(J-408) Development of a Crosslinked Protein-Based Microgel Delivery System for Cardiac Tissue Regeneration

Introduction:: Heart decellularized extracellular matrix (dECM) has been investigated as a therapeutic biomaterial that improves cardiac function, promotes cell proliferation, and reduces fibrosis in the infarcted heart model. While injecting dECM hydrogel in the sol state has shown encouraging results, we observe limited retention at the site of injection and burst release of loaded biologics. In this research, we demonstrated electrospray emulsion generated microparticles loaded with VEGF exhibited extended release, resulting in increased tube diameter of endothelial cells in the in vitro tube formation assay, but were challenged by particle polydispersity and batch-to-batch variability. To improve upon this, we designed a microfluidic droplet generator and investigated determinant parameters of droplet diameter by adjusting driving pressure (flow rate) and microfluidic dimensions, observing increased droplet diameter with increased nozzle diameter and input pressure independently using both food dye for visualization and Gel-MA, a modified, UV-crosslinkable gelatin, as an analogue for more viscous dECM. When emulsified and crosslinked in a modified generator-crosslinker inline setup, Gel-MA forms microgels with improved particle size distribution at a relatively high yield, paving the way for tightly controlled dECM microgel synthesis. The droplet and crosslinked microgel sizes were measured from microscope images using CellProfiler and will be later confirmed with dynamic light scattering and scanning electron microscopy. Future studies will evaluate the microfluidic generation and UV crosslinking of dECM microgels, and investigate their in vitro and in vivo bioactivity, including its releasing profile and regenerative effects.


Materials and Methods:: To investigate dECM microgels as a drug delivery platform, 2.5 ug/ml of recombinant VEGF was loaded prior to electrospray microparticle generation. VEGF release from microgels was determined using dot blot and bioactivity assessed using HUVEC tube formation assay. Tube formation assay was conducted by seeding HUVEC cells on GelTrex for 12 hours in the presence or absence of dECM microgels with or without VEGF. Phalloidin and DAPI stained HUVECs were analyzed using ImageJ for mesh number, size, tube thickness, length, and nodes.

Flow-focusing PDMS droplet generator chips were cast using standard soft lithography techniques and mounted on glass slides. Chips are mounted on a light microscope and imaged by an Amscope camera, while pressures are controlled by custom control software. To investigate changes in droplet size with changing pressures, the oil driving pressure was set constant, while water driving pressure was varied within a range such that the low forms no droplets while the high produces a constant aqueous stream. Average particle diameter was recorded by microscopy and analyzed using ImageJ (FIJI) and CellProfiler software. Gel-MA microgels were synthesized as a proof-of-concept using a 30 um nozzle chip and fluorinated oil-surfactant mix. Crosslinking of Gel-MA with an Irgacure photoinitiator was performed in-line on the microfluidic outlet tubing with a 365 nm UV light source. Microgels were extracted by solvent evaporation and then washed in PBS prior to analysis.


Results, Conclusions, and Discussions:: VEGF-loaded dECM microgels showed increased thickness in HUVEC tubes and smaller mesh size compared to controls and free VEGF. This indicates greater maturation of vessels, possibly by providing additional nucleation sites for vessel formation and growth. In showing desirable bioactivity when loaded with VEGF, we have demonstrated electrospray-generated dECM microparticles are a promising delivery vehicle for protein-based payloads, with the potential for extension to others; however, they are limited by high polydispersity and batch-batch variability.



With consideration to the limitations of electrospray emulsification, a microfluidic droplet generator was developed to remedy these deficits. Device characterization using food dye and mineral oil demonstrated droplet size increases linearly in response to higher input driving pressure. Gel-MA droplets exhibited the same response in emulsion studies without crosslinking. The food dye droplets range in diameter from 23 um to 91 um while the Gel-MA droplets range from 11 um to 69 um. These results indicate that we can produce tightly controlled protein-based droplets using an in-house designed and fabricated microfluidic device.



In order to produce microgels rather than emulsified droplets, UV crosslinking was introduced in the outflow channel of the microfluidic device. Gel-MA droplets were emulsified in the droplet generator using fluorinated oil with FluoSurfactant and crosslinked into microgels under UV light. Solvent evaporation was found to be effective at separating microgels from the fluorinated oil phase. Crosslinked microgel particles were recovered and remained stable in aqueous solution. Phase microscopy of the suspended particles revealed improved consistency in diameter and reduced aggregation compared to previous methods.



Future studies will evaluate the microfluidic generation and UV crosslinking of ECM-based microparticles, and investigate the in vitro and in vivo bioactivity of dECM particles, including their release profiles of both intrinsic and loaded proteins. Bioactivity and regenerative effects will also be assessed. High intensity sonication as an emulsion technique will also be investigated as a method for further miniaturizing this delivery system. Our microfluidic control system is also amenable to use with other chip designs, leaving the potential for nanoemulsion approaches with further chip revisions.


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