Full-cycle study on developing a novel structured micromixer and evaluating the nanoparticle products as mRNA delivery carriers.

Achieving precise control of nanoparticle size while maintaining consistency and high uniformity is of paramount importance for improving the efficacy of nanoparticle-based therapies and minimizing potential side effects. Although microfluidic technologies are widely used for reliable nanoparticle synthesis, they face challenges in meeting critical homogeneity requirements, mainly due to imperfect mixing efficiency. Furthermore, channel clogging during continuous operation presents a significant obstacle in terms of quality control, as it progressively impedes the mixing behavior necessary for consistent nanoparticle production for therapeutic delivery and complicates the scaling-up process. This study entailed the development of a 3D-printed novel micromixer embedded with hemispherical baffle microstructures, a dual vortex mixer (DVM), which integrates Dean vortices to generate two symmetrical counter-rotating intensified secondary flows. The DVM with a relatively large mixer volume showed rapid mixing characteristics even at a flow rate of several mL min-1 and produced highly uniform lipids, liposomes, and polymer nanoparticles in a size range (50-130 nm) and polydispersity index (PDI) values below 0.15. For the evaluation of products, SARS-CoV-2 Spike mRNA-loaded lipid nanoparticles were examined to verify protein expression in vitro and in vivo using firefly luciferase (FLuc) mRNA. This showed that the performance of the system is comparable to that of a commercial toroidal mixer. Moreover, the vigorous in-situ dispersion of nanoparticles by harnessing the power of vortex physically minimizes the occurrence of aggregation, ensuring consistent production performance without internal clogging of a half-day operation and facilitating quality control of the nanoparticles at desired scales.

Facile scalable one-flow synthesis of ionizable cationic lipid library as precursors of nanoparticle carriers.

A variety of ionizable and cationic lipids have been synthesized as precursors for nanoparticle carriers. However, the laborious synthetic routes in batch reactors often involve the use of toxic and carcinogenic agents, as well as challenge of removing gaseous byproducts. In this study, we present facile one-flow micro-reaction process that enables the synthesis of 11 ionizable lipids as well as 7 cationic lipids, including the well-known DODAP and DOTAP. These lipids can be scaled up to produce approximately ∼10g/h by using a straightforward size-up approach. The development of the lipid library was involved generating highly moisture-sensitive acyl chloride at 25 °C for 1.5 min. The toxic byproducts such as HCl, CO2 and CO were subsequently removed using a liquid-gas separator. The esterification with dimethylamino-1,2-diol at 25 °C for 3 min, monitored in-line with FTIR, completed the process. Additionally, the synthesized ionizable lipids were converted to cationic lipids with methyl sulfate, chloride ions via dimethyl sulfate and Steglich esterification in a continuous flow system. Finally, the produced DODAP was transformed into a uniform-sized LNPs (64 nm, PDI 0.07) and liposomal nanoparticles (72 nm, PDI 0.05) while DOTAP was converted to liposomes (55 nm, PDI 0.08) using a custom micro-mixer. This efficient platform for lipid synthesis significantly contributes to the practical applications of lipid-based nanomedicines.

Flash precipitation of random copolymers in a micro-mixer for controlling the size and surface charge of nanoparticles.

Precisely controlling the size and surface chemistry of polymeric nanoparticles (P-NPs) is critical for their versatile engineering and biomedical applications. In this work, various NPs of amphipathic random copolymers were comparatively produced by the flash nanoprecipitation (FNP) method using a tube-in-tube type of micro-mixer up to 330 mg min-1 in production scale in a kinetically controlled manner. The NPs obtained from poly(styrene-co-maleic acid), poly(styrene-co-allyl alcohol), and poly(methyl methacrylate-co-methacrylic acid) were concurrently controlled in the range 51-819 nm in size with narrow polydispersity index (<0.1) and -44 to -16 mV in zeta potential, by depending not only on the polymeric chemistry and the concentration but also the mixing behavior of good solvents (THF, alcohols) and anti-solvent (water) under three flow regimes (laminar, vortex and turbulence, turbulent jet). Moreover, the P(St-MA) derived NPs under turbulent jet flow conditions were post-treated in the initial solution mixture for up to 16 h, resulting in lowering of the zeta potential to -52 mV from the initial -27 mV and decreasing size to 46 nm from 50 nm by further migration of hydrophilic segments with -COOH groups on the outer surface, and the removal of THF trapped in the hydrophobic core.

Auto-Optimized Electro-Flow Reactor Platform for the in-situ Reduction of P(V) Oxide to P(III) and Their Application.

Trivalent phosphine catalysis is mostly utilized to activate the carbon-carbon multiple bonds to form carbanion intermediate species and is highly sensitive to certain variables. Random manual multi-variables are critical for understanding the batch disabled regeneration of trivalent phosphine chemistry. We need the artificial intelligence-based system which can change the variable based on previously conducted failed experiment. Herein, we report an auto-optimized electro-micro-flow reactor platform for the in-situ reduction of stable P(V) oxide to sensitive P(III) and further utilized the method for Corey-Fuchs reaction.

Toward microfluidic continuous-flow and intelligent downstream processing of biopharmaceuticals.

Biopharmaceuticals have emerged as powerful therapeutic agents, revolutionizing the treatment landscape for various diseