Advanced Materials

The endothelialization of organ-on-chip platforms and vascular implants is often limited by slow cell attachment and unstable monolayer formation. This work presents a scalable workflow that imprints micro- and nano-gratings into elastin-like recombinamer (ELR)-based hydrogels, enabling rapid endothelial cell capture and accelerating monolayer formation within 14 days. Three gelatin-ELR formulations are engineered, with {superscript 1}H-NMR confirming incorporation of sequences designed to modulate bioactivity (ELR1: inert; ELR2: uPA-responsive; ELR3: RGD-adhesive). ELR incorporation generates fibrillar microstructures and enhances mechanical performance, yielding elastic-dominant networks suitable for high-fidelity pattern transfer and stable culture. Using this library, the combined effects of ELR bioactivity and groove geometry on human iPSC-derived endothelial cells (iPSC-ECs) are systematically evaluated. In a 15-minute attachment assay, patterned ELR composites markedly improve cell retention compared to gelatin, with ELR2 on [~]350 nm and [~]4 {micro}m grooves performing best, consistent with controlled, cell-mediated interfacial remodeling. This early advantage persists, as ELR2 and ELR3 hydrogels support rapid alignment and reach confluence by day 14, whereas gelatin remains sub-confluent. Cytoskeletal analysis confirms F-actin alignment. By combining enhanced early capture with protease-regulated remodeling, ELR2 identifies a favorable design window. These results establish a materials design framework linking programmable ELR chemistry with surface topography to engineer endothelial interfaces, providing a versatile platform for vascular biomaterials and microphysiological systems.

We present a proof-of-concept platform in which amyloids are displayed on the surface of engineered Bacillus subtilis spores for bioengineered materials. Amyloids possess high tensile strength, elasticity, and tunable assembly, but their use is limited by inaccessible native sources and low-yield or toxic heterologous expression. Here, spores were engineered to display the native amyloid TasA and Humboldt squid suckerins 9 and 10 as fusions to the spore coat protein CotY. Amyloid production and fibril formation were confirmed by Western blot and X-34 staining, and quantitative analysis indicated mg/L-level yields. Atomic force microscopy revealed altered stiffness and surface ultrastructure, and incorporation of amyloid-displaying spores into resin-based 3D printing modified tensile strength. These findings highlight spore-based amyloid display as a scalable, modular platform for materials applications, leveraging established industrial spore production.

The human vasculature is a complex, multiscale system comprising hierarchical networks of macroscale to microscopic vessels. Existing in vitro fabrication techniques often fail to bridge these disparate scales, as high-resolution methods like multiphoton ablation are too slow for replicating larger vessels, while 3D printing lacks the resolution for fine microscale features. Here, we report a "twisted wire templating" strategy capable of generating perfusable bifurcating hydrogel networks that seamlessly transition from the macro- to the micro-scale (2.3 mm to 140 {micro}m) through seven orders of bifurcations. By optimizing wire-twisting geometries and polyurethane dip-coating, we overcame instability-driven bead formation to ensure replication fidelity across the networks. Fabrication rigs were reconfigured from existing 2D planar layouts to 3D reconfigurable architectures to better replicate 3D vessel geometries which simultaneously reducing the laboratory footprint and fabrication times by 47%. Using a Taguchi orthogonal array, we further optimized surface chemistry and hydrogel composition to inhibit structural failure during template extraction, resulting in fully patent, perfusable networks. This method provides a robust, low-cost, and scalable foundation for creating physiologically representative vascular models for investigating multiscale disease mechanisms and organ-level tissue engineering.

