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Bio-Orthogonal Nanogels for Multiresponsive Release.

ABSTRACT: Responsive nanogel systems are interesting for the drug delivery of bioactive molecules due to their high stability in aqueous media. The development of nanogels that are able to respond to biochemical cues and compatible with the encapsulation and the release of large and sensitive payloads remains challenging. Here, multistimuli-responsive nanogels were synthesized using a bio-orthogonal and reversible reaction and were designed for the selective release of encapsulated cargos in a spatiotemporally controlled manner. The nanogels were composed of a functionalized polysaccharide cross-linked with pH-responsive hydrazone linkages. The effect of the pH value of the environment on the nanogels was fully reversible, leading to a reversible control of the release of the payloads and a "stop-and-go" release profile. In addition to the pH-sensitive nature of the hydrazone network, the dextran backbone can be degraded through enzymatic cleavage. Furthermore, the cross-linkers were designed to be responsive to oxidoreductive cues. Disulfide groups, responsive to reducing environments, and thioketal groups, responsive to oxidative environments, were integrated into the nanogel network. The release of model payloads was investigated in response to changes in the pH value of the environment or to the presence of reducing or oxidizing agents.

SUBMITTER: Alkanawati MS

PROVIDER: S-EPMC8278386 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hydrogels are facile architectures for the controlled presentation of proteins with far-reaching applications, from fundamental biological studies in three-dimensional culture to new regenerative medicine and therapeutic delivery strategies. Here, we demonstrate a versatile approach for spatially-defined presentation of engineered proteins within hydrogels through i) immobilization using bio-orthogonal strain-promoted alkyne-azide click chemistry and ii) dynamic protease-driven protein release using exogenously applied enzyme. Model fluorescent proteins were expressed using nonsense codon replacement to incorporate azide-containing unnatural amino acids in a site-specific manner toward maintaining protein activity: here, cyan fluorescent protein (AzCFP), mCherry fluorescent protein (AzmCh), and mCh decorated with a thrombin cut-site. (AzTMBmCh). Eight-arm poly(ethylene glycol) (PEG) was modified with dibenzylcyclooctyne (DBCO) groups and reacted with azide functionalized PEG in aqueous solution for rapid formation of hydrogels. Azide functionalized full-length fluorescent proteins were successfully incorporated into the hydrogel network by reaction with PEG-DBCO prior to gel formation. Temporal release and removal of select proteins (AzTMBmCh) was triggered with the application of thrombin and monitored in real-time with confocal microscopy, providing a responsive handle for controlling matrix properties. Hydrogels with regions of different protein compositions were created using a layering technique with thicknesses of hundreds of micrometers, affording opportunities for the creation of complex geometries on size scales relevant for controlling cellular microenvironments.Statement of significanceControlling protein presentation within biomaterials is important for modulating interactions with biological systems. For example, native tissues are composed of subunits with different matrix compositions (proteins, stiffness) that dynamically interact with cells, influencing function and fate. Toward mimicking such temporally-regulated and spatially-defined microenvironments, we utilize bio-orthogonal click chemistry and protein engineering to create hydrogels with distinct regions of proteins and modify them over time. Through nonsense codon replacement, we site-specifically functionalize large proteins with i) azides for covalent conjugation and ii) an enzymatic cleavage site for user-defined release from hydrogels. Our results exemplify not only the ability to create unique bio-functionalized hydrogels with controlled mechanical properties, but also the potential for creating interesting interfaces for cell culture and tissue engineering applications.

Project description:Background: Protein theranostics integrate both diagnostic and treatment functions on a single disease-targeting protein. However, the preparation of these multimodal agents remains a major challenge. Ideally, conventional recombinant proteins should be used as starting materials for modification with the desired detection and therapeutic functionalities, but simple chemical strategies that allow the introduction of two different modifications into a protein in a site-specific manner are not currently available. We recently discovered two highly efficient peptide ligases, namely butelase-1 and VyPAL2. Although both ligate at asparaginyl peptide bonds, these two enzymes are bio-orthogonal with distinguishable substrate specificities, which can be exploited to introduce distinct modifications onto a protein. Methods: We quantified substrate specificity differences between butelase-1 and VyPAL2, which provide orthogonality for a tandem ligation method for protein dual modifications. Recombinant proteins or synthetic peptides engineered with the preferred recognition motifs of butelase-1 and VyPAL2 at their respective C- and N-terminal ends could be modified consecutively by the action of the two ligases. Results: Using this method, we modified an EGFR-targeting affibody with a fluorescein tag and a mitochondrion-lytic peptide at its respective N- and C-terminal ends. The dual-labeled protein was found to be a selective bioimaging and cytotoxic agent for EGFR-positive A431 cancer cells. In addition, the method was used to prepare a cyclic form of the affibody conjugated with doxorubicin. Both modified affibodies showed increased cytotoxicity to A431 cells by 10- and 100-fold compared to unconjugated doxorubicin and the free peptide, respectively. Conclusion: Bio-orthogonal tandem ligation using two asparaginyl peptide ligases with differential substrate specificities is a straightforward approach for the preparation of multifunctional protein biologics as potential theranostics.

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Bio-Orthogonal Nanogels for Multiresponsive Release.

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