ACS Applied Bio Materials

Extracellular vesicles (EVs) are promising nanocarriers for therapeutic delivery; however, the factors governing EV uptake by recipient cells remain incompletely understood. In this study, we investigated whether EV internalization is primarily influenced by donor-cell origin or recipient-cell phenotype. Fluorescently labeled EVs derived from HEK293T, or SKBR-3 cells were incubated with a range of human epithelial, immune, and murine cancer cell lines at different doses and time points. HEK293T-derived EVs showed highly variable uptake across recipient cells, with hepatocellular carcinoma cell lines Huh7 and HepG2 exhibiting the highest internalization, while parental HEK293T cells showed the lowest. THP-1 immune cells also demonstrated strong uptake, whereas Jurkat cells showed moderate uptake. In murine melanoma models, Yummer cells internalized more EVs than B16F10 cells. Importantly, similar uptake trends were observed using SKBR-3-derived EVs, where Huh7 and HepG2 again displayed the highest uptake despite originating from a different donor cell source. EV internalization increased with dose and incubation time until saturation at higher concentrations. Together, these results demonstrate that EV uptake is predominantly determined by recipient-cell characteristics rather than EV source. These findings provide important mechanistic insight for the development of EV-based therapeutics and suggest that optimizing recipient-cell targeting is essential for efficient vesicle-mediated delivery. Graphical abstractEV uptake is determined by cell membrane properties rather than by the source of the EVs. The image was created by Biorender. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/726167v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f5c1cborg.highwire.dtl.DTLVardef@860962org.highwire.dtl.DTLVardef@1d20239org.highwire.dtl.DTLVardef@9003af_HPS_FORMAT_FIGEXP M_FIG C_FIG

Surface functionalization of biomaterials enables the immobilization of proteins and other molecules and can be utilized to direct the biological response to devices and implants. Fetuin-A is a blood plasma protein involved in numerous physiological processes, including the regulation of mineralization. Notably, many investigations of fetuin-A have explored its cellular interaction when in solution, but limited studies report the role of fetuin-A when used as a surface modifier. The present investigation explores the response elicited by fetuin-A on Saos-2 cells when it is immobilized on a model gold surface through the covalent reaction with dithiobis(succinimdyl propionate) (DSP). Comparative surface characterization using x-ray photoelectron spectroscopy (XPS), atomic force microscopy - infrared spectroscopy (AFM-IR) and surface plasmon resonance (SPR) confirmed the surface modifications but indicate partial inhomogeneity in the functionalizer surface coverage. The interaction of albumin and fetuin-A with the surface was quantified by radiolabeling, quartz crystal microbalance with dissipation (QCM-D) and SPR, demonstrating a higher mass of fetuin-A bound to the surface in comparison to serum albumin. Over 7 days, cells bound to the surfaces with immobilized fetuin-A showed significantly hindered proliferation of osteoblast-like cells compared to the positive control (fibronectin), presumably due to a decrease in cell metabolism. This study provides new insights into the role of fetuin-A in regulating Saos2 cell response and elucidates its potential use in combination with chemical functionalizers for biomedical applications requiring surface modification.

Tetrahedral DNA nanostructures (TDNs) are promising nanocarriers due to their structural precision, biocompatibility, and efficient cellular uptake. However, their stability under physiological conditions remains a key challenge. In this study, TDNs were synthesized via a one-pot thermal annealing method and characterized using native PAGE, dynamic light scattering (DLS), and zeta potential analysis, confirming uniform size ([~]13 nm) and negative surface charge. Their stability was systematically evaluated across different biological media (DMEM complete, serum-free DMEM, and E3), temperatures (4 {degrees}C, 25 {degrees}C, and 37 {degrees}C), and pH conditions (4.0, 7.0, and 8.5) over 24 h. Results revealed rapid degradation in serum-containing medium, increased instability at higher temperatures, and reduced stability under acidic conditions, while serum-free, lower-temperature, and neutral to mildly basic environments enhanced structural integrity. These findings highlight the strong environmental dependence of TDN stability and provide insights for optimizing their design for biomedical applications.

mRNA-lipid nanoparticles (LNP) have proven their potential as a rapidly adaptable vaccine platform and promise to revolutionize numerous therapeutic areas. A major hurdle towards the widespread adoption of mRNA-LNP vaccines and therapeutics is their limited liquid shelf-life compared to more established modalities currently necessitating an ultralow temperature cold-chain to enable their distribution and storage. While ongoing efforts aim to improve liquid stability through chemical modification of mRNA and lipid components, complementary strategies that are broadly applicable across chemistries may further accelerate translation. Here, we present an approach to improve the liquid shelf-life of mRNA-LNPs that does not rely on modifications to the mRNA or LNP chemistry. In particular, we show that bleb formation induced by high ionic strength acidic citrate buffers during LNP formation reduces mRNA degradation and retains in vitro activity during extended liquid storage. We observed an increase in the in vitro activity storage half-life from 2.8 to 18.9 days at 25{degrees}C when prepared using high ionic strength buffers translating into a [~]7-fold improvement in the liquid shelf-life of MC3-LNPs. This enhanced stability of LNPs with large amount of bleb formation was mainly attributed to reduced rates of lipid-mRNA adduct formation and mRNA fragmentation. Furthermore, the acidic buffer dependent stabilization was observed across different ionizable lipids with the extent dependent on the ionizable lipid head group. We envision that the induction of bleb formation via selection of appropriate acidic mixing buffers may represent a universal approach to enhance mRNA-LNPs stability and enable extended long-term refrigerated storage.

Membrane models of scaffolded discoidal lipid bilayers called nanodiscs have proven to be a valuable tool for the study of membrane proteins in a native environment. DNA-scaffolded membrane model has emerged as an alternative tool for membrane protein studies. Taking advantage of the designability of DNA nanostructure, we created a double-decker double-stranded DNA ring (DDring) to self-assemble DNA-based nanodiscs (DNA-ND). The DDring is 17 nm wide and 4 nm high, and equipped with 28 alkyl chains on the inside that can interact with each hydrophobic leaflet of the lipid bilayer. We further demonstrate the functionality of DNA-ND membrane model with the assembly of membrane proteins. DDrings are suited to neutral or cationic charged phospholipids and detergents. This study provides more insights into the potential use of DNA- assisted nanodiscs for membrane protein characterization.

The combination of synthetic biology and additive manufacturing has driven major changes in production of biomaterials, especially through the use of three-dimensional (3D) bioprinting to create engineered living materials. However, current fabrication methods can be limited by prohibitive hardware costs and the inability to maintain structural fidelity in complex, free-form living architectures. This work demonstrates how to build a low-cost, open-source 3D bioprinting platform that can make complicated bacterial structures with complex geometry and high dimensional accuracy. A commercially available, conventional fused deposition modeling 3D printer was modified to create a bioprinting system that is simple to build. The modified bioprinter, which costs around $450, is less expensive than many commercial bioprinters. This 3D-printing technology uses slurry-based support bath methods featuring low-cost gelatin and agarose microparticles, resulting in structures with a high aspect ratio (>8:1) and feature sizes as small as 260 m. The optimization of critical printing settings, including the ability of the bioink to retract during non-print movements, resulted in a reduction of unwanted bacterial deposition by nearly two orders of magnitude. Long-term viability experiments showed that bacteria in the bioprints could survive for at least 28 days with nutrient supplementation. Additionally, 3D-printed engineered biofilms revealed that incubation conditions and extracellular matrix composition significantly impacted the mechanical properties of printed constructs, with tradeoffs between matrix production and mechanical integrity. This study showcases an accessible 3D bioprinting platform for advanced bioprinting technologies, enabling development of engineered living materials with potential applications in synthetic biology, biotechnology, and tissue engineering.

Cancer cell membrane coated biomimetic nanoparticles have been shown to be highly efficient in cellular uptake, homotypic tumour targeting, and the ability to suppress tumour growth compared to uncoated nanoparticles. Long duration anti-cancer treatment regimens require highly stable cancer cell membrane coated biomimetic nanoparticle. To manufacture such highly stable cancer cell membrane coated biomimetic nanoparticle, we used "Click-chemistry" to encapsulate cancer cell membrane on nanoparticles. In situ characterization was done to confirm the functionality of the novel Click-chemistry based formulation to encapsulate cancer cell membrane on nanoparticles. Gold nanoparticles were encapsulated with the cell membranes of cell lines of lung adenocarcinoma, malignant melanoma, high-grade serous epithelial ovarian cancer, colorectal cancer, oral cancer, esophageal adenocarcinoma, adenoid cystic carcinoma of salivary gland, and breast cancer. Functional group analysis, size, morphology, and surface charge confirmed long-stability of the biomimetic nanoparticles after incubating in complete growth medium for 12-months.

