Materials Today Bio

Gelatin methacrylate (GelMA) hydrogels offer many advantageous properties such as excellent biocompatibility, tunable stiffness, and rapid fabrication, making them attractive materials for neural systems-on-a-chip devices used to study axonal outgrowth in three-dimensional (3D) environments. In this study, we evaluate properties of GelMA hydrogels crosslinked with visible blue light and application of these hydrogels for encapsulation of primary dorsal root ganglion (DRG) explants inside a custom microscale device. We selected low GelMA polymer concentrations of 3 % and 6 % w/v for hydrogel preparation to create soft materials and demonstrate the effect of degree of functionalization (DoF) on stiffness of GelMA in a blue light-induced crosslinking system. Additionally, a decrease in crosslinking efficiency and stiffness of GelMA was observed upon reconstitution of the polymer in DMEM cell culture media compared to PBS. To evaluate the axonal outgrowth, DRG explants were encapsulated in various GelMA formulations and cultured for 7 days. Following incubation, the explants were fixed and immunochemically labelled with anti-beta-III-tubulin antibody. We further describe an application of custom-developed semi-automated image analysis tool together with linear mixed effects modeling to evaluate axonal outgrowth across different GelMA formulations. Among the formulations tested in our study, 3 % GelMA 95 % DoF hydrogels supported the longest neurite outgrowth suggesting that matrix properties influenced neurite extension.

Biofilm mechanics depend strongly on growth history, hydration, nutrient availability, and interfacial attachment, yet conventional rheology requires harvesting and transfer that can perturb the as-grown structure. Here, we introduce bioRheoFlow, a 3D-printed rheometer-mounted platform for cultivation and mechanical characterization without sample transfer. The platform combines evaporation compensation with static or axial nutrient flow, and benchmark measurements validate its rheometric performance. Applied to Pseudomonas putida and Pantoea sp., bioRheoFlow reveals that biofilms cultivated in situ under axial flow exhibit substantially higher elastic moduli than conventionally grown and scraped samples. Despite similar linear-viscoelastic stress ranges, the species show distinct nonlinear responses: P. putida exhibits a lower crossover stress and more abrupt failure, whereas Pantoea sp. sustains larger stresses and deformations. Creep suggests contributions from bulk deformation and biofilm-substrate interactions. bioRheoFlow provides a controlled framework for probing growth-conditioned mechanics in living biofilms.

The growing demand for three-dimensional (3D) in vitro models that accurately replicate human tissue to investigate disease mechanisms, enable tissue replacement, and support drug testing is driving the need for more advanced manufacturing platforms. Mesenchymal stem/stromal cells (MSCs) are promising candidates for tissue engineering and stem cell therapies due to their regenerative potential and ethical advantages. However, the reproducible and scalable production of 3D models that truly mimic the human complexity and function poses a major challenge. To address these challenges, we have developed a scalable and semi-automated 3D differentiation process for generating osteogenic microtissue derived from MSCs encapsulated in core-shell capsules (CSCs). To validate material-dependent, osteo-inductive effects of hyaluronic acid (HA), we systematically characterised our encapsulation process with regard to CSC integrity, spheroid formation and viability, as well as diffusion and shear thinning properties. Osteogenic differentiation was performed in static and dynamic culture, using either conventional 6-well plate format or a rotating wall vessel bioreactor. HA-CSCs were compared with inert carboxymethyl cellulose (CMC)-CSCs. In addition, hypoxic culture condition and the implementation of human platelet lysate (hPL) further supported physiological relevance. HA led to the formation of larger spheroids with high cell viability and increased metabolic activity compared to CMC. Calcium phosphate deposition within the extracellular matrix (ECM) and elevated ALP activity confirmed successful osteogenesis in all conditions. Furthermore, both HA and dynamic culture promote osteogenesis, and when combined, synergistically enhance the osteogenic differentiation. In conclusion, we present a semi-automated, high-throughput and xeno-free platform process for the 3D differentiation of MSCs towards osteogenic lineage. By integrating HA, hypoxia and dynamic culture conditions, this capsule-based system enhances the physiological relevance, enabling advanced in vitro disease modelling, drug testing, and scalable microtissue generation while reducing reliance on animal models.

