(A-15) Injectable hybrid hydrogel microparticles for biomacromolecule delivery via droplet-based microfluidic templating

Introduction:: Hydrogel microparticles ranging from 1-1000µm, referred to as microgels, are attractive for biological applications afforded by their injectability and modularity which allows for facile delivery of mixed populations for tailored combination of therapeutics. Significant efforts have been made to broaden methods for microgel production specifically via the materials and chemistries by which they are made. Based on previous work we have developed a method for producing click poly (ethylene) glycol (PEG)-based microgels via droplet-based-microfluidics as well as microgels containing chemically crosslinked liposomes (lipo-microgels) via the same method. These two microgel populations allow for the encapsulation and release of cargo of varying size including hydrophobic and hydrophilic small molecules, growth factors, and biologically relevant proteins.

Materials and Methods::
PEG-microgels and lipo-microgels were formed by crosslinking four-arm thiol-functionalized PEG (ƒ=4, M_n 5,000 g/mol) with either four-arm maleimide functionalized PEG (ƒ=4, M_n 5,000 g/mol) or four-arm vinyl sulfone functionalized PEG (ƒ=4, M_n5,000 g/mol), and 10mM maleimide-functionalized liposomes. Liposome formulations were composed of a 4:1:5 ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide] (MPB-PE). The size and polydispersity of the liposomes was determined via DLS. Microgels were formed via a microfluidic device with three aqueous inlets controlled via pressure pumps allowing the monomer streams to remain separate until meeting with the oil phase for subsequent droplet formation. Microgel size distribution was determined via brightfield and confocal microscopy and mechanical properties of individual microgels as well as microgel suspensions were investigated using AFM as well as oscillatory shear rheology respectively. Fluorescent labeling followed by super-resolution confocal microscopy was used to investigate the presence of liposomes within the lipomicrogels. Spectrophotometric analysis, ELISA, and BCA assays were used to generate cumulative release profiles for target/model cargo. Circular dichroism (CD) and electrospray-ionization mass spectrometry (ESI-MS) were used to confirm release of intact protein cargo.




Results, Conclusions, and Discussions::
Maleimide-functionalized liposomes were synthesized for chemical conjugation into hydrogel microparticles. Monodisperse spherical populations of microgels and lipomicrogels were generated via microfluidic methods using the Michael-type addition reaction. Preliminary investigation cargo loading and release indicates prolonged release with protection of cargo.Synthesis of both microgels and lipomicrogels resulted in the generation of homogenous spherical populations with diameters of 74+/-16µm and 82+/-25µm respectively. Super-resolution imaging performed via Airyscan confirmed the presence of liposomes throughout the microgels and indicated an increased concentration of liposomes toward the core of the microgel particle. Nanomechanical mapping experiments conducted via AFM for bulk hydrogels, microgels, and lipomicrogels, indicated a reduction in modulus between the bulk hydrogel and microgels which could be indicative of an increased mesh size and informative for molecule release. Microgel suspensions of increasing concentration were tested via stead shear viscosity experiments and all concentrations tested exhibited shear thinning behavior. Additionally, microgels recovered following injection through a 27G needle showed no size or morphological changes. The release of 3.5kDa FITC-Dextran as a model molecule was sustained over 3 weeks for both microgels and lipomicrogels. To determine the ability of these microgels to deliver intact protein cargo, myoglobin was loaded and released from microgel populations and CD as well as ESI-MS confirmed no change in structure or molecular weight following release.




Acknowledgements (Optional): :

References (Optional): :