Polymeric 3D printing of microfluidic devices for biosensing is an appealing fabrication alternative for rapid manufacturing of biosensing devices with complex geometry in a streamlined, repeatable and cost-effective manner without the need for expensive instrumentation such as those employed in photochemical etching and soft lithography. Hybrid 3D printed paper-based microfluidics is an emerging area which harnesses the unique properties of both, merging the construction of microfluidic structures and the inherent capillary-driven flow within paper substrates. In this work, we have fabricated hydrophobic barriers by 3D printing a single layer of machinable wax, thermoplastic polyurethane, polylactic acid and polypropylene directly on chromatography paper to create open microchannels and determine the most suitable material. Characterization of each open microchannel using the four materials revealed polypropylene as the most reliable material with high hydrophobic barrier integrity and resolution. Polypropylene achieved functional microchannels with a resolution of 621 {+/-} 33{micro}m, hydrophobic barrier integrity of (93.75 {+/-} 9.16%), wicking speed of 0.38mm/s and optimal hydrophilicity of channels (51.4 {+/-} 8.36 {degrees}) with minimal embedding during thermal curing. To demonstrate proof of principle, a fluorescence assay demonstrating the formation of a dimeric g-quadruplex structure from a g-rich sequence which significantly enhances fluorescence of thioflavin T was implemented.

Shiga toxins (Stx) are key virulence factors of Shiga toxin-producing Escherichia coli (STEC), which are responsible for severe foodborne infections that can progress to hemolytic-uremic syndrome (HUS). Currently, no specific anti-toxin therapies are available. In this study, we devised a glycoengineering strategy utilizing Functional-Spacer-Lipid (FSL) conjugates to create small extracellular vesicles (sEVs)-based decoy receptors for Shiga toxin type 1 (Stx1). sEVs isolated from human Caco-2 and HEK293T cells were functionalized with Gb3 trisaccharide (Gal1[->]4Gal{beta}1[->]4Glc)-containing FSL conjugates, yielding Gb3-decorated vesicles, displaying the Gal1[->]4Gal epitope. Characterization of FSL-modified sEVs confirmed that FSL incorporation did not adversely affect sEV morphology, size distribution, or surface charge. Western blotting and bead-assisted flow cytometry verified the presence of exosomal markers (CD9 and CD63) and the Gb3 epitope on modified vesicles. Gb3-tagged sEVs from both cell types exhibited high specificity in binding Stx1B, while control vesicles carrying Galili epitope (Gal1[->]3Gal{beta}1[->]4GlcNAc), lacking Stx1B binding, demonstrated negligible binding. Gb3-expressing Caco-2 cell-based assays revealed that Gb3-decorated sEVs markedly reduced Stx1B binding to Caco-2 cells, indicating effective competition with cellular receptors. Furthermore, glycoengineered sEVs did not impair Caco-2 cell viability at concentrations sufficient for Stx1B sequestration. These findings establish FSL-mediated glycoengineering as a rapid and versatile approach for generating sEV-based decoy receptors that effectively bind Stx1B. Gb3-containing human sEVs may serve as an agent for neutralizing Stx1B and potentially other glycan-binding toxins, supporting the development of promising next-generation anti-toxin therapeutics.