Bone formation and remodeling depend on dynamic biochemical and biomechanical signaling from the collagen-rich osteoid that precedes mineralization, yet the role of osteoid nonlinear mechanics in regulating osteocyte-like network formation remains poorly understood. Here, we engineered a synthetic bottlebrush polymer hydrogel (BB) that mimics key mechanical features of osteoid and compared it to collagen type-I (Col1) matrices with matched shear modulus (~70 Pa) and strain-stiffening behavior. Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) were cultured for 28 days in growth (GM) or osteogenic media (OM) to examine network formation, and functional connectivity using live cell fluorescence recovery after photobleaching. hMSCs cultured in BB networks and OM showed upregulation of early osteocyte markers compared to Col1. We find that strain-stiffening materials with minimal stress relaxation promote osteocyte-like cell differentiation with functional connectivity, establishing osteoid-mimetic BB hydrogels as a promising matrix to study human osteocytogenesis and osteocyte mechanotransduction in vitro.

Oxygen (O2) transport is a major limitation in high-density cell culture, particularly as three-dimensional (3D) spheroids are increasingly used to better mimic in vivo tissue environments. Here, we developed a poly(4-methyl-1-pentene) (PMP)-based microwell platform, leveraging its lower molecular absorption and greater mechanical stability than the commonly used O2-permeable polydimethylsiloxane (PDMS), while retaining high O2 permeability. We measured O2 permeability in 50-m-thick PMP membranes and found approximately 200-fold greater O2 permeability than the conventional 1-mm polystyrene culture-plate material. PMP membrane-based microwell plates with 350-m-diameter wells maintained spatial separation of pre-formed hepatocyte spheroids while supplying O2 from the plate bottom, synergistically enhancing spheroid oxygenation. Compared with non-O2-permeable plates, the platform reduced localized hypoxia and hypoxia-associated cell injury while improving spheroid viability and growth. Collectively, PMP-based O2-permeable microwell plates provide a promising, scalable, high-throughput platform for spheroid culture with multiple applications in tissue engineering, drug screening and beyond.

Microfluidics-assisted encapsulation of cells in hydrogel microbeads has found diverse applications in tissue engineering, disease modelling, and drug testing. However, upscaled production of submillimeter-sized microbeads, each encapsulating optimally tens to hundreds of living cells, remains a challenge. Excessive viscous stresses or non-homogeneous crosslinking reactions hamper droplet monodispersity, hydrogel integrity, and/or cell viability. Herein, we present a device consisting of (i) tens of parallel step-emulsifiers arranged radially around the hydrogel inlet and (ii) an in-flow UV crosslinking system capable of continuous generation and crosslinking of up to around 45000 microbeads/min, each encapsulating N~50 cells, at cell viability exceeding 80%. We use gelatin methacryloyl (GelMA) as the biomaterial of choice and establish a unique sweet spot in terms of UV wavelength, irradiance, and exposure time- the latter controlled via the net applied flow rate and the length of the UV-exposed microchannel- at which we achieve an optimal balance between long-term microbead stability and cell viability. In particular, we observe a strong correlation between reduced cell viability and poor crosslinking efficiency, the latter attributed to the presence of a spontaneous core-shell structure. We further study the impact of added methacrylated hyaluronic acid (HAMA) and extracellular matrix (ECM) on cell proliferation and microtissue formation in the case of triple-negative breast cancer (TNBC) cells for future application in drug testing. The upscaled production of the encapsulated microtissues demonstrated here provides new perspectives for both drug testing and cell therapies where the precision in microenvironment formulation as well as production throughput remain of primary importance.

