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1.
Proc Natl Acad Sci U S A ; 121(6): e2309457121, 2024 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-38289949

RESUMEN

Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.


Asunto(s)
Materiales Biocompatibles , Hidrogeles , Animales , Hidrogeles/química , Biopolímeros , Mamíferos
2.
Bioconjug Chem ; 35(6): 750-757, 2024 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-38815180

RESUMEN

Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (ß-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.


Asunto(s)
Inteínas , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , beta-Lactamasas/metabolismo , beta-Lactamasas/química , Proteínas Luminiscentes/química , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/química , Proteína Fluorescente Roja , Proteínas/química
3.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33468675

RESUMEN

Hydrogel biomaterials derived from natural biopolymers (e.g., fibrin, collagen, decellularized extracellular matrix) are regularly utilized in three-dimensional (3D) cell culture and tissue engineering. In contrast to those based on synthetic polymers, natural materials permit enhanced cytocompatibility, matrix remodeling, and biological integration. Despite these advantages, natural protein-based gels have lagged behind synthetic alternatives in their tunability; methods to selectively modulate the biochemical properties of these networks in a user-defined and heterogeneous fashion that can drive encapsulated cell function have not yet been established. Here, we report a generalizable strategy utilizing a photomediated oxime ligation to covalently decorate naturally derived hydrogels with bioactive proteins including growth factors. This bioorthogonal photofunctionalization is readily amenable to mask-based and laser-scanning lithographic patterning, enabling full four-dimensional (4D) control over protein immobilization within virtually any natural protein-based biomaterial. Such versatility affords exciting opportunities to probe and direct advanced cell fates inaccessible using purely synthetic approaches in response to anisotropic environmental signaling.


Asunto(s)
Productos Biológicos/química , Técnicas de Cultivo de Célula/métodos , Hidrogeles/química , Proteínas/química , Productos Biológicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula , Humanos , Hidrogeles/farmacología , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Polímeros/química , Ingeniería de Tejidos/métodos
4.
Circ Res ; 127(10): 1306-1322, 2020 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-32883176

RESUMEN

RATIONALE: Myocardial infarction causes spatial variation in collagen organization and phenotypic diversity in fibroblasts, which regulate the heart's ECM (extracellular matrix). The relationship between collagen structure and fibroblast phenotype is poorly understood but could provide insights regarding the mechanistic basis for myofibroblast heterogeneity in the injured heart. OBJECTIVE: To investigate the role of collagen organization in cardiac fibroblast fate determination. METHODS AND RESULTS: Biomimetic topographies were nanofabricated to recapitulate differential collagen organization in the infarcted mouse heart. Here, adult cardiac fibroblasts were freshly isolated and cultured on ECM topographical mimetics for 72 hours. Aligned mimetics caused cardiac fibroblasts to elongate while randomly organized topographies induced circular morphology similar to the disparate myofibroblast morphologies measured in vivo. Alignment cues also induced myofibroblast differentiation, as >60% of fibroblasts formed αSMA (α-smooth muscle actin) stress fibers and expressed myofibroblast-specific ECM genes like Postn (periostin). By contrast, random organization caused 38% of cardiac fibroblasts to express αSMA albeit with downregulated myofibroblast-specific ECM genes. Coupling topographical cues with the profibrotic agonist, TGFß (transforming growth factor beta), additively upregulated myofibroblast-specific ECM genes independent of topography, but only fibroblasts on flat and randomly oriented mimetics had increased percentages of fibroblasts with αSMA stress fibers. Increased tension sensation at focal adhesions induced myofibroblast differentiation on aligned mimetics. These signals were transduced by p38-YAP (yes-associated protein)-TEAD (transcriptional enhanced associate domain) interactions, in which both p38 and YAP-TEAD (yes-associated protein transcriptional enhanced associate domain) binding were required for myofibroblast differentiation. By contrast, randomly oriented mimetics did not change focal adhesion tension sensation or enrich for p38-YAP-TEAD interactions, which explains the topography-dependent diversity in fibroblast phenotypes observed here. CONCLUSIONS: Spatial variations in collagen organization regulate cardiac fibroblast phenotype through mechanical activation of p38-YAP-TEAD signaling, which likely contribute to myofibroblast heterogeneity in the infarcted myocardium.


