(441e) Injectable Co-Assembled Peptide Hydrogels for Sustained Local Delivery of Therapeutic Enzymes

Background and Motivation: Functional biomaterials fabricated from supramolecular self-assembly are of interest for many biomedical applications. However, to be useful for applications such as antigen display, catalysis, or molecular recognition, whole protein must be installed to provide the biomaterial with these functional capabilities, which cannot be accomplished with unfolded peptides or small molecules. This can be challenging because covalently grafting proteins onto post-assembled biomaterials can alter protein bioactivity and is often non-specific and irreproducible. Instead, the functional moiety can be installed via recombinant covalent fusion to the assembly peptide in the pre-assembled state. Fusion proteins provide many advantages over post-assembly modification, including: i) stable functional integration, ii) reproducible concentration of the integrated functional molecule, iii) diverse library of possible fusion peptides, and iv) tunable and modular synthesis of multi-component biomaterials via simple mixing of multiple fusion peptide variants. The CATCH (Co-Assembly Tags based on CHarge complementarity) biomaterial system consists of a pair of oppositely charged variants of the synthetic b-sheet fibrillizing peptide, Q11. These CATCH peptides can then be functionalized with folded protein into fusion peptides. Separately, the CATCH(+/-) peptides do not aggregate due to electrostatic repulsion, but once combined, they co-assemble into nanofiber-based hydrogels. This occurs under near-physiological conditions, which is a critical design criterion for fusion peptides to maintain proper protein folding and function.

The overall goal of this work is to show the versatility of this biomaterial system to be utilized in localized and sustained delivery of enzyme-based therapeutics. Since 1960, enzymes have been used as therapeutics and have been approved by the FDA for various applications. However, systemically delivered therapeutic enzymes are often rapidly cleared from circulation by renal filtration, degradation by proteases, and recognition by the immune system, limiting their use for biomedical applications. Therefore, immobilizing these proteins to CATCH nanofiber-based hydrogels can locally prolong availability and functional efficiency of enzymes. This is especially beneficial for applications aiming to treat localized pathologies, such as arthritis, solid cancers, and areas of chronic inflammation. Localized delivery can decrease the rate of renal clearance and improve therapeutic outcomes, whilst also reducing off-target effects and administered dose or dose frequency. Previous works from our lab have shown that proteins can be successfully immobilized within the CATCH platform, and that the CATCH hydrogel vehicle itself is minimally immunogenic in vivo. This makes CATCH a suitable vehicle for therapeutic protein cargo.

Methods:

Protein Expression and Purification: Three fusion proteins, CATCH-NanoLuciferase, CATCH-Uricase, and CATCH-AdsA, were expressed in an E. coli host system and purified via nickel affinity chromatography.

CATCH Hydrogel Fabrication: CATCH peptides were dissolved in aqueous buffer and combined in equimolar ratios, with the addition of fusion protein, to final gel volumes of 10-40uL and peptide concentration of 12mM. The gels were left to incubate 1 hour, either in a 96-well plate or inside the barrel of a syringe. The gels fabricated in vitro were washed with PBS to remove any free protein or peptide not incorporated into the gel.

CATCH-NanoLuc Retention/Activity in vivo: 50nM CATCH-NL was injected into the subcutaneous space in the top of the hind paw in female C57BL/6J mice, either in a soluble formulation or in a hydrogel. Furimazine was administer shortly after and in vivo imaging on the IVIS was conducted to measure bioluminescence. Furimazine injections and IVIS measurements were repeated over a 22-day time period. Total flux of the paw was calculated and graphed over time.

CATCH-Uricase Hydrogel Activity in vitro: Blank hydrogels and CATCH-Uricase (16.5mM) functionalized hydrogels were formed and incubated with either soluble uric acid, or monosodium urate crystals. Soluble uric acid depletion was recorded at 293nm, and crystal depletion was monitored via microscopy. Enzymatic retention within the hydrogels was probed using tryptophan fluorescence. Hydrogels formed with either wild type uricase or CATCH-uricase were made and then incubated with PBS for 48 hours. The tryptophan fluorescence from protein in the supernatant and inside the hydrogel were measured.

CATCH-Uricase Hydrogel Activity in vivo: Female C57BL/6 mice were treated with either 20uL of PBS, wild type uricase, or hydrogels with or without uricase injected subcutaneously into the top of the hind paw. Two days later, 0.5mg MSU was subcutaneously injected into the same paw. Paw inflammation was subsequently assessed with digital calipers and neutrophil activity was assessed using the IVIS after intraperitoneal luminol injections.

