Poster Y16 - Baculoviral Growth Factor Eluting Nanocomposite Hydrogel Fabricated via Core-Shell Microfluidics to Enhance Cellular Uptake in Wound Healing Applications

Introduction: To maintain homeostasis, the human body is equipped with a robust set of mechanisms that respond to even the slightest injury with a precisely-timed signal cascade that initiates healing. In some bodies, co-morbid conditions or medical interventions dysregulate these pathways which severely impairs healing. Within wound healing applications, chronic and ischemic conditions along with rapid environmental clearance necessitate the design of a system that favors both rapid cellular uptake and sustained release within the wound bed, edges, and peri-wound area. Hydrogel delivery systems based on core-shell structure exploit the advantageous properties of multi-layer materials to offer enhanced protection, encapsulation, and controlled release of biologics. To reduce reliance on a dysfunctional signaling cascade, we have developed a localized gene therapy that can instruct cells to produce the proteins needed for recovery, directly at the site of injury by using a hierarchical encapsulation strategy for the delivery of engineered recombinant baculovirus expressing wound healing growth factor vascular endothelial growth factor A (VEGFA) to injured tissues. Baculovirus, encapsulated in PLGA nanoparticles is used as the core material in core-shell microfluidic device to fabricate nano-embedded gelatin hydrogel microparticles with tunable physiochemical and release properties. Both sustained release of baculovirus and cellular uptake of the virus-loaded nanoparticles are optimized to provide prolonged growth factor expression throughout the wound healing cascade to overcome the challenges of impaired wound healing.

Materials and

Methods: Gene Cloning and Baculovirus Production:
Recombinant baculovirus expressing VEGFA was generated by incorporating the genes for human VEGFA and eGFP into a pOET6 plasmid to generate a VEGFA-eGFP-pOET6 plasmid. This plasmid was amplified by chemical transformation in DH5alpha E.Coli. Recombinant baculovirus was produced by transfection in Sf21 cells. Viral titer was determined by observation of cytopathic effects in Sf21 cells using end-point dilution method.
Nanoparticle Synthesis:
Nanoparticles were created by double emulsion solvent evaporation. First using the homogenizer, the virus was slowly injected into a PLGA-dichloromethane (DCM) solution. The resultant dispersion was then quickly injected into 20mL PVA under ultrasonication. The final nanoparticle suspension was stirred for 2 hours at 700 rpm to evaporate the DCM and used immediately to prevent aggregation.
Hydrogel Synthesis:
To create the hydrogels, various concentrations of gelatin were dissolved in deionized water. These concentrations were combined in various ratios with the encapsulated baculovirus nanoparticle suspension using a core-shell microfluidic device. Flow conditions within the device were tuned to achieve successful hydrogel nanocomposite production.
Characterization:
In vitro characterization of nanoparticles was done by DLS, TEM, and cellular uptake by fluorescence microscopy using coumarin-6 tagging in human umbilical vein endothelial cells (HUVECs). Efficacy and safety of the virus was investigated in HUVEC cells by MTT assay and GFP expression. Hydrogels were characterized by SEM, swelling, and virus release profile.

Results, Conclusions, and Discussions: Current wound healing dressings are effective in promoting a wound-healing environment, but are limited by their ability to regulate the wound healing cascade and promote later stages of revascularization. By exploiting nanocomposite materials, we offer a bioactive hydrogel for localized gene therapy to promote wound healing with sustained virus release, and nanoparticle-mediated cellular uptake. Preliminary data suggests that the PLGA nanoparticles prepared were spherical with a particle size of 388 nm. Successful cellular uptake of PLGA nanoparticles eluted from gelatin hydrogels was confirmed via fluorescence microscopy which showed nanoparticle localization within the cytoplasm of HUVEC cells, indicating cellular uptake of the nanoparticles. By embedding the nanoparticles within the gelatin hydrogel, sustained release of the nanoparticles was observed with prolonged GFP fluorescence in transduced cells, indicating successful transduction and expression of the growth factor gene of interest. By providing sustained release and enhanced cellular uptake, this technology opens the door to clinical applications of gene therapy using baculovirus to support tissue regeneration.

Acknowledgements (Optional): Funding provided by the Natural Sciences and Engineering Research Council of Canada, Fonds de recherche du Québec – Nature et technologies, Canadian Institute of Health Research, and the Ministry of Higher Education of the Arab Republic of Egypt.