Asunto(s)
Diferenciación Celular , Colágeno/química , Infarto del Miocardio/metabolismo , Miofibroblastos/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Colágeno/metabolismo , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/citología , Fibras de Estrés/metabolismo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Señalizadoras YAP
5.
Nat Mater ; 18(9): 1005-1014, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31110347

RESUMEN

Protein-modified biomaterials can be used to modulate cellular function in three dimensions. However, as the dynamic heterogeneous control over complex cell physiology continues to be sought, strategies that permit a reversible and user-defined tethering of fragile proteins to materials remain in great need. Here we introduce a modular and robust semisynthetic approach to reversibly pattern cell-laden hydrogels with site-specifically modified proteins. Exploiting a versatile sortase-mediated transpeptidation, we generate a diverse library of homogeneous, singly functionalized proteins with bioorthogonal reactive handles for biomaterial modification. We demonstrate the photoreversible immobilization of fluorescent proteins, enzymes and growth factors to gels with excellent spatiotemporal resolution while retaining native protein bioactivity. Localized epidermal growth factor presentation enables dynamic regulation over proliferation, intracellular mitogen-activated protein kinase signalling and subcellularly resolved receptor endocytosis. Our method broadly permits the modification and patterning of a wide range of proteins, which provides newfound avenues to probe and direct advanced cellular fates in four dimensions.


Asunto(s)
Materiales Biocompatibles , Geles , Proteínas/química , Diferenciación Celular , Ingeniería de Tejidos/métodos
6.
Annu Rev Biomed Eng ; 21: 241-265, 2019 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-30857392

RESUMEN

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.


Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Sistemas de Liberación de Medicamentos , Hidrogeles/química , Polímeros/química , Ingeniería de Tejidos/tendencias , Adenosina Trifosfato/química , Glucosa/química , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Campos Magnéticos , Ácidos Nucleicos/química , Oxidación-Reducción , Resistencia al Corte , Temperatura , Ultrasonido
7.
J Am Chem Soc ; 141(39): 15619-15625, 2019 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-31525979

RESUMEN

Given the critical role that proteins play in almost all biological processes, there is great interest in controlling their presentation within and release from biomaterials. Despite such outstanding enthusiasm, previously developed strategies in this regard result in ill-defined and heterogeneous populations with substantially decreased activity, precluding their successful application to fragile species including growth factors. Here, we introduce a modular and scalable method for creating monodisperse, genetically encoded chimeras that enable bioactive proteins to be immobilized within and subsequently photoreleased from polymeric hydrogels. Building upon recent developments in chemoenzymatic reactions, bioorthogonal chemistry, and optogenetics, we tether fluorescent proteins, model enzymes, and growth factors site-specifically to gel biomaterials through a photocleavable protein (PhoCl) that undergoes irreversible backbone photoscission upon exposure to cytocompatible visible light (λ ≈ 400 nm) in a dose-dependent manner. Mask-based and laser-scanning lithographic strategies using commonly available light sources are employed to spatiotemporally pattern protein release from hydrogels while retaining their full activity. The photopatterned epidermal growth factor presentation is exploited to promote anisotropic cellular proliferation in 3D. We expect these methods to be broadly useful for applications in diagnostics, drug delivery, and regenerative medicine.


Asunto(s)
Materiales Biocompatibles , Proteínas Inmovilizadas/química , Procesos Fotoquímicos , Proteínas/química , Liberación de Fármacos , Modelos Moleculares , Conformación Proteica
8.
Prog Polym Sci ; 65: 53-82, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-28522885

RESUMEN

Human tissues are sophisticated ensembles of many distinct cell types embedded in the complex, but well-defined, structures of the extracellular matrix (ECM). Dynamic biochemical, physicochemical, and mechano-structural changes in the ECM define and regulate tissue-specific cell behaviors. To recapitulate this complex environment in vitro, dynamic polymer-based biomaterials have emerged as powerful tools to probe and direct active changes in cell function. The rapid evolution of polymerization chemistries, structural modulation, and processing technologies, as well as the incorporation of stimuli-responsiveness, now permit synthetic microenvironments to capture much of the dynamic complexity of native tissue. These platforms are comprised not only of natural polymers chemically and molecularly similar to ECM, but those fully synthetic in origin. Here, we review recent in vitro efforts to mimic the dynamic microenvironment comprising native tissue ECM from the viewpoint of material design. We also discuss how these dynamic polymer-based biomaterials are being used in fundamental cell mechanobiology studies, as well as towards efforts in tissue engineering and regenerative medicine.