Animal-derived surface coatings present the gold standard in stem cell culture. However, they come with major drawbacks such as lot-to-lot inconsistency and an ethically problematic production. Approaches to develop synthetic coating alternatives have largely failed to recapitulate biological functions essential to PSC-self renewal and differentiation. In this study, we have investigated the suitability of a fully synthetic, chemically defined extracellular matrix-mimicking coating named BIOINSTRUCT for hiPSC culture. BIOINSTRUCT was employed during maintenance culture of IMR90-4 cells, as well as directed differentiation of HIHCNi008-A, ACTN2-GFP, and UKKI-011-A cells. BIOINSTRUCT preserved hiPSC characteristics over the experimental period of 10 passages similar to Matrigel. Spontaneous differentiation resulted in the formation of ectodermal, mesodermal and endodermal derivatives, confirming the retention of pluripotency. Directed differentiation on BIOINSTRUCT enabled the generation of neuronal, cardiac and pancreatic endocrine cells. Altogether, our data underscore BIOINSTRUCT as a promising alternative to animal-derived extracellular matrix coatings.

Developing biomaterials that faithfully recapitulate the complex architecture of native tissues is central to tissue engineering. Conventional hydrogels typically exhibit nanoscale porosity, which limits mass transport and cell migration and thereby constrains tissue development. Here, we exploit liquid-liquid phase separation (LLPS) to generate interconnected macroporous gelatin methacryloyl (GelMA) scaffolds with tunable pore sizes ranging from 2 to 50 m under physiological pH conditions. By mapping the GelMA/dextran phase diagram, we identify a composition range in which LLPS occurs through spinodal decomposition, resulting in a spontaneous bicontinuous, trabecular-like architecture. Pore size can be further tuned by varying polymer concentrations and temperature. We advantageously combined thermal gradients and photopatterning to enable spatial control over pore morphology to fabricate hierarchically structured materials. The biological relevance of these architectures is assessed using fibroblasts cultured within controlled porosity gradients. We show that pore size strongly influences cell infiltration, growth, and spatial organization, with larger pores providing sufficient space for cell migration while reducing the dependence on matrix degradation. Pore size of 7-15 m is identified for maximizing fibroblast growth. Overall, our approach provides a versatile platform for porosity screening, enabling the engineering of biomimetic hydrogels with optimized microarchitectures tailored to specific biological contexts for three-dimensional cell culture.

Macromolecular release from synthetic cells often relies on membrane pores or channels. We explored whether internally generated osmotic stress could increase membrane permeability and promote release. Using double-emulsion microfluidics, we encapsulated an Escherichia coli cell-free expression system in Pluronic L121 polymersomes to co-express the fluorescent protein mScarlet3 and the dextranase PsDex1711 in the presence of dextran. PsDex1711-mediated dextran hydrolysis produced a greater increase in bulk osmolality than reactions without PsDex1711 DNA. Tracking hundreds of individual polymersomes showed that PsDex1711-containing populations expanded, whereas control populations contracted. The distribution of normalized fluorescence revealed a subset with lower residual fluorescence than the main population. In selected expanded polymersomes, integrated internal mScarlet3 fluorescence declined to 18-22% of peak levels, without visible membrane disruption, consistent with protein release. These findings support a proof of concept for using internal gene expression to promote macromolecular release through osmotic actuation without introducing dedicated membrane pores.

Digital micromirror device (DMD)-based bioprinting enables high-resolution fabrication of hydrogel-based tissue constructs, but print fidelity and hydrogel mechanics are typically coupled through polymer concentration, light exposure, and crosslinking density. This creates a materials constraint for vascular bioprinting, where constructs must preserve microscale architecture during fabrication while supporting multicellular remodeling, lumen formation, and long-term tissue organization. To meet these requirements, we developed a 4D GelMA-Cad/gelatin hydrogel with time-dependent mechanics that maintains print fidelity while providing a soft, cell-permissive matrix. GelMA-Cad provides a covalently photocrosslinked, bioactive network containing an N-cadherin-mimetic peptide, while uncrosslinked gelatin serves as a transient matrix-modifying component that diffuses from the hydrogel after fabrication, producing further softening without bulk degradation of the GelMA-Cad network. We characterized hydrogel chemistry with NMR, quantified time-dependent changes in stiffness, visualized pore morphology with cryo-SEM, and evaluated the platform in retinal and pulmonary constructs. The 4D hydrogel supports 3D-patterned accuracy, cell viability, inner blood-retinal barrier vasculature, and vascularized lung-alveolar tissue models. Single-cell RNA-sequencing of the pulmonary vascular-alveolar model demonstrated compartment-specific transcriptional responses, including enrichment of lung capillary-associated endothelial features and AT1-like epithelial states. Overall, this platform decouples DMD print fidelity from post-print matrix mechanics for vascularized soft tissue bioprinting and tissue modeling.