Biomaterials with highly tunable mechanical properties and tissue-mimetic structural features are critical for diverse biomedical applications. Photopolymerizable citrate-based polymers (CBP), such as methacrylate polydiolcitrate (mPDC), enable high-resolution fabrication of biodegradable scaffolds via light-based 3D printing for regenerative engineering. However, mPDC scaffolds typically exhibits substantial brittleness due to the formation of highly crosslinked and heterogeneous polymer network, an intrinsic limitation of many acrylate-based polymers, thereby restricting their use across a broad range of tissue types. Herein, we report facile network-engineering strategies to modulate crosslinking density and network topology of CBPs through the incorporation of acrylate-based reactive diluents and/or a thiol-based chain transfer agent, 3,6-dioxa-1,8-octanedithiol (DOD). These approaches enabled significantly improved and broadly tunable mechanical properties, with Youngs modulus spanning 6.8-134 MPa, ultimate tensile strength ranging from 1.8 MPa to 18 MPa, and strain at break varying from 14% to 61%. Notably, incorporation of isobornyl acrylate (IBOA) alone significantly enhanced toughness, yielding a 3.6-fold increase in Youngs modulus (50 MPa vs. 14 MPa) and a 2.8-fold increase in strain at break (39% vs. 14%). Moreover, combined incorporation of IBOA and DOD remarkably improved ductility, achieving a 4-fold increase in strain at break to 61% while maintaining comparable stiffness. All mPDC composites exhibited tunable biodegradability, good cytocompatibility, and excellent 3D printability. Using these composite inks, 3D-printed meniscus scaffolds supported the human chondrocyte growth and fibrochondrogenic matrix deposition, while 3D-printed vascular stents supported endothelial monolayer formation. Collectively, this study establishes a versatile photopolymerizable citrate-based biomaterial platform with broadly tunable mechanical performance, controllable biodegradability, good cytocompatibility, and high printability, offering strong potential for customized biomedical applications ranging from load-bearing to soft tissue engineering.

Engineering functional tissue constructs requires not only replicating their 3D architecture but also capturing their dynamic biochemical and mechanical environments. While 3D bioprinting technologies enable spatial control over cell and biomaterial deposition, post-fabrication modulation of material properties remains limited. Photografting approaches allow for spatiotemporal functionalization of certain 3D matrices by chemically binding bioactive factors onto spatially determined regions of a material, but current methods often rely on specialized chemistries with narrow material compatibility. Here, we introduce AddGraft, a biocompatible, off-the-shelf additive designed for semi-orthogonal thiol-ene photografting in vinyl-functionalized hydrogels. AddGraft, a heterobifunctional polyethylene glycol, carries an acrylate moiety for network incorporation during photocrosslinking and a norbornene group for post-crosslinking functionalization. AddGraft integrates into the polymer network during gel crosslinking without altering bulk mechanics, enabling precise modification at any time post-fabrication. We demonstrate compatibility with multiple acrylated biomaterial platforms and light-based volumetric photopatterning technology. Photopatterning achieves high spatial resolution and gradient formation in 3D, while grafting of multi-thiolated crosslinkers allows localized stiffening of hydrogels. Encapsulated human mesenchymal stromal cells exhibit high viability and undergo morphological changes in response to the dynamic tuning of their microenvironment. By decoupling structural and functional roles, AddGraft enables on-demand spatial and temporal control over hydrogel properties. This approach expands the biofabrication toolkit for engineering cell-instructive, 4D tissue environments with translational relevance in regenerative medicine.

Providing long-term (>6 months) zero-order drug release from easily administered formulations is a key challenge in improving patient adherence and facilitating access. Herein, we report the design and development of an injectable, biodegradable, long-acting polymeric microparticle-embedded hydrogel platform for prolonged, zero-order release of therapeutics. This "soft implant" is injectable for ease of administration and can be retrieved via a small incision, allowing for discontinuation of therapy if desired. Central to the platform are surface-eroding poly(orthoester) (POE) microparticles, which were molecularly tailored to tune zero-order drug release across a wide range of timeframes. We demonstrate the clinical potential of the "soft implant" using levonorgestrel, a contraceptive agent requiring sustained dosing. In vitro, we observed zero-order release for 300 days, projected for >12 months, with behavior consistent with surface erosion further supported through Raman chemical mapping. In vivo studies confirmed zero-order release for six months, projected to 12 months, from a subcutaneous injection in rats. We envision that our platform could transform therapies that require long-term, regular drug dosing, significantly improving compliance and therapy outcomes.

