Versatile Tissue-Injectable Hydrogels with Extended Hydrolytic Release of Bioactive Protein Therapeutics.

Hydrogels generally have broad utilization in healthcare due to their tunable structures, high water content, and inherent biocompatibility. FDA-approved applications of hydrogels include spinal cord regeneration, skin fillers, and local therapeutic delivery. Drawbacks exist in the clinical hydrogel space, largely pertaining to inconsistent therapeutic exposure, short-lived release windows, and difficulties inserting the polymer into tissue. In this study, we engineered injectable, biocompatible hydrogels that function as a local protein therapeutic depot with a high degree of user-customizability. We showcase a PEG-based hydrogel functionalized with bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) handles for its polymerization and functionalization with a variety of payloads. Small-molecule and protein cargos, including chemokines and antibodies, were site-specifically modified with hydrolysable "azidoesters" of varying hydrophobicity via direct chemical conjugation or sortase-mediated transpeptidation. These hydrolysable esters afforded extended release of payloads linked to our hydrogels beyond diffusion; with timescales spanning days to months dependent on ester hydrophobicity. Injected hydrogels polymerize in situ and remain in tissue over extended periods of time. Hydrogel-delivered protein payloads elicit biological activity after being modified with SPAAC-compatible linkers, as demonstrated by the successful recruitment of murine T-cells to a mouse melanoma model by hydrolytically released murine CXCL10. These results highlight a highly versatile, customizable hydrogel-based delivery system for local delivery of protein therapeutics with payload release profiles appropriate for a variety of clinical needs.

Logic-Based Delivery of Site-Specifically Modified Proteins from Environmentally Responsive Hydrogel Biomaterials.

The controlled presentation of proteins from and within materials remains of significant interest for many bioengineering applications. Though "smart" platforms offer control over protein release in response to a single external cue, no strategy has been developed to trigger delivery in response to user-specified combinations of environmental inputs, nor to independently control the release of multiple species from a homogenous material. Here, a modular semisynthetic scheme is introduced to govern the release of site-specifically modified proteins from hydrogels following Boolean logic. A sortase-mediated transpeptidation reaction is used to generate recombinant proteins C-terminally tethered to gels through environmentally sensitive degradable linkers. By varying the connectivity of multiple stimuli-labile moieties within these customizable linkers, YES/OR/AND control of protein release is exhaustively demonstrated in response to one and two-input combinations involving enzyme, reductant, and light. Tethering of multiple proteins each through a different stimuli-sensitive linker permits their independent and sequential release from a common material. It is expected that these methodologies will enable new opportunities in tissue engineering and therapeutic delivery.

Programming Stimuli-Responsive Behavior into Biomaterials.

Stimuli-responsive materials undergo triggered changes when presented with specific environmental cues. These dynamic systems can leverage biological signals found locally within the body as well as exogenous cues administered with spatiotemporal control, providing powerful opportunities in next-generation diagnostics and personalized medicine. Here, we review the synthetic and strategic advances used to impart diverse responsiveness to a wide variety of biomaterials. Categorizing systems on the basis of material type, number of inputs, and response mechanism, we examine past and ongoing efforts toward endowing biomaterials with customizable sensitivity. We draw an analogy to computer science, whereby a stimuli-responsive biomaterial transduces a set of inputs into a functional output as governed by a user-specified logical operator. We discuss Boolean and non-Boolean operations, as well as the various chemical and physical modes of signal transduction. Finally, we examine current limitations and promising directions in the ongoing development of programmable stimuli-responsive biomaterials.

Logical stimuli-triggered delivery of small molecules from hydrogel biomaterials.

Stimuli-responsive biomaterials are useful platforms for environmentally triggered drug delivery. By varying the molecular architecture of orthogonal stimuli-labile linkages between small molecules and non-degradable materials, we demonstrate the Boolean logic-based release of model therapeutics from gels. Programmable responses are demonstrated for materials sensitive to input combinations involving enzymes, chemical reductants, and light via YES, OR, and AND logic gates.

Engineered modular biomaterial logic gates for environmentally triggered therapeutic delivery.

The successful transport of drug- and cell-based therapeutics to diseased sites represents a major barrier in the development of clinical therapies. Targeted delivery can be mediated through degradable biomaterial vehicles that utilize disease biomarkers to trigger payload release. Here, we report a modular chemical framework for imparting hydrogels with precise degradative responsiveness by using multiple environmental cues to trigger reactions that operate user-programmable Boolean logic. By specifying the molecular architecture and connectivity of orthogonal stimuli-labile moieties within material cross-linkers, we show selective control over gel dissolution and therapeutic delivery. To illustrate the versatility of this methodology, we synthesized 17 distinct stimuli-responsive materials that collectively yielded all possible YES/OR/AND logic outputs from input combinations involving enzyme, reductant and light. Using these hydrogels we demonstrate the first sequential and environmentally stimulated release of multiple cell lines in well-defined combinations from a material. We expect these platforms will find utility in several diverse fields including drug delivery, diagnostics and regenerative medicine.

Multicellular Vascularized Engineered Tissues through User-Programmable Biomaterial Photodegradation.

A photodegradable material-based approach to generate endothelialized 3D vascular networks within cell-laden hydrogel biomaterials is introduced. Exploiting multiphoton lithography, microchannel networks spanning nearly all size scales of native human vasculature are readily generated with unprecedented user-defined 4D control. Intraluminal channel architectures of synthetic vessels are fully customizable, providing new opportunities for next-generation microfluidics and directed cell function.