Analyzing the differentiation potential of cells in contact with newly developed materials is essential for assessing their ability to integrate into biological tissues and promote functional regeneration. Material properties such as rigidity, topography, and wettability significantly influence stem cell differentiation and are therefore optimized in implants. In this context, surface potential has been repeatedly, albeit inadvertently, shown to enhance osteogenesis. Here, we demonstrate that this surface property modulates cellular mechanosensing by altering the cells perception of substrate rigidity. Specifically, we show that human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on surfaces with a net zero charge, coated with collagen type I, exhibit characteristics typical of cells adhering to compliant substrates. Conversely, mesenchymal stem cells on polarized surfaces activate mechanoresponsive pathways that promote osteogenesis, as evidenced by large spreading areas, enhanced contractility, and Yes-associated protein (YAP) translocation into the nucleus. Furthermore, our data suggest that negative net surface potentials lead to the local accumulation of calcium ions, which further facilitates osteogenic differentiation. Collectively, our findings reveal that biomaterials surface potential, a previously uncharacterized mediator of cellular mechanotransduction, should be considered in the design of next-generation biomaterials for tissue regeneration applications.

Lipid nanoparticles (LNPs) have demonstrated strong potential in COVID-19 mRNA vaccines nevertheless they still face the challenges in low mRNA delivery efficacy. Virus-like porous silica (VLPSi) nanoparticles (NPs) represent a promising biomimetic delivery platform because their spiked morphology may enhance cellular internalization and promote endosomal membrane disruption. However, the application of VLPSi for mRNA has been rarely explored. In this study, hybrid lipid-VLPSi NPs were developed by combining VLPSi with either lipoplexes (LPs) or LNPs. The effects of lipid types, mass ratio of different compositions, and amine modifications of VLPSi on mRNA delivery were studied. The results demonstrated that both LP and LNP could be successfully integrated with VLPSi to form hybrid delivery systems for mRNA transfection. VLPSi could significantly enhance mRNA delivery of both LPs and LNPs due to improved cellular uptake, structural stabilization of the mRNA complex, and enhanced endosomal escape mediated by the rigid virus-like surface architecture. Among the tested lipid formulations, the ionizable lipid ALC-0315 and helper lipid DOPE with mass ratio of 5:3 was the most effective lipid composition to be integrated with VLPSi, showing the highest mRNA delivery performance. In addition, amino modification of VLPSi was found to be a critical factor for efficient mRNA delivery. Hybrid LNPs containing amino-modified VLPSi showed significantly higher transfection efficiency than those containing unmodified VLPSi. Notably, amino-modified LNP-VLPSi achieved up to fivefold higher gene expression than conventional LNPs. Overall, this study establishes VLPSi as an efficient platform for amplifying lipid-mediated mRNA delivery. Owing to its straightforward integration into widely used LNP systems, VLPSi offers an adaptable and effective strategy for advancing next-generation mRNA therapeutics.

In vitro models are increasingly critical for interrogating cancer biology and therapeutic response, however, accurately recapitulating the tumor microenvironment (TME) remains a persistent challenge, particularly in head and neck cancers (HNC) characterized by complex cell-matrix interactions and heterogeneity. Current models often lack independent tunability of biochemical and biophysical cues, limiting systematic investigation of microenvironmental cues in a high-throughput format. Here, we establish a 3D droplet-based bioprinting platform for the fabrication of customizable in vitro TME models using poly(ethylene glycol) (PEG) hydrogels. Human HNC cell lines (FaDu and 2A3) with differing HPV statuses were bioprinted into PEG matrices spanning physiologically relevant stiffnesses (0.7-4.8 kPa) and compositions, including non-functionalized PEG and peptide-functionalized PEG (PEGfnc: RGD, YIGSR, CNYYSNS) and cultured for 7 days. Cluster growth, cell viability, and cluster morphology were assessed across multiple time points, matrix compositions, and matrix stiffnesses. Proliferation and endpoint phenotype expression were visualized using confocal microscopy through immunofluorescence. Results indicated enhanced cell viability in PEGfnc matrices, compared to non-functionalized matrices, while effect of matrix stiffness was less prominent. Median cluster size reached 40-50 m by day 7, and linear mixed-effects modeling identified how changes in cluster surface area, volume, and tumoroid complexity varied with cell type, matrix, and stiffness. By decoupling and systematically varying key TME parameters, this approach provides a robust and scalable framework for dissecting tumor-matrix interactions and advancing physiologically relevant in vitro models for cancer research and therapeutic screening.