Autonomous soft materials that can actuate, perform a function, and then self-terminate without external intervention remain difficult to realize. Here, a bilayer hydrogel actuator fabricated by digital light processing-based 3D bioprinter is introduced that couples rapid thermoresponsive deformation with slower enzyme-programmed mechanical feedback to achieve self-regulated shape transformation and autonomous recovery. The system integrates a poly(N-isopropylacrylamide) actuation layer with a bovine serum albumin-poly(ethylene glycol) diacrylate enzyme-programmed layer loaded with trypsin. Above the lower critical solution temperature, deswelling of the actuation layer generates a strain mismatch across the bilayer and drives rapid closure. In parallel, proteolytic cleavage of albumin domains progressively softens the enzyme-programmed layer, reduces interlayer constraint, and acts as an intrinsic mechanical off-switch that relaxes curvature and restores the open state. This materials logic enables sustained enzyme release, time-dependent modulus loss, and autonomous shape recovery without staged external triggers. As a proof-of-concept, this platform is implemented as a gastrointestinal-retentive hydrogel gripper for localized intestinal enzyme delivery, where it exhibits thermally triggered gripping, millinewton-scale gripping force, autonomous reopening, and robust ex vivo retention on porcine small intestine under dynamic motion. These findings establish enzyme-programmed mechanical feedback as a general design strategy for self-regulated soft actuators and therapeutic materials with built-in functional lifetimes.

Engineering physiologically relevant breast in vitro models remains challenging due to the glands complex three-dimensional microanatomy, together with the need for epithelial polarity and hormone responsiveness. To overcome these challenges, fabrication methods are needed that rapidly create alveoli-scale structures with efficient diffusion and sustained hormonal stimulation. Here, Filamented Light (FLight) biofabrication is leveraged to print highly porous, ECM-based hydrogel scaffolds directly within standard Transwell inserts with separate apical and basal access. FLights speckle-patterned laser generates multiscale scaffold architectures that integrate filament-derived microchannels ([~]15 m) to promote diffusion with alveoli-inspired cylindrical microwell arrays (O100, O150, O200 m) that impose geometric constraints to guide epithelial organization. Each insert is printed in <10 s and incorporates slow-release prolactin microcrystals to provide lactogenic stimulation in situ. Primary human milk-derived mammary epithelial cells (milk MECs) were seeded onto the constructs. There, milk MECs line the printed microwells, establish zona occludens-1-positive tight junctions, and express lactation-associated markers (prolactin receptor and {beta}-casein), alongside milk fat globules and intracellular lipid droplets. Collectively, this rapidly reconfigurable FLight platform enables high-throughput generation of hormone-responsive human mammary microtissues for lactation-focused studies and is adaptable to other lumen-forming epithelia.

Direct ink writing is compatible with an expansive materials palette. While enabling diverse applications, this materials versatility brings significant bottlenecks in ink formulation, often requiring the mixing, printing, and testing of dozens to hundreds of ink compositions over the course of a project. To accelerate ink-space exploration, we introduce gradient embedded multinozzle (GEM) printheads that combine the high-throughput parallelized printing of multinozzles with combinatorial ink mixing. These printheads allow simultaneous mixing of two-, three-, and four-input inks which are distributed to printer nozzles to create complex 3D structures with graded compositions of inks. Using a two-way GEM printhead, we vali-date cell compatibility by printing scaffolds containing various concentrations of fibroblasts and observing non-linear compaction behaviours. We next test a three-way GEM multinozzle to print ten compositions of di- and multi-functionalized poly(ethylene-glycol) diacrylate hydrogel tri-leaflet valves, optimizing for stiffness, swelling ratio, and toughness. Our GEM multinozzles are compatible with open-source printers and either pressure- or volume-driven extrusion systems and promise to accelerate iterative ink design and testing.