In breast cancer, collagen fiber alignment and the second harmonic generation forward-to-backward ratio (F/B), an optical measurement sensitive to fibril-scale organization within collagen fibers, have each been associated with metastatic outcome. Although both are measured at the tumor-stromal interface, in vitro studies have largely examined them separately, because experimental systems have not allowed the two to be varied independently. To address this technical gap, we used a microfluidic biofabrication approach to generate paired aligned and unaligned regions within 3D collagen hydrogels via extensional strain and varied measured F/B across gels by changing the pre-gel pH. The resulting library covered alignment values reported in breast tumors and F/B values associated with poorer metastatic outcome in invasive ductal carcinoma at matched matrix stiffness, pore diameter, and fiber fraction. In MDA-MB-231 cells, alignment biased migration along the fiber axis without changing migration speed, as expected, but the magnitude of the contact guidance response varied with measured F/B. In contrast, MCF-7 cells showed little overall contact guidance, yet migrated ~30% faster in high F/B than low F/B gels in both aligned and unaligned regions. Together, these results show that cells migrated differently in matrices with similar fiber alignment. Thus, including measured F/B may provide additional information to help anticipate how cells migrate in collagen environments that appear similar based on alignment alone.

Granular hydrogels, made of jammed soft microparticles, are of great interest for 3D (bio)printing, as they combine ideal rheological properties and extensive modularity, yielding favorable microstructures for tissue engineering. Typically, the yield-stress properties of these materials, which facilitate printability, are defined by the preparation state and the initial particle content of the hydrogel. Here, we propose a granular hydrogel whose yield-stress and, consequently, printability and printed scaffolds shape retention can be controlled not only by the initial weight fraction of particles but also by their stimuli-responsive swelling. The responsive microgels are prepared from poly(N-isopropylacrylamide) crosslinked by dynamic covalent disulfide bonds. The particles swelling is triggered by reduction-induced cleavage of the disulfide bonds into two pendant thiol groups, enabling printing of multilayer scaffolds with high shape fidelity. Without chemically induced swelling, the same formulation requires higher particle concentrations to be printable. Importantly, the printed structures can be annealed by oxidizing the thiol units back into disulfide inter-particle bonds. The printed scaffolds are compatible with human dermal fibroblasts (HDF) and support cell-material interactions. This system offers a unique opportunity for on-demand modulation of both printability and scaffold microstructure, enabling unique control over local stiffness, packing density, and porosity in granular scaffolds.

Recent efforts to reconstruct critical-sized craniomaxillofacial defects using biomaterials have increasingly sought strategies to accelerate proangiogenic processes immediately after surgical implantation to improve host-graft integration. A class of mineralized collagen scaffolds has recently been identified to promote osteogenesis of bone marrow and adipose-derived mesenchymal stem cells both in vivo and in vitro, motivating efforts to identify strategies to promote osteogenic-angiogenic coupling in order to support regenerative healing. Here, we report the first-time use of a glycoengineering approach to generate a population of engineered ASCs (eASCs) from human adipose-derived stem cells (hASCs). This effort inserts an azido sugar onto the cell membrane that can be selectively targeted using DBCO-tagged biomolecules to selectively boost eASCs activity. We report the effects of azido sugar labeling conditions (concentration and incubation time) on eASCs labeling efficiency. We show proliferation and pro-regenerative gene expression activity of eASCs in mineralized collagen scaffolds are largely similar to conventional hASCs. And we demonstrate that molecular cargo functionalized with dibenzocyclooctyne (DBCO) can be conjugated to azido groups on eASCs via click chemistry. Together, this work shows metabolically labeled eASCs have the potential to be used for selective delivery of activity-inducing bioactive cues to enhance regenerative potential.

