Designing DNA nanostar hydrogels with programmable degradation and antibody release

Technical Summary: Designing DNA Nanostar Hydrogels with Programmable Degradation and Antibody Release

Problem Statement
DNA nanostar (DNAns) hydrogels are promising materials for in vivo applications, including tissue regeneration and the delivery of drugs and antibodies. While the mechanical properties of these hydrogels can be tuned via chemical and physical methods, a systematic and quantitative understanding of the design principles controlling their enzymatic degradation is lacking. Previous studies have comprehensively examined the enzymatic degradation of DNAns liquid droplets, but the degradation of the "gel" regime remains unexplored. Specifically, it is unclear how the dense, entangled network of a hydrogel affects the accessibility of restriction enzymes (REs) to their target sites, and whether this degradation can be programmed to control the release of encapsulated cargo.

Methodology
The authors investigated the enzymatic degradation of DNAns hydrogels using a combination of gel electrophoresis, time-resolved particle tracking microrheology (PTMR), and enzyme-linked immunosorbent assay (ELISA).

• Material Design: Four distinct DNAns variants (Designs A–D) were engineered with varying structural parameters:
  • Design A: Features self-complementary sticky ends for direct hybridization and includes flexible joints (unpaired adenines) at the core and before the sticky ends.
  • Designs B–D: Utilize non-self-complementary sticky ends that hybridize with double-stranded DNA linkers to bridge nanostars. These designs vary in arm length, linker length, and the presence or absence of flexible joints (Design C lacks a flexible core).
  • Enzyme Sites: The nanostars and linkers were engineered to contain recognition sequences for specific restriction enzymes (EcoRI, BtgI, MspA1I, EcoRV) and were compared against non-specific endonuclease DNaseI.
• Experimental Conditions: Hydrogels were formed at high DNA concentrations (~250 µM). Degradation was monitored by incubating samples with enzymes at 37°C.
  • Microrheology: Mean-squared displacement (MSD) of tracer beads (200 nm or 500 nm) was measured to calculate viscosity and viscoelasticity changes over time.
  • Electrophoresis: Native agarose gel electrophoresis was used to visualize DNA fragmentation and confirm network cleavage.
  • Cargo Release: Humanized monoclonal IgG (Bevacizumab) was encapsulated in Design D hydrogels. Release was quantified via ELISA of the supernatant over 73 hours.

Key Contributions and Results

1. Resistance of Linker-Free Hydrogels to Site-Specific Enzymes:
   Contrary to observations in DNAns liquid droplets, Design A hydrogels (linker-free) displayed strong resistance to sequence-specific restriction enzymes (EcoRI, MspA1I), even at high enzyme-to-DNA ratios. While DNaseI rapidly degraded the hydrogel (reducing viscosity ~33-fold in 6 hours), site-specific REs showed no measurable degradation over 16–24 hours. Gel electrophoresis and microrheology confirmed the network remained intact.

2. Steric Hindrance vs. Pore Size:
   The authors tested whether increasing pore size or network fluidity would enhance RE accessibility.

   • Temperature: Raising temperature up to 58°C (approaching the sticky-end melting temperature) did not induce degradation.
   • Blocking Strands: Adding oligonucleotides to block sticky ends and disrupt network formation (creating a liquid-like state) did not enable EcoRI cleavage.
   • Linker Expansion: Design B, which incorporates a dsDNA linker to increase the mesh size (pore size ~30 nm vs. ~20 nm in Design A), also showed no degradation by EcoRI.
   • Conclusion: The resistance is not due to pore size exclusion but rather to steric and geometric hindrance imposed by the nanostar architecture, which prevents enzymes from accessing recognition sites within the dense network.

3. Programmable Degradation via Site Relocation:
   Degradation efficiency was significantly improved by relocating the restriction site from the nanostar arms to the linker strand.

   • Linker vs. Arm: When the recognition site was moved to the linker (Design C, NS(V)+L(I)), EcoRV (a blunt-end cutter) successfully cleaved the network. In contrast, EcoRI (a sticky-end cutter) showed minimal cleavage even on the linker.
   • Design Impact: Comparing Designs B and D (which differ in arm and linker lengths) revealed that while both could be degraded by EcoRV when the site was on the linker, their mechanical responses differed. Design D (longer arms) exhibited a more elastic initial state and a 21-fold greater viscosity reduction upon degradation compared to Design B. This demonstrates that the temporal evolution of viscoelasticity can be programmed through nanostar geometry.

4. Controlled Antibody Release:
   The study demonstrated that sequence-specific degradation can trigger the release of encapsulated cargo.

   • Retention: Design D hydrogels retained Bevacizumab with negligible passive release (<2 ng/mL) over 48 hours.
   • Triggered Release: Upon treatment with EcoRV, the hydrogel network degraded, resulting in a pronounced increase in antibody release, reaching 64 ng/mL after 73 hours.
   • Correlation: The release profile closely mirrored the appearance of low-molecular-weight DNA cleavage products in the supernatant, confirming that sequence-specific cleavage enables sustained cargo release.

Significance
The paper establishes that the degradability and mechanical response of DNAns hydrogels can be rationally tuned through molecular design and sequence-specific enzymatic cleavage. The findings reveal that while dense DNAns networks provide intrinsic protection against sequence-specific cleavage due to steric hindrance, this barrier can be overcome by strategic placement of restriction sites on linker strands. This provides a design-based strategy for engineering DNA materials with programmable degradation and release profiles. The authors conclude that these results advance the development of stimuli-responsive scaffolds for applications in tissue regeneration, 3D cell culture, and drug delivery systems requiring continuous time-varying rheology.