Ingestible electronics enable the tracking and treatment of gastrointestinal and systemic diseases. However, bulky batteries and circuit boards require large capsules that can result in bowel obstruction, a medical emergency. Here, we engineered a 9 x 26 mm electronic pill capable of triggered severing into tiny pieces with sizes clinically proven to reduce obstruction risk. Our capsule enables multicomponent circuit boards to connect with separately encapsulated powering elements via conductive, interlocking connections. Heat induced softening of polyethylene glycol/polycaprolactone channels activates a spring to separate encapsulated components into inert 9 x 15 mm segments, facilitating intestinal passage. Separation triggers included closed-loop sensors and time-delay circuits. In vivo swine studies demonstrate the ability of our capsules to sense luminal oxygen changes via an optoelectronic sensor, locally trigger upadacitinib delivery, and facilitate safe excretion.

Premature clearance and limited organ targeting remain major barriers for nanoparticle (NP) drug delivery. Hitchhiking NPs on red blood cells (RBCs) can enhance circulation and organ-selective accumulation, but most approaches require ex vivo RBC extraction and reinfusion, limiting clinical translation. Here, we report an in situ RBC-hitchhiking strategy, named i-Bind, which employs polyphenol surface functionalization to enable spontaneous NP attachment to RBCs directly in the bloodstream. Driven by strong interactions of phenolic motifs with RBC membranes, i-Bind NPs exhibited markedly enhanced and more stable hitchhiking onto RBCs under flowing whole blood conditions. In both healthy and diseased mice, i-Bind NPs selectively target the lungs, resulting in an over 20-fold increase in lung-to-liver deposition ratio compared to unmodified NPs. Additionally, i-Bind NPs show preferential targeting to distinct lung immune cell subsets in a pathology-dependent manner, including cDC2s in healthy lungs, neutrophils in acute lung injury, and cDC1s in lung metastases. In a melanoma lung metastasis model, delivery of the STING agonist diABZI via i-Bind NPs significantly inhibited lung metastasis progression by reprogramming the lung immune microenvironment. Collectively, i-Bind provides a simple and versatile platform for organ-selective drug delivery and immune reprogramming. TeaserSurface functionalization of nanoparticles enables in situ red blood cell hitchhiking, unlocking new paths for organ-selective immune reprogramming

Advanced nanocarrier technologies have reshaped treatment paradigms for inflammatory and degenerative disorders by facilitating cell-specific delivery of bioactive molecules, including nucleic acids. Despite this progress, therapeutic application of microRNAs (miRs) has been hindered by rapid degradation, limited stability in circulation, and suboptimal cytosolic delivery within complex biological environments. In this study, we engineered and validated a macrophage-directed lipid nanoparticle (LNP) system designed to efficiently deliver the anti-inflammatory microRNA miR-146a (MacLNP-miR146a). Mannose-functionalized LNPs were generated through a scalable lipid injection formulation approach, producing highly uniform nanoparticles with strong physicochemical integrity across diverse pH conditions and in serum-rich environments. The optimized four-lipid composition supports efficient miR-146a encapsulation, promotes endosomal escape, and enhances intracellular trafficking, leading to effective cellular uptake and favorable tissue distribution in both in vitro and in vivo models. Notably, MacLNP-miR146a demonstrates strong biocompatibility in primary cell systems and animal studies. Together, these findings position MacLNP-miR146a as a robust and translational nanotherapeutic strategy for modulating macrophage-driven inflammation, including biomaterial-associated foreign body responses and related inflammatory pathologies.

The dynamics of the tumor microenvironment (TME) are a key determinant of cancer progression and therapeutic resistance through complex interactions between tumor, stromal and immune cell populations. Among these, tumor-associated macrophages (TAMs) play a central role in promoting tumor growth and immune suppression. However, the specific contributions of TAMs remain poorly understood due to the lack of tools enabling selective genetic manipulation in three-dimensional (3D) tumor models. Here, we present a gold nanoparticle-assisted optoporation approach that enables spatially selective plasmid-based gene delivery to TAMs within intact heterocellular 3D pancreatic ductal adenocarcinoma (PDAC) spheroids, thereby modulating the TME. In two-dimensional (2D) TAM cultures, conventional transfection of IRF5- and IKBKB-encoding plasmids validated their capacity to induce TAM repolarization, as evidenced by activation of interferon signaling. Extending this approach to 3D PDAC spheroids, nanoparticle-assisted optoporation achieved selective transfection of TAMs with IRF5- and IKBKB-encoding plasmids by transiently generating nanoscale membrane pores in illuminated cells. TAMs transfection elicited a robust interferon response, marked by transcriptional upregulation of IFNA, IFNB1, and CXCL10, and increased protein levels of IFNB1, IFNL1, and CXCL13, together with downregulation of pro-tumorigenic markers CEACAM5, IL19, and IL32. These coordinated changes indicate a shift towards an anti-tumorigenic TME. By enabling minimally invasive, TAM-specific gene delivery in complex multicellular 3D spheroids, this strategy allows precise modulation of the TME and opens new avenues for modeling its dynamics in cancer progression and therapeutic response.

