Injectable Co-Assembled Peptide Hydrogels for Sustained Local Delivery of Therapeutic Enzymes

Introduction:: Functional biomaterials fabricated from supramolecular self-assembly are of interest for many biomedical applications. However, to be useful for applications such as catalysis, whole protein must be installed to provide the biomaterial with this functionality. 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 protein can be installed via recombinant covalent fusion to an assembly peptide in the pre-assembled state. 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. Separately, the CATCH(4K+/6E-) peptides do not aggregate due to electrostatic repulsion, but once combined, they co-assemble into nanofiber-based hydrogels. CATCH peptides recombinantly fused to folded proteins can be incorporated into the nanofibers during assembly, endowing the hydrogels with functionality. Immobilizing therapeutic enzymes to CATCH nanofiber-based hydrogels can locally prolong availability and functional efficiency of enzyme therapeutics. Localized delivery can decrease the rate of renal clearance and improve therapeutic outcomes, whilst also reducing off-target effects and administered dose. 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. Specifically, we show the functional incorporation of NanoLuciferase for reporter activity, uricase for the treatment of gout, and Adenosine synthase A (AdsA) for immunomodulation.


Materials and Methods:: Protein Expression/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.

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 IVIS imaging was conducted to measure bioluminescence. Furimazine injections and IVIS measurements were repeated over a 22-day time period.

CATCH-Uricase Hydrogel: CATCH-uricase hydrogels, or blank hydrogels, were incubated with uric acid crystals and imaged on the microscope for crystal depletion. 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.

CATCH-AdsA Hydrogel: Female C57BL/6 mice were treated with 40uL 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 was collected 24 hours after LPS challenge, processed for anti-neutrophil (Li6G) immunohistochemistry, and analyzed for neutrophil infiltration.


Results, Conclusions, and Discussions:: First, we used NanoLuciferase (NanoLuc), which generates bioluminescence upon catalysis of its substrate, as a reporter enzyme for catalytic activity. After injection into mouse paws, we saw no enzymatic activity from the soluble NanoLuc group after 24 hours, suggesting it had been cleared from the injection site. 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 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. In vitro we show that the CATCH-uricase hydrogels can deplete uric acid crystals. 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. 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.

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). Using this enzyme in the CATCH platform, we hope to locally modulate the immune system. First, we show that AdsA immobilized within the hydrogel is still able to convert ATP into adenosine through measuring free phosphates. 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, we show 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.


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