9.
J Am Chem Soc ; 139(11): 3958-3961, 2017 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-28264159

RESUMEN

Combining bioorthogonal chemistry with the use of proteins engineered with adhesive and morphogenetic solid-binding peptides is a promising route for synthesizing hybrid materials with the economy and efficiency of living systems. Using optical sensing of chloramphenicol as a proof of concept, we show here that a GFP variant engineered with zinc sulfide and silica-binding peptides on opposite sides of its ß-barrel supports the fabrication of protein-capped ZnS:Mn nanocrystals that exhibit the combined emission signatures of organic and inorganic fluorophores. Conjugation of a chloramphenicol-specific DNA aptamer to the protein shell through strain-promoted azide-alkyne cycloaddition and spontaneous concentration of the resulting nanostructures onto SiO2 particles mediated by the silica-binding sequence enables visual detection of environmentally and clinically relevant concentrations of chloramphenicol through analyte-mediated inner filtering of sub-330 nm excitation light.


Asunto(s)
Cloranfenicol/química , ADN/química , Proteínas Fluorescentes Verdes/química , Manganeso/química , Dióxido de Silicio/química , Sulfuros/química , Compuestos de Zinc/química , Sitios de Unión , Química Clic , Modelos Moleculares , Tamaño de la Partícula , Ingeniería de Proteínas , Propiedades de Superficie
10.
Nat Mater ; 14(5): 523-31, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25707020

RESUMEN

Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.


Asunto(s)
Materiales Biomiméticos/química , Diferenciación Celular , Hidrogeles/química , Proteínas Inmovilizadas/química , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Procesos Fotoquímicos , Andamios del Tejido/química , Vitronectina/química
11.
bioRxiv ; 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38766128

RESUMEN

Hydrogel biomaterials offer great promise for 3D cell culture and therapeutic delivery. Despite many successes, challenges persist in that gels formed from natural proteins are only marginally tunable while those derived from synthetic polymers lack intrinsic bioinstructivity. Towards the creation of biomaterials with both excellent biocompatibility and customizability, recombinant protein-based hydrogels have emerged as molecularly defined and user-programmable platforms that mimic the proteinaceous nature of the extracellular matrix. Here, we introduce PhoCoil, a dynamically tunable recombinant hydrogel formed from a single protein component with unique multi-stimuli responsiveness. Physical crosslinking through coiled-coil interactions promotes rapid shear-thinning and self-healing behavior, rendering the gel injectable, while an included photodegradable motif affords on-demand network dissolution via visible light. PhoCoil gel photodegradation can be spatiotemporally and lithographically controlled in a dose-dependent manner, through complex tissue, and without harm to encapsulated cells. We anticipate that PhoCoil will enable new applications in tissue engineering and regenerative medicine.

12.
Nat Chem Eng ; 1(1): 2-5, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-39015819

RESUMEN

Focusing on engineering control over cell function and fate, this article examines the critical balance of 'outside-in' and 'inside-out signaling in tissue development and regeneration. It highlights emerging strategies to manipulate these interactions, including biomaterial design and synthetic biology to influence this delicate equilibrium and fine tune cellular responses.

13.
Nat Protoc ; 19(4): 1015-1052, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38253657

RESUMEN

The conditional assembly of split-protein pairs to modulate biological activity is commonly achieved by fusing split-protein fragments to dimerizing components that bring inactive pairs into close proximity in response to an exogenous trigger. However, current methods lack full spatial and temporal control over reconstitution, require sustained activation and lack specificity. Here light-activated SpyLigation (LASL), based on the photoregulation of the covalent SpyTag (ST)/SpyCatcher (SC) peptide-protein reaction, assembles nonfunctional split fragment pairs rapidly and irreversibly in solution, in engineered biomaterials and intracellularly. LASL introduces an ortho-nitrobenzyl(oNB)-caged lysine into SC's reactive site to generate a photoactivatable SC (pSC). Split-protein pairs of interest fused to pSC and ST are conditionally assembled via near-ultraviolet or pulsed near-infrared irradiation, as the uncaged SC can react with ST to ligate appended fragments. We describe procedures for the efficient synthesis of the photocaged amino acid that is incorporated within pSC (<5 days) as well as the design and cloning of LASL plasmids (1-4 days) for recombinant protein expression in either Escherichia coli (5-6 days) or mammalian cells (4-6 days), which require some prior expertise in protein engineering. We provide a chemoenzymatic scheme for appending bioorthogonal reactive handles onto E. coli-purified pSC protein (<4 days) that permits LASL component incorporation and patterned protein activation within many common biomaterial platforms. Given that LASL is irreversible, the photolithographic patterning procedures are fast and do not require sustained light exposure. Overall, LASL can be used to interrogate and modulate cell signaling in various settings.