Nanomaterial-enabled delivery systems have transformed therapeutic strategies for treating inflammatory and degenerative diseases by enabling targeted delivery of small molecules and nucleic acids. However, the clinical translation of microRNA (miR) therapeutics remains limited by instability, enzymatic degradation, and inefficient intracellular delivery in biological environments. Here, we present the design and validation of a next-generation lipid nanoparticle (LNP) platform optimized for the stable and effective delivery of the anti-inflammatory microRNA miR-146a. This LNP system is produced using a scalable lipid injection-based formulation method and yields nanoparticles with uniform size distribution and exceptional physicochemical stability across a wide pH range (2.5-8) and in serum-containing conditions. The four-component lipid architecture enables high miR-146a loading efficiency, efficient endo/lysosomal escape, and robust cellular internalization, resulting in effective tissue uptake and biodistribution both in vitro and in vivo. Importantly, LNP-mediated delivery of miR-146a exhibits excellent biocompatibility and potent anti-inflammatory activity in primary cells and animal models. Collectively, these results suggest this LNP-miR146a platform as a stable, efficient, and translatable approach for modulating inflammation and addressing biomaterial-associated inflammatory responses.

Vascularization remains a major obstacle in tissue engineering. Here, we introduce a developmentally inspired bioprinting strategy to generate centimeter-scale, self-organising "mother vessel" constructs from iPSC-derived human mesodermal progenitor cells (hiMPCs). By systematically optimizing the bioink composition, we identified a formulation that combines high print fidelity, mechanical stability and cell compatibility within a single-step bioprinting process. Within the first week after printing, hiMPCs in the "mother vessel" constructs underwent spontaneous differentiation and morphogenesis, forming intima-, media-, and adventitia-like layers containing CD31 endothelial, SMA mural and CD34/CD150 progenitor cells. Remarkably, Iba1 macrophage-like cells appeared despite their absence in the initial population, indicating intrinsic differentiation into both vascular and non-vascular lineages essential for angiogenesis, remodeling and tissue homeostasis. Surrounding the newly formed vessel wall-like structure was a broad, vascularized mesodermal tissue compartment that also contained the above-mentioned progenitors. Co-culture with prevascularized mesodermal organoids resulted in early structural interconnection of microvessels with the printed wall, representing a prerequisite for subsequent hierarchical vascular network formation. As a proof-of-concept, the mother vessel withstood controlled flow conditions in a bioreactor without detectable leakage, demonstrating its principal suitability for perfusion analyses. Together, these findings establish a biologically driven platform that bridges macro- and microvascularization. This may pave the way toward perfusable, vascularized larger tissue constructs, a major bottleneck in regenerative biofabrication.

Understanding the tumor microenvironment (TME) requires experimental platforms that faithfully recapitulate its key components. Here, we present an innervated and vascularized head and neck squamous cell carcinoma (HNSCC)-on-a-chip platform built with fully defined and tunable engineered extracellular matrices (eECMs). In a stepwise increase of complexity, we first co-cultured patient-derived HNSCC cells, cancer-associated fibroblasts, and endothelial cells within tailored eECMs, revealing matrix-dependent differences in self-organization and chemotherapeutic sensitivity. We then integrated these 3D constructs into a cancer-vasculature-interface, which enabled analysis of eECM-dependent directional collective migration and metastatization. Finally, we incorporated HNSCC-specific innervation through injectable 3D human bioengineered trigeminal ganglia, establishing a chip-based innervation-tumor-vasculature tri-interface. Together, this all-human platform captures fundamental determinants of HNSCC progression, including a fully defined ECM, vasculature, and innervation, within a single modular system that is broadly adaptable for interrogating how the tumor microenvironment shapes solid tumor behavior and therapeutic responses. TeaserHNSCC-on-a-chip integrates defined ECM, vasculature, and innervation to investigate tumor behavior and therapeutic responses.