Dynamic covalent hydrogels formed through reversible acylhydrazone crosslinking have emerged as promising injectable biomaterials. However, a fundamental gap remains in understanding how the macromolecular structure of oxidized polysaccharides (OxPs) governs gelation kinetics and how these kinetics pathways translate into material properties and cellular responses. We address this question by developing an acylhydrazone hydrogel library composed of alginate adipohydrazide crosslinked with oxidized alginate (OxA) or oxidized dextran (OxD), two reactive aldehyde-bearing polymers with comparable chemical functionality but fundamentally distinct backbone structure. By varying polysaccharide type, oxidation degree, and reaction pH, we decoupled the effects of chemical functionality from macromolecular structure and established quantitative structure-kinetics-property-function relationships. OxD-based hydrogels undergo rapid, largely pH-independent gelation, whereas OxA-based systems display pronounced pH-dependent kinetics with significantly delayed network formation under physiological pH. These differences in gelation kinetics and OxPs macromolecular structures lead to marked variations in hydrogel mechanics, including stiffness, stress relaxation, stability, injectability, and post-injection recovery. Importantly, differences in gelation kinetics modulate cell-matrix interactions in three-dimensional culture. Slowly forming OxA hydrogels maintained rounded chondrocyte shape, while rapidly gelling OxD networks induced transient cell elongation. Mesenchymal stem cells displayed similar shapes regardless of gelation kinetics, indicating cell-type-specific responses to matrix formation dynamics.

A major challenge in vascular tissue engineering is the development of scalable fabrication methods capable of producing vascular grafts with suitable mechanical properties and hemocompatibility for both preclinical evaluation and future clinical translation. Using a recently developed method to generate acellular vascular grafts from densified collagen hydrogel, we present a proof-of-concept in vivo evaluation of the scalable collagen graft technology. Thick-walled, genipin-crosslinked grafts, representative of a fabrication approach applicable to clinically relevant vessel sizes, were implanted as interposition grafts in the rat abdominal aorta. These grafts supported rapid neointima formation and re-endothelialisation of the luminal surface but developed late thrombosis, highlighting the challenges of evaluating clinically scalable grafts in small-animal models. To enable assessment of the platform in millimetre-scale vessels, we developed a thin-walled (~100 {micro}m), non-crosslinked graft using a constrained densification approach and hyaluronic acid-mediated modulation of collagen fibril assembly to improve compliance matching and hemocompatibility, with the latter providing a matrix-level approach that may complement surface-bound antithrombotic coatings. These grafts remained patent for up to 17 weeks and exhibited neointima formation, re-endothelialisation, and early cellular infiltration into the graft wall. Together, these findings demonstrate in vivo functionality of densified collagen vascular grafts and establish a foundation for future evaluation in larger animal models.

Biological systems such as extracellular vesicles (EVs) are highly sophisticated and functionally diverse. Despite growing recognition of EVs' roles in health and disease through mediating intercellular communication, their inherent heterogeneity and challenges in purity and yield hinder their mechanistic research and clinical translation. To address this gap, we sought to translate essential EV properties into a controllable and simplified platform by engineering biomimetic synthetic vesicles (SVs) incorporating defined proteins and lipids. Using microfluidics, we generated SVs loaded with bulk membrane and cytosolic proteins from cancer cells, as well as individual organotropism-associated integrins. Bulk membrane protein incorporation into SVs reduced surface charge, modified lipid profiles, and enhanced cellular uptake, while integrin incorporation altered biodistribution. To investigate protein loading, we profiled input materials and SVs by mass spectrometry, identifying features linked to high incorporation, such as low molecular weight, positive charge, high isoelectric point, and predicted stability, globularity, lipidation, and amphipathic helices. This work presents a synthetic platform for dissecting the roles of EV-associated features and cargo, while providing insights into protein properties relevant to the optimization of delivery systems.