Asunto(s)
Escherichia coli , Ingeniería de Proteínas , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Ingeniería de Proteínas/métodos , Aminoácidos , Mamíferos
14.
bioRxiv ; 2024 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-38645065

RESUMEN

Biomechanical contributions of the ECM underpin cell growth and proliferation, differentiation, signal transduction, and other fate decisions. As such, biomaterials whose mechanics can be spatiotemporally altered - particularly in a reversible manner - are extremely valuable for studying these mechanobiological phenomena. Herein, we introduce a poly(ethylene glycol) (PEG)-based hydrogel model consisting of two interpenetrating step-growth networks that are independently formed via largely orthogonal bioorthogonal chemistries and sequentially degraded with distinct bacterial transpeptidases, affording reversibly tunable stiffness ranges that span healthy and diseased soft tissues (e.g., 500 Pa - 6 kPa) alongside terminal cell recovery for pooled and/or single-cell analysis in a near "biologically invisible" manner. Spatiotemporal control of gelation within the primary supporting network was achieved via mask-based and two-photon lithography; these stiffened patterned regions could be subsequently returned to the original soft state following sortase-based secondary network degradation. Using this approach, we investigated the effects of 4D-triggered network mechanical changes on human mesenchymal stem cell (hMSC) morphology and Hippo signaling, as well as Caco-2 colorectal cancer cell mechanomemory at the global transcriptome level via RNAseq. We expect this platform to be of broad utility for studying and directing mechanobiological phenomena, patterned cell fate, as well as disease resolution in softer matrices.

15.
Adv Sci (Weinh) ; 11(24): e2301708, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38477407

RESUMEN

While direct cell transplantation holds great promise in treating many debilitating diseases, poor cell survival and engraftment following injection have limited effective clinical translation. Though injectable biomaterials offer protection against membrane-damaging extensional flow and supply a supportive 3D environment in vivo that ultimately improves cell retention and therapeutic costs, most are created from synthetic or naturally harvested polymers that are immunogenic and/or chemically ill-defined. This work presents a shear-thinning and self-healing telechelic recombinant protein-based hydrogel designed around XTEN - a well-expressible, non-immunogenic, and intrinsically disordered polypeptide previously evolved as a genetically encoded alternative to PEGylation to "eXTENd" the in vivo half-life of fused protein therapeutics. By flanking XTEN with self-associating coil domains derived from cartilage oligomeric matrix protein, single-component physically crosslinked hydrogels exhibiting rapid shear thinning and self-healing through homopentameric coiled-coil bundling are formed. Individual and combined point mutations that variably stabilize coil association enables a straightforward method to genetically program material viscoelasticity and biodegradability. Finally, these materials protect and sustain viability of encapsulated human fibroblasts, hepatocytes, embryonic kidney (HEK), and embryonic stem-cell-derived cardiomyocytes (hESC-CMs) through culture, injection, and transcutaneous implantation in mice. These injectable XTEN-based hydrogels show promise for both in vitro cell culture and in vivo cell transplantation applications.


Asunto(s)
Materiales Biocompatibles , Hidrogeles , Hidrogeles/química , Humanos , Materiales Biocompatibles/química , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Elasticidad , Animales , Viscosidad , Ratones , Elastina/genética , Elastina/química , Elastina/metabolismo
16.
bioRxiv ; 2024 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-38979380

RESUMEN

Integrin α5ß1 is crucial for cell attachment and migration in development and tissue regeneration, and α5ß1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5ß1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5ß1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine. One-Sentence Summary: A de novo-designed fibronectin substitute, NeoNectin, is specific for integrin α5ß1 and can be incorporated into biomaterials for regenerative medicine.