Extracellular matrix (ECM) mechanical properties regulate tissue homeostasis and disease progression, with persistent ECM stiffening serving as a hallmark of fibrosis; yet, the early transition from healthy to diseased tissue remains poorly understood. Dynamic three-dimensional (3D) tissue models that capture early-stage stiffening are needed to investigate cellular responses during disease initiation. This work presents an innovative platform for studying cell responses in 3D environments undergoing active matrix stiffening. A bioinspired synthetic ECM incorporates collagen-mimetic peptides and employs sequential, non-terminal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to enable controlled increases in matrix stiffness over physiologically relevant timescales. Alternating polymer incubations produce a 2.5-fold increase in storage modulus over 72 hours, modeling the mechanical transition from healthy to early-stage fibrotic lung tissue. Live-cell reporter fibroblasts enable real-time monitoring of alpha-smooth muscle actin (SMA) expression, revealing significant upregulation during matrix stiffening that remains transient and difficult to detect via traditional endpoint assays. Active stiffening also modulates fibroblast motility, transiently increasing migration speed while persistently enhancing directional persistence. Complementary computational reaction-diffusion modeling provides mechanistic insight into modulus gradient formation and reaction kinetics. This versatile toolbox enables investigation of early mechanobiological responses to matrix stiffening and may aid identification of markers of fibrotic disease onset.

Functional networks of wired neurons comprise the basis for neuronal computation and processing. Within neuronal networks, activation of unique ensembles is an important identity of neuronal processing. However, dissociated neuronal networks form homogeneous functional structures with minimal variety in ensemble dynamics. To reintroduce such dynamics, we propose structuring the networks to follow multi-connectivity (micro- and meso-network) paradigms. Here, we use agarose microembossing to physically pattern dissociated neuronal networks across these scales. To perform agarose microembossing, we impress features with poly-dimethyl-siloxane (PDMS) stamps into liquid agarose to emboss features which hold under cold gelation. We validate the viability of primary neurons within the hydrogel patterns and interrogate circuit dynamics through calcium imaging. Patterned features presented with robust ensemble dynamics that are dependent on connectivity paradigms. Altogether, this work establishes a platform for investigating how engaging multi-scale features in the physical network informs neuronal ensemble dynamics. Clinical RelevanceThis work enables further dissociated studies to probe dynamics. We expect that this platform would be especially useful in early-stage drug development or personalized medicine pipelines that need to investigate circuit dynamics.