Nanoplastics generated from plastic waste in our ecosystems are becoming increasingly prevalent as bulk plastics exposed to natural factors like water and sunlight fragment to the nanoscale over time. These incidental nanoplastics span a wide range of physicochemical properties, which makes studying nanoplastic interactions in biological systems difficult. Here, we characterized the behavior of incidental nanoplastics generated through mechanical abrasion within coacervate droplets to probe the surface properties of the nanoplastics. We used elastin-like polypeptides (ELPs) to create hydrophobic or charged coacervate microenvironments. Using optical microscopy and fluorescence quantification, we observed that nanoplastics made from polyethylene terephthalate (nPET), nylon 6 (nPA), and polystyrene (nPS) exhibited distinct partitioning behavior with more favorable interactions with hydrophobic droplets. This indicated that the hydrophobic polymer backbone was the predominate surface feature despite exposed functional groups of the incidental nanoplastics, in contrast to findings with model carboxylated latex nanospheres (nPS-COOH). Furthermore, the selective partitioning of incidental nanoplastics into the hydrophobic droplets was able to capture over 80% of nPET in solution, and after recovery of the protein droplet, was able to cumulatively capture over 75% of the nPET feedstock across multiple cycles. This work explores the nuanced surface characteristics of incidental nanoplastics, expands the application of coacervates as chemical probes, and demonstrates a biopolymer approach for effective nanoplastic removal.

Detection of micro- and nanoplastics (MNPs) in human tissues has raised growing concern about their biological effects on tissue and cell function. While previous studies have examined MNP-cell interaction, most focused on limited cell and plastic types. Here, we present a comprehensive, quantitative investigation into how different types of nanoplastics (NPs) associate with and affect diverse cell types under physiologically relevant conditions. Using microfluidic-calibrated fluorescence microscopy, we quantify NP accumulation in cells in vitro and match cellular NP concentrations to levels reported in human tissues. While cell-associated NPs could be gradually released in vitro, they persist in vivo for over one month without detectable reduction in a mouse model. We discover that NP exposure at these levels broadly impairs cell proliferation across epithelial, endothelial, fibroblast, and immune cells, with cell type-dependent sensitivity. NP exposure also reduces motility in T cells and fibroblasts, with more complex effects observed in macrophages. Mechanistically, NP-cell association and trans-epithelial transport involved not only classical endocytic regulators but also pathways related to ion and water transport. Notably, NP association and release were highly sensitive to the extracellular fluid environment within the physiological range. By testing inhibitors of these pathways, we identified molecules that reduce NP-cell association and promote release. We further compared common NPs found in human samples and widely used in research: polystyrene (PS), polyethylene (PE), and polypropylene (PP). Although these NPs similarly impaired proliferation and motility, they showed markedly different cellular association and release dynamics. These findings reveal the impact of NPs on tissue cell functions and uncover novel regulatory pathways, establishing a quantitative framework for studying NP-cell interactions in biologically relevant conditions.

To achieve a therapeutic effect, nanoparticles delivering nucleic acids must facilitate endosomal escape (EE) of their cargo. Despite extensive research, the mechanisms that lead to an effective EE are not sufficiently understood. Herein, we utilized Molecular Dynamics (MD) simulations in All Atom (AA) and Coarse Grained (CG) resolutions to differentiate the interaction of four polymeric formulations (polyplexes) and one lipid nanoparticle (LNP) with endosomal membranes. On the one hand, the results emphasize the benefit of hydrophobic residues in the nanoparticles. On the other hand, the role of anionic lipids in the biological membranes is demonstrated. Furthermore, the identified interaction patterns were successfully correlated to the in vitro performance of the formulations. For the first time, different EE mechanisms of polyplex formulations are visualized in simulation and therefore distinguishable from one another. Hence, this work highlights the power of MD simulations for taking a big step towards better understanding EE efficiency. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/711661v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@abba74org.highwire.dtl.DTLVardef@5e2b8eorg.highwire.dtl.DTLVardef@7db144org.highwire.dtl.DTLVardef@1034e_HPS_FORMAT_FIGEXP M_FIG C_FIG

