Poster O17 - Co-delivery of enzyme and substrate in a supramolecular hydrogel loaded with poly-lactic-glycolic acid microspheres for sustained enzymatic catalysis

Introduction: Hydrogels and polymer-shell particles are widely used for sustained release and localization of biomedical therapeutic and diagnostic cargoes. Synthetic supramolecular peptide hydrogels enable facile incorporation of proteins into the gel via the recombinant addition of a peptide tag to a protein of interest, permitting that protein to co-assemble into the hydrogel. This can spare sensitive biomolecules like enzymes from extensive chemical processing that can be damaging to the enzymes. In contrast, polymer microparticles (MPs) can provide a method of controlled delivery of molecules that are poorly soluble in aqueous solutions, limiting their bioavailability, or that rapidly hydrolyze in aqueous environments. Encapsulation of these substrates in PLGA MPs can enable high concentration delivery in a less toxic manner, and co-delivery of MPs within an enzyme functionalized gel can provide co-localization of enzyme in the gel and substrate in the MPs, maximizing enzymatic function. Thus, incorporation of substrate-loaded MPs into hydrogels functionalized with an enzyme can enable ready interaction between the enzyme and substrate due to spatial co-localization.
One co-assembling peptide based hydrogel that has shown the ability to incorporate active enzymes is the Co-assembly Tags based on CHarge complementarity (CATCH) system developed by the Hudalla lab. Through recombinant expression of the enzyme of interest, nanoluciferase in this study, with a CATCH tag, the CATCH-tagged fusion enzyme can co-assemble into the CATCH gels. In this work, substrate (furimazine) was encapsulated in PLGA MPs that were assembled into the CATCH gels to provide sustained release of substrate for continuous enzyme activity.

Materials and

Methods: CATCH Gel formation: CATCH gels were composed of equimolar aqueous mixtures of CATCH(4K+) and CATCH(6E-) peptides at final concentrations of 12mM peptide. Nanoluciferase enzyme tagged with an N-terminal CATCH(6E-) tag was included in the mixture at specified concentrations to produce enzyme-functinoalized gels. PLGA MPs were added into the CATCH(6E-) mixtures before mixing with CATCH(4K+) peptide.
Confocal Microscopy: 12mM CATCH(4K/6E) gels prepared with rhodamine-loaded PLGA MPs were labeled with thioflavin T and imaged on a Zeiss 880 confocal microscope.
Cryo-SEM: 12mM CATCH(4K/6E) gels loaded with blank MPs were freeze fractured and imaged by the UF ICBR EM core using a Hitachi SU5000 SEM.
Rheology: 12mM CATCH(4K/6E) gels loaded with 0-2 mg/mL MPs were prepared and rheological properties were evaluated with an Anton Parr rheometer.
In vitro: 96-well plates were loaded with CATCH-nanoluciferase enzyme either in a 12 mM gel or as soluble enzyme, with furimazine MPs loaded at 0.5% w/w in PLGA, 0.05% w/w in PLGA, or blank MPs. Luminescence was measured on a spectrophotometer and area under the curve over 18 hours was calculated.
Mouse cadavers: Fresh cadavers with injected with 200uL 12mM CATCH gels functionalized with 25-100 nM of CATCH-nanoluciferase enzyme and 2-5 mg/mL of furimazine (nanoluciferase substrate) MPs. No additional injections of furimazine or gel were performed on cadavers. Cadavers were imaged with IVIS immediately after injection and then once per day for one week using 60 s exposure time. Cadavers were stored at 4C in the dark between measurements.

Results, Conclusions, and Discussions: Microscopy of MP-loaded CATCH(4K/6E) showed that MPs were well distributed throughout the bulk of the CATCH hydrogels and across all planes (Figure 1). Cryo-SEM showed that MPs were integrated into the fiber walls of the gels and thus could be maintained in the gel for an extended duration (Figure 1B).
Rheology was performed to evaluate the bulk mechanical properties of the CATCH-MP gels as a function of MP concentration (Figure 2). Between 0-2 mg/mL MP loading, no difference was seen in the storage (G') and loss (G'') moduli of CATCH-MP gels across angular frequencies (Figure 2A). Further, the damping factor of all measured CATCH-MP groups was between 0.1 and 0.2, indicating weak elastic gels were formed from 0-2 mg/mL MP (Figure 2B). The step-shear flow rate of CATCH-MP gels further showed no difference based on MP concentration after high shear rate is ended (Figure 2C). High strain disruption of the CATCH-MP gels at 1000% strain demonstrated a delay in the recovery rate of the gels ranging from ~30 seconds at 0 mg/mL MP to ~120 seconds at 2 mg/mL MP, but all gels regained a positive G'/G'' ratio immediately after high strain was ended, indicating that CATCH-MP gels are self-healing and shear-thinning (Figure 2D).
The sustained enzymatic activity of CATCH(4K/6E) gels loaded MPs was evaluated via luminescence from the reaction of nanoluciferase (NL) enzyme with furimazine. CATCH-MP gels with with CATCH-NL (CATCH-NL-MP) and furimazine MPs showed high luminescence over 18 hours compared to wells with soluble NL and/or blank MPs (Figure 3).
In a pilot study to be expanded in vivo, CATCH-NL-MP gels with furimazine-loaded MPs injected into the flank of mouse cadavers demonstrated sustained enzymatic activity for up to 1 week after injection (Figure 4). Luminescence was directly related to both the concentration of CATCH-NL and the concentration of MPs delivered. After 1 week, all combinations of functionalized CATCH-MP gels except for the lowest concentration (2 mg/mL MP + 25 nM CATCH-NL) showed signal above the baseline. This reaction persists longer than after delivery of free furimazine which is typically cleared within 1 day of injection in vivo.

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