Cellular heterogeneity is a fundamental determinant of precision medicine, governing transcriptional plasticity, lineage commitment, and adaptive programs that drive disease progression and therapeutic resistance. However, most molecular interrogation technologies remain inherently destructive, relying on bulk measurements that average cellular signals and systematically obscure rare but functionally decisive subpopulations. As a result, current approaches provide only static snapshots of complex cellular systems, preventing longitudinal analyses of dynamic molecular states and masking the contributions of relapse-initiating, phenotypically plastic, or therapy-resistant cells that ultimately dictate population fate. Here, we present magnetic nanobiopsy, a simple and scalable platform for minimally invasive, repeatable intracellular molecular sampling from living cells. Magnetically actuated nanocomposites (200 nm-1.4 {micro}m) enable efficient intracellular access and stable biomolecular capture while preserving cellular structural and functional integrity. High-resolution imaging and biochemical analyses confirm robust internalization and anchoring of intracellular biomolecules, while flow cytometry demonstrates that the retrieved cargo remains cell-specific and quantitatively representative of the parental heterogeneous population. By enabling longitudinal, single-cell-resolved molecular profiling within intact living populations, magnetic nanobiopsy bridges the gap between static bulk analyses and technically complex single-cell methods. This platform establishes a new framework for real-time investigation of cellular heterogeneity, adaptive responses, lineage diversification, and transient cell-state transitions, with broad applicability in cell biology, oncology, and biobanking. HighlightsO_LIMagnetic nanobiopsy enables non-destructive, longitudinal molecular sampling of living cell populations with high throughput and minimal operational complexity. C_LIO_LIThe nanoprobes bypass endo-lysosomal sequestration, directly interfacing with the cytosol to capture representative protein and RNA cargo via a membrane-preserving budding exit. C_LIO_LIQuantitative validation confirms that the retrieved molecular profiles faithfully mirror the heterogeneity and relative abundance of the parental cell population. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/705970v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e0bee0org.highwire.dtl.DTLVardef@13a5b6org.highwire.dtl.DTLVardef@1e48f5dorg.highwire.dtl.DTLVardef@805b08_HPS_FORMAT_FIGEXP M_FIG C_FIG A magnetic nanobiopsy platform is presented for the longitudinal, non-destructive molecular sampling of living cell populations. Magnetically actuated nanocomposites directly access the cytosol--bypassing endo-lysosomal sequestration--to recruit a representative cargo of proteins and RNA through surface interactions and mechanical dragging. Guided by a changing external magnetic field, the molecularly loaded nanoparticles reversibly traverse the plasma membrane and exit via a controlled, budding-like mechanism that ensures cellular integrity. By preserving cell viability and population-level heterogeneity, this approach enables the retrieval of representative biomolecular cargo and the continuous monitoring of dynamic cellular states.

Following the successes of the messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines during the COVID-19 pandemic, mRNA-LNPs are being explored for many critical disease indications including infectious disease vaccination, cancer immunotherapies, and protein replacement therapies. LNPs require a polymer coating to provide stability in storage, and to minimise clearance from the body by reducing protein adsorption after injection. Poly(ethylene glycol)-lipids (PEG-lipids) have fulfilled this role to date, however increasing prevalence of antibodies against PEG in the general population jeopardises the efficacy of future PEGylated LNP doses and increases the likelihood of adverse pseudo-allergic responses. There is, therefore, an urgent unmet need to develop LNPs with new surfaces of PEG-alternative polymers which can evade anti-PEG antibodies, particularly where repeat dosing is required. Here, we present a family of polymer lipids, poly(acrylamido) (PAM) lipids, which effectively replace conventional PEG-lipids in mRNA-LNP formulations. We identify key design parameters to show that PAM-lipid monomer chemistry, molar mass and end-group all have critical effects on LNP size, polydispersity and in vitro transfection efficiencies, while having little impact on LNP morphology or internal structure. We determine that side-group (monomer) chemistry is a key mediator in alleviating anti-polymer antibody cross-reactivity. Compared to clinical benchmark PEGylated LNPs, several PAM-LNPs displayed improved transfection efficacy across multiple mRNA cargos in diverse cell types, organs, and routes of administration, both in vitro and in vivo. In particular, mRNA transfection improved in immune cells both in vitro (up-to 120-fold), and in vivo (up-to 5-fold), including superior mRNA expression in lymph nodes (2.5-fold). In part, this is likely because PAMs increase LNP uptake/association with primary immune cells (BMDCs), and increase biodistribution to the lymphoid tissues (LNs, spleen). Crucially, PAM-LNPs avoid circulating anti-PEG antibodies to recover lost mRNA efficacy after repeated dosing in vivo, 300% higher than PEG-LNPs. Overall, our findings establish the PAM-lipid family as a versatile platform of chemically varied PEG-alternatives, towards the next generation of therapeutic mRNA-LNP technologies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/708093v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@107de1aorg.highwire.dtl.DTLVardef@186b6eforg.highwire.dtl.DTLVardef@1542371org.highwire.dtl.DTLVardef@e2d47f_HPS_FORMAT_FIGEXP M_FIG C_FIG