibited the expected morphology of activated macrophages. Interestingly, somewhat similar morphological changes were observed when 1:100 ECnVs were added to RAW264.7 cells. When AZ647 labeled ECnV were added to RAW264.7 cells, fluorescent ECnVs were found to be internalized by both activated and inactivated cells, with the greatest uptake observed in media containing both 1:1000 LPS and 1:100 ECnVs. Dex treatment of LPS-activated RAW264.7 cells(at 10nM, 100nM, and 1000nM) reduced the production of inflammatory markers(nitric oxide, NO) and restored cell morphology. Future work will seek to expand this data using ELISA assessment(IL-1B) and investigate the effects of Dex-loaded ECnVs on both activated and inactivated macrophages.