In this paper, we aim to present a new intravital cells visualization method, which is based on use of a dye called ABDS ("A Beautiful dye for staining"), which can be prepared using a marker pen and is useful for eukaryotic cell research. Using a wide range of instruments, including optical measurements, microscopy studies and wet biology techniques, we have shown that ABDS is close by properties to Rhodamine 6G dye (R6G), which is well known as endoplasmic reticulum stainer. However, by the careful examination of the ABDS and R6G images (ABDS/R6G), we have proved for the first time that these dyes also stain the cytoplasmic membranes. The significant contrast between ABDS/R6G signal from cell membrane and endoplasmic reticulum allows them to be distinguished in the fluorescence photographs. Other important properties of ABDS are its availability, simplicity in manufacturing, safety for living cells in vitro, and bright stable fluorescence, which in contrast to commercial dye like DiBAC allows us to study cells in space and time with high detalization. The paper includes a method for preparing ABDS, a data set with its characteristics, comparison with other commercial dyes, as well as examples of ABDS usage in cells research. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/717455v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@f1ceacorg.highwire.dtl.DTLVardef@137abd2org.highwire.dtl.DTLVardef@1f19efcorg.highwire.dtl.DTLVardef@1fcbc9e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA protocol for high-resolution vital staining of the cells using an inexpensive dye based on permanent marker ink is proposed. C_LIO_LIThe absorption, emission and Raman spectra of the proposed dye are presented, and a direct comparison with commercial dyes Rhodamine 6G, DiBAC and Deep Red Cell Mask dye is made. C_LIO_LIThe main characteristics of the proposed dye are low toxicity, long-term fluorescence, and the ability to separately stain the endoplasmic reticulum and cytoplasmic membrane. C_LIO_LIThe ability of the Rhodamine 6G dye to stain cell membranes also has been proved. C_LI

Cell-penetrating peptides (CPPs) can deliver biomacromolecular cargos into cells, potentially enabling a new mode of intracellular drug delivery. However, a major problem with CPP-mediated delivery is entrapment of CPPs within endosomes and covalent linkages ensure CPPs and cargos share a common fate. We previously developed a CPP-adaptor system based on reversible, calcium-dependent cargo binding that produces cargo release from adaptors as complexes dissociate following internalization and Ca2+ efflux from early endosomes. Having employed CPP-adaptors with an array of protein cargos of differing charges, it became apparent that positively charged cargos often appeared to dominate internalization and that association with the adaptor had little effect. To systematically address the effects of cargo charge and CPP function, we tested the ability of several adaptors to increase internalization of a set of adaptor binding GFP cargos having net charges of +9, +15, +20, +25 and +36. Intrinsic internalization of these cargos reproduced reported patterns showing that positive charge increases internalization. However, labeling these cargos with a chemical fluorophore revealed that GFP fluorescence grossly underestimated total internalization. Internalization was charge and concentration dependent with more positive cargos showing apparent saturation of internalization at 100-400 nM, well below the concentrations at which covalently linked CPP-cargos are dosed. We tested the ability of 5 adaptors to internalize these cargos. Our prototype adaptor, TAT-CaM, was completely ineffective with the +9 cargo, but internalized moderately charged cargos extremely efficiently at concentrations far below the {micro}M range. A derivative adaptor, TAT-LAH4-CaM, was highly effective with all cargos and produced similar maximal internalization at 100-400 nM. However, two adaptors specifically designed with increased positive charge inhibited internalization of the most positive cargos. One of these, GFP-CaM, based on the supercharged GFP with net charge of +36, did increase internalization of the least positive cargos, demonstrating an adaptor with high affinity for the cell surface can increase internalization of a neutral cargo at very low concentration. The common maximal level of intrinsic GFP cargo internalization correlated with surface loading of these cargos, suggesting a limit to the beneficial effects of increased plasma membrane association. However, TAT-CaM further increased internalization via an apparently distinct mechanism. In this limited study of the interaction of cargo charge and adaptor efficacy, we found diverse behaviors that hint at the power and flexibility possible with adaptor/cargo internalization.