The ability to generate vascularized tissue constructs supplied by surgically accessible vessels remains a major limitation in reconstructive tissue engineering. Previous work demonstrated that positioning porous biomaterials around a vein can induce extensive vascular sprouting and generate a vascularized tissue volume without creation of an arteriovenous loop. However, this phenomenon has principally been demonstrated using rigid calcium-phosphate scaffolds, limiting its direct relevance to soft-tissue reconstruction. Here, we investigated whether a flexible decellularized dermal matrix could similarly be vascularized around a single unmodified vein and whether autologous dermal fibroblasts could further enhance this response. Decellularized rat dermis, with or without autologous dermal fibroblast seeding, was implanted either subcutaneously in the dorsal region or around the femoral vein for 8 weeks. Following Microfil perfusion, three-dimensional micro-CT demonstrated an extensive branching vascular network within perivenous constructs. Vessel volume relative to tissue volume was 0.2 +/- 0.1% in subcutaneous constructs without fibroblasts (n = 11) and 0.6 +/- 0.2% with fibroblasts (n = 9), compared with 6.+/- 1.4 % in perivenous constructs without fibroblasts (n = 10) and 7.3+/- 1.2% with fibroblasts (n = 10). Histology and immunohistochemistry similarly showed greater cellular repopulation and substantially higher CD31- and -SMA-positive vessel densities following perivenous implantation. Integrated two-way ANOVA of the same 8-week data confirmed a dominant effect of implantation site across the vascular outcomes, while fibroblast seeding significantly affected vascular volume, dermal remodeling, and nuclear density. The effect of fibroblast seeding on vascular volume was site dependent, with a significant implantation site fibroblast interaction. At 8 weeks, the vascularized dermal construct was completely dissected free from the surrounding tissues while maintaining its connection to the femoral vein. A small superficial incision into the isolated dermis produced visible bleeding, supporting an acute functional connection of the intradermal vascular network to the host circulation while the femoral venous connection was preserved. After flap elevation, perivenous constructs retained substantially greater vascular volume and showed more complete surface healing and organized skin architecture than subcutaneous constructs. Fibroblast seeding accelerated wound closure, while its effect on post-elevation vascular volume depended on implantation site: VV/TV decreased in subcutaneous constructs but increased in perivenous constructs. These findings show that single-vein vascularization can extend from rigid calcium-phosphate biomaterials to a compliant biological extracellular-matrix scaffold and may provide a simplified axial vascularization strategy without arterialization or incorporation of an arterial vessel.

Photovoltaic subretinal prostheses replace the lost photoreceptors in patients blinded by atrophic age-related macular degeneration (AMD) and provide central vision by electrically stimulating the inner retinal neurons. Subretinal debris in geographic atrophy separate the inner nuclear layer from the implant by tens of micrometers, increasing the stimulation threshold and reducing the pattern contrast. Absence of this separating layer in animal models diminishes the accuracy of preclinical testing of the next-generation implants, especially with smaller pixels. We developed three-dimensional biomimetic scaffolds nanoprinted onto photovoltaic arrays to provide a precisely controlled separation between the implants and target neurons in animal models of retinal degeneration. Gap sizes of the scaffolds were adjusted to meet the following requirements: they should allow light penetration to the implant, provide unobstructed flow of electric current, but preclude migration of cells into their 3-D structure. Gaps of 3 m were found to be optimal as they restricted cellular migration and maintained the inner nuclear layer above both planar and pillar electrode arrays. Scaffolds did not measurably alter the photovoltaic response and accurately reproduced the distance dependence of stimulation threshold. Following implantation, the initially fluid-filled intra-scaffold space was progressively filled by cellular matter matching the density of the subretinal debris. This transition is expected to increase resistivity of the scaffold to that of tissue, which was evidenced by concurrently improved grating acuity. These results establish a model of the controlled separation between the implant and the target neurons reproducing the features of subretinal debris in geographic atrophy of human patients, thereby providing a platform for accurate preclinical evaluation of the subretinal implants.

