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Drug Delivery

Characterization of VEGF Specific Affibodies for Use in Complex Wound Healing

(L-448) Characterization of VEGF Specific Affibodies for Use in Complex Wound Healing

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row L - Poster # 448

Presenting Author(s)

MF

Madeleine Ford (she/her/hers)

Undergraduate Researcher
Hettiaratchi Lab at the University of Oregon Knight Campus
Eugene, Oregon, United States

Co-Author(s)

JS

Justin E. Svendsen, BS

PhD Candidate
University of Oregon
eugene, Oregon, United States

Primary Investigator(s)

Marian H. Hettiaratchi

Assistant Professor
University of Oregon Knight Campus for Accelerating Scientific Impact, United States

Co-Author(s)

SO

Simon C. Oh

Post-bacc
University of Oregon, United States

Introduction:

Angiogenesis, the formation of new blood vessels, is a critical part of all wound healing. Angiogenesis functions via a protein cascade, where different growth factors are released at different rates and at different times. This cascade can be interrupted or diminished through physiological conditions (like diabetes) or wound size and complexity. Current approaches are unable to maintain sustained growth factor delivery, resulting in suboptimal healing and an increase in detrimental side effects. Therefore, there is a critical need for improved protein delivery vehicles to restore angiogenic protein presentation during injury. My specific research focuses on the angiogenic protein, vascular endothelial growth factor (VEGF). VEGF plays a role in the healing of multiple tissue types making it important for complex musculoskeletal injuries. To improve healing of wounds like these we aim to deliver VEGF in a temporally controlled manner. We have bioengineered tunable affinity affibodies, that will be conjugated to a hydrogel, for this purpose. Affinity-based hydrogels have shown promise in the delivery of other therapeutic proteins. We currently have three different strength affibodies that bind to VEGF with high, moderate, and low affinities. The goal of this study was to further characterize the affinities of these affibodies their ability to control the release of VEGF from an affinity-based hydrogel.

Materials and Methods:

Fluorescence and magnetic activated cell sorting (FACS/MACS) were used to select for functionality during yeast surface display and find a preliminary affinity value, measuring only dissociation constant. Soluble affibodies were expressed in E. coli containing a pET28b+ plasmid. His-tagged affibody was purified from lysed bacteria cells via metal affinity chromatography. Gel electrophoresis was used to verify purity of all affibodies. Biolayer interferometry (BLI) was used to determine the rates of dissociation and association between VEGF and the affibody, beyond what was found in yeast surface display. Knowing both association and dissociation rate constants allows us to predict release profile. We measured VEGF release from affibody-conjugated hydrogels. Affibodies were conjugated to polyethylene-glycol maleimide (PEG-mal) through the C-terminal cysteine on the affibody and crosslinked with dithiothreitol to form a 5% w/v affibody-conjugated hydrogel (see Figure 1A). Hydrogels are loaded with VEGF protein at a 500:1 molar ratio of affibody to VEGF and placed in a buffer solution of phosphate buffered saline with 0.1% w/v bovine serum albumin. The supernatant was collected and replaced with fresh BSA in PBS at specific time points over 21 days. A VEGF-specific enzyme-linked immunoassay (ELISA) was used to measure the amount of protein released at each time point.

Results, Conclusions, and Discussions:

Results: Preliminary affinity values were measured using affinity ramps (Figure 1B) and three affibodies were successfully expressed and purified at the following affinities: high (dissociation constant, KD = 58.3nM), moderate (KD = 307 nM), and low (KD = 6470 nM) affinities (Figure 1C). BLI data was collected for our high-affinity affibody and fit to a binding kinetics model (Figure 1D). BLI showed a koff of 3.55x10-3 min-1 (representing rate of dissociation), a kon of 1.43x105 M-1 min-1 (representing rate of association), and a combined dissociation constant, KD of 24.9 nM. This data was well fit to the model (R2 = 0.96 and a relative X2 = 1.90). Additionally, KD measurements collected via BLI were within one order of magnitude of the preliminary affinity ramp measurement. Controlled release experiments from affibody-conjugated hydrogels were performed, and ELISAs are ongoing.

Conclusion: VEGF-specific affibodies at different affinities were successfully generated and purified. Yeast surface display demonstrated variable affinities suitable for a controlled release platform. Affinity kinetics of the high-affinity affibody were successfully characterized using BLI. This work shows the promise of affibody-hydrogel release platforms for the temporally controlled delivery of VEGF.

Discussion: Future directions include completing ELISAs for all controlled release time points and conducting BLI on moderate- and low-affinity affibodies. We will also use our hydrogels to deliver VEGF to mouse micro vascular fragments in vitro and measure cellular response to released VEGF. This work is also being expanded to other angiogenic growth factors including platelet derived growth factor (PDGF) and fibroblast growth factor 2 (FGF-2).