CATCH-Adenosine Synthase A Hydrogel Activity in vitro: The formation of free phosphates were measured using a malachite green assay kit after an hour incubation for the following groups: ATP alone, soluble AdsA alone, ATP + soluble AdsA, and ATP + AdsA hydrogel. The ability of the AdsA functionalized hydrogel to prevent macrophage activation was also explored. THP-1 cells were differentiated into macrophages with PMA. The macrophages were seeded at 1.5 million cells/mL in a 96-well plate and stimulated with LPS overnight. The cells were then treated with the supernatants from the following groups: ATP; ATP + soluble AdsA; ATP + CATCH hydrogel with AdsA; ATP + blank CATCH hydrogel; or PBS buffer. Cultures were incubated for 1 hour before collecting media for further analysis of IL-1β and CXCL1 production using ELISA kits.

CATCH-AdsA Hydrogel Activity in vivo: Female C57BL/6 mice were treated with 20uL hydrogels with or without AdsA injected subcutaneously into the top of the hind paw. 24 hours later LPS was injected. Luminol was administered and IVIS images were obtained to assess neutrophil activation. Paw tissue from a separate cohort of mice was collected 24 hours after LPS challnege, processed for anti-neutrophil (Li6G) immunohistochemistry, and analyzed for neutrophil infiltration.

Results and Discussion: First, we used NanoLuciferase (NanoLuc), which generates bioluminescence upon catalysis of its substrate, as a reporter enzyme for catalytic activity. After injection into the subcutaneous space on the top of the hind paw, we saw no enzymatic activity from the soluble NanoLuc group after 24 hours, suggesting it had been cleared from the injection site. However, the catalytic activity from the NanoLuc immobilized within the hydrogel persisted for over 21 days. This shows that the enzyme remains active and localized to the injection site for over 3 weeks, making the CATCH vehicle attractive for sustained delivery of enzyme-based therapeutics.

Next, we moved into using enzymes with therapeutic potential: uricase and adenosine synthase A (AdsA). Uricase converts uric acid into a more soluble end-product, making it an attractive therapeutic to treat gout, which is caused by the crystallization of uric acid within the joint space. We show here that the CATCH-uricase functionalized hydrogels are able to deplete soluble uric acid and monosodium urate crystals, whereas the blank hydrogels are not. Furthermore, 85% of the CATCH-uricase remains within the hydrogel after 48 hours, whereas only 30% of the wild type enzyme remains, showing that only the CATCH-tagged enzyme is retained and immobilized within the gel. In a mouse model of gout, only the CATCH-uricase functionalized hydrogels are able to decrease the inflammation associated with the crystals. This is shown through caliper measurements, where wild type uricase and blank hydrogels are comparable to the positive control group (PBS) and only CATCH-uricase hydrogels are able to decrease paw inflammation. These data show that the uricase is only efficacious if it is localized to the area in the CATCH vehicle, and that the vehicle itself has minimal effects in this model. Similar results are shown with the luminol data. The luminol emission as a reporter for neutrophil activity peaked at 24 hours and was much higher in the mice that received soluble WT uricase, as compared to the CATCH-uricase hydrogels.

Finally, AdsA is explored as a novel therapeutic for inflammation. AdsA is a bacterial enzyme that converts extracellular ATP (inflammatory signal) into adenosine (anti-inflammatory signal). This enzyme helps the bacteria to evade raising the host’s immune system. Using this enzyme in the CATCH platform, we hope to locally modulate the immune system. This will be advantageous for treating autoimmune diseases where systemic immunosuppressants often leave patients susceptible to infection and other complications. First, we show that AdsA immobilized within the hydrogel is still able to convert ATP into adenosine through measuring free phosphates. Next, we show that this reaction from the hydrogel is able to prevent activation of LPS-stimulated macrophages, in terms of decreasing the production of both pro-inflammatory IL-1B and neutrophil chemoattractant CXCL1. Lastly, in a mouse model of LPS-induced inflammation, we are able to show that the AdsA functionalized hydrogel can decrease neutrophil activation, as measured through luminol, as well as decreased neutrophil infiltration, as measured by Li6G immunohistochemistry. Overall, these data look promising for localized immune modulation.

Conclusion: Through this work we have shown stable functional integration of three very different proteins into the CATCH hydrogel platform. This biomaterial system provides a modular, tunable, and reproducible method for the localized and sustained delivery of enzyme-based therapeutics.