Paper-based colorimetric sensors offer a low-cost and accessible platform for point-of-care (POC) analysis, but enzyme activity loss during coating and drying can weaken analytical signals and require high enzyme loadings or complex immobilization procedures. Although surfactants are widely used to improve wettability in paper-based assays, their potential contribution to colorimetric performance beyond these effects remains unclear. Here, we investigated surfactant-assisted colorimetric signal enhancement in a glucose assay implemented on a 96-puddle paper plate (96-PPP) and identified Tween 20 as the most effective surfactant. Its effect on detection performance became more pronounced as glucose oxidase (GOx) loading decreased; at 0.1 mg/mL GOx, Tween 20 lowered the limit of detection (LoD) from 0.113 to 0.034 mg/mL (approximately 3.3-fold) over a working range of 0-5 mg/mL, despite no statistically significant change in the measured contact angle at this loading. Tween 20 had no appreciable effect on the reaction in solution but preserved 95% of the apparent reaction rate constant after drying, compared with 11% without it, and atomic force microscopy (AFM) revealed a more dispersed dried enzyme morphology on mica. Tween 20-containing sensors also showed slower signal decay during repeated wetting-drying cycles and thermal stress, retained 77% (vs 26%) of the response at 400 mM NaCl, and exhibited within-PPP and between-batch coefficients of variation (CVs) below 10% (vs 12.3-19.5%), while maintaining glucose selectivity over potentially interfering molecules. These results indicate that Tween 20 enhances paper-based glucose sensing beyond wettability, in part by retaining enzyme cascade activity during drying, although the contributions of the individual enzymes and the underlying mechanism remain to be established.

Glial cells play essential roles in maintaining neural tissue homeostasis and limiting the spread of injury-induced neuroinflammation, yet their dysfunction can also exacerbate neurodegenerative, neuroimmune, and neurodevelopmental disorders. Therapeutic targeting of specific glial cell populations would open up new treatment possibilities, but currently available viral and non-viral delivery vehicles are limited by carrier toxicity as well as poor delivery efficiency and specificity. To work towards new glia-targeted, non-viral delivery vehicles, we performed an in silico analysis on neural cell specific RNA-Seq datasets in mice to identify surface molecules differentially enriched on neurons, astrocytes, and microglia. We identified Slc2a5 (Glut5) as a microglia-enriched transporter and validated its microglia specific expression in healthy murine neural tissue using RNA in situ hybridization. By employing a sequential thiol-ene and enzymatic catalyzed reaction scheme, we synthesized polyelectrolytic dendrimers with multivalent presentation of fructose, the natural ligand for Glut5, and formulated nano-sized polyionic complexes (PICs) loaded with protein or nucleic acid cargo by mixing oppositely charged dendrimers. Fructose functionalized PICs lacking cationic surface charge enabled fructose-dependent intracellular protein delivery to Glut5-expressing human breast cancer cells (MCF-7) and mouse fibroblasts, while restricting uptake by Glut5-negative mouse astrocytes, confirming transporter-dependent selectivity in vitro. Fructose functionalized PICs resulted in minimal neuroinflammation when injected into mouse striatum at 1 mg/ml but elicited concentration-dependent neural tissue toxicity at higher doses. Fructose-PICs failed to achieve microglia specific intracellular delivery of functional proteins when injected into healthy neural tissue but aided in directing protein delivery to microglia and astrocytes at ischemic stroke lesions. Together, our findings demonstrate proof-of-principle for using in silico RNA-seq screening workflows to identify cell specific surfaceome targets and incorporating that insight into a modular biomaterial platform for targeted drug delivery.