17.
Nat Commun ; 14(1): 5250, 2023 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-37640707

RESUMEN

Photodynamic hydrogel biomaterials have demonstrated great potential for user-triggered therapeutic release, patterned organoid development, and four-dimensional control over advanced cell fates in vitro. Current photosensitive materials are constrained by their reliance on high-energy ultraviolet light (<400 nm) that offers poor tissue penetrance and limits access to the broader visible spectrum. Here, we report a family of three photolabile material crosslinkers that respond rapidly and with unique tricolor wavelength-selectivity to low-energy visible light (400-617 nm). We show that when mixed with multifunctional poly(ethylene glycol) macromolecular precursors, ruthenium polypyridyl- and ortho-nitrobenzyl (oNB)-based crosslinkers yield cytocompatible biomaterials that can undergo spatiotemporally patterned, uniform bulk softening, and multiplexed degradation several centimeters deep through complex tissue. We demonstrate that encapsulated living cells within these photoresponsive gels show high viability and can be successfully recovered from the hydrogels following photodegradation. Moving forward, we anticipate that these advanced material platforms will enable new studies in 3D mechanobiology, controlled drug delivery, and next-generation tissue engineering applications.


Asunto(s)
Materiales Biocompatibles , Hidrogeles , Biofisica , Sistemas de Liberación de Medicamentos , Luz
18.
Matter ; 6(12): 4195-4244, 2023 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-38313360

RESUMEN

There is a growing interest in the development of technologies to probe and direct in vitro cellular function for fundamental organoid and stem cell biology, functional tissue and metabolic engineering, and biotherapeutic formulation. Recapitulating many critical aspects of the native cellular niche, hydrogel biomaterials have proven to be a defining platform technology in this space, catapulting biological investigation from traditional two-dimensional (2D) culture into the 3D world. Seeking to better emulate the dynamic heterogeneity characteristic of all living tissues, global efforts over the last several years have centered around upgrading hydrogel design from relatively simple and static architectures into stimuli-responsive and spatiotemporally evolvable niches. Towards this end, advances from traditionally disparate fields including bioorthogonal click chemistry, chemoenzymatic synthesis, and DNA nanotechnology have been co-opted and integrated to construct 4D-tunable systems that undergo preprogrammed functional changes in response to user-defined inputs. In this Review, we highlight how advances in synthetic, semisynthetic, and bio-based chemistries have played a critical role in the triggered creation and customization of next-generation hydrogel biomaterials. We also chart how these advances stand to energize the translational pipeline of hydrogels from bench to market and close with an outlook on outstanding opportunities and challenges that lay ahead.

19.
RSC Adv ; 13(35): 24487-24490, 2023 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-37588975

RESUMEN

The extracellular matrix (ECM) undergoes constant physiochemical change. User-programmable biomaterials afford exciting opportunities to study such dynamic processes in vitro. Herein, we introduce a protein-polymer hydrogel whose stiffness can be pharmacologically and reversibly regulated with conventional antibiotics. Specifically, a coumermycin-mediated homodimerization of gel-tethered DNA gyrase subunit B (GyrB) creates physical crosslinking and a rheological increase in hydrogel mechanics, while competitive displacement of coumermycin with novobiocin returns the material to its softened state. These unique platforms could potentially be modulated in vivo and are expected to prove useful in elucidating the effects of ECM-presented mechanical signals on cell function.

20.
Adv Mater ; 35(19): e2209904, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-36808641

RESUMEN

Stimuli-responsive biomaterials show great promise for modeling disease dynamics ex vivo with spatiotemporal control over the cellular microenvironment. However, harvesting cells from such materials for downstream analysis without perturbing their state remains an outstanding challenge in 3/4-dimensional (3D/4D) culture and tissue engineering. In this manuscript, a fully enzymatic strategy for hydrogel degradation that affords spatiotemporal control over cell release while maintaining cytocompatibility is introduced. Exploiting engineered variants of the sortase transpeptidase evolved to recognize and selectively cleave distinct peptide sequences largely absent from the mammalian proteome, many limitations implicit to state-of-the-art methods to liberate cells from gels are sidestepped. It is demonstrated that evolved sortase exposure has minimal impact on the global transcriptome of primary mammalian cells and that proteolytic cleavage proceeds with high specificity; incorporation of substrate sequences within hydrogel crosslinkers permits rapid and selective cell recovery with high viability. In composite multimaterial hydrogels, it is shown that sequential degradation of hydrogel layers enables highly specific retrieval of single-cell suspensions for phenotypic analysis. It is expected that the high bioorthogonality and substrate selectivity of the evolved sortases will lead to their broad adoption as an enzymatic material dissociation cue and that their multiplexed use will enable newfound studies in 4D cell culture.


Asunto(s)
Materiales Biocompatibles , Peptidil Transferasas , Animales , Péptidos , Hidrogeles , Ingeniería de Tejidos/métodos , Mamíferos
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