The tumor microenvironment (TME) is a complex ecosystem composed of tumor cells, cancer-associated fibroblasts (CAFs), immune suppressive cells, and the extracellular matrix (ECM), playing a crucial role in tumor development and CAR-T cell therapy efficacy. CAR-T therapy has shown promise in hematological malignancies but faces challenges in solid tumors due to the TMEs ability to suppress CAR-T cell infiltration, proliferation, and cytotoxicity. Traditional drug evaluation models, such as 2D cell cultures and animal models, have significant limitations due to oversimplification of the in vivo environment or physiological differences between species. Organoid models offer a more biomimetic approach but often fail to fully recapitulate the TMEs complexity and heterogeneity. Our research developed a tumor organoid and CAF co-culture model using the IBAC co-culture chip, demonstrating that CAFs significantly impact CAR-T cell therapy efficacy by forming physical (e.g., fibronectin) and chemical (e.g., IL-10) barriers that prevent CAR-T cell infiltration and cytotoxicity. This model provides a high-biomimetic platform for investigating the TMEs effects on CAR-T therapy and highlights the importance of incorporating a comprehensive stromal component into in vitro models to enhance their predictive power for cancer treatment.

Predicting high-concentration viscosity of monoclonal antibodies such as IgG1 is crucial for their development as therapeutics for subcutaneous delivery. Unfortunately, traditional experimental rheometry methods for assessing viscosity are low-throughput. This study evaluates Self-Interaction Nanoparticle Spectroscopy (SINS) assays--specifically charge-stabilized SINS (CS-SINS) and PEG-stabilized SINS (PS-SINS)--for high-throughput viscosity prediction. We characterized 96 IgG1 antibodies, assessing SINS against in silico descriptors and dynamic light scattering (DLS) data. CS-SINS showed strong correlation with charge, offering limited additional utility. In contrast, PS-SINS provided orthogonal information; integrating it with in silico data and DLS significantly improved random forest model accuracy for binary viscosity classification. PS-SINS measurements in multiple buffers captured complementary information, achieving comparable accuracy without DLS. Importantly, PS-SINS scores exhibited a strong logarithmic relationship (r=0.98) with high-concentration viscosity in Fc variants of clinical antibodies, suggesting a direct mechanistic link. Furthermore, PS-SINS performed reliably with one column purified (protein A) samples, supporting its early-stage application. These findings establish PS-SINS as a high-throughput tool to accelerate the developability assessment of antibody candidates.

Biocompatible fluorosurfactants are essential for many droplet microfluidic workflows but are often obtained from commercial sources because published syntheses of perfluoropolyether (PFPE)-based surfactants typically require acid chloride intermediates and chemistry-oriented purification methods. These requirements can limit access for biology and clinical laboratories seeking low-cost or customizable surfactant systems. Here we describe a practical method for preparing functional PFPE-based fluorosurfactant materials by direct carbodiimide coupling of functionalized PFPE carboxylic acids(Krytox 157 FSH) to amine-containing head groups under laboratory-accessible conditions. Using this approach, we prepared a PFPE-polyethylene-glycol (PFPE-PEG) material from Jeffamine ED900 and a PFPE-Tris material from Tris base. Because these products were not fully structurally characterized, we present them as functional reaction products and evaluate them by use in biomicrofluidic workflows rather than by definitive compositional assignment. PFPE-Tris was useful for generating relatively uniform small droplets, whereas the PFPE-PEG preparation supported a broader range of biological applications. These materials were used in genomic library screening for {beta}-glucosidase activity, thermocycling-associated droplet workflows, and protein crystallization experiments. In addition, the PFPE-PEG preparation improved emulsion behavior in many protein crystallization screens that were unstable with a commercial droplet oil used in our laboratory. This method reduces the practical barrier to in-house fluorosurfactant preparation and allows biology-focused laboratories to explore head-group chemistry, oil composition, and operating conditions without complete reliance on commercial reagents. The results support this workflow as a useful entry point for biomicrofluidics laboratories, while also highlighting the need for careful interpretation of thermocycled droplet assays and for future analytical characterization of the resulting materials. Significance statementDroplet microfluidics relies on fluorosurfactants that are often costly and difficult to synthesize outside of chemistry-focused settings. We describe a simple, biology-laboratory-compatible approach for generating functional perfluoropolyether-based fluorosurfactant materials using direct carbodiimide coupling and straightforward cleanup. The resulting materials supported multiple biomicrofluidic workflows in our laboratory, including enzymatic screening and protein crystallization, and provide a practical route for groups seeking lower-cost and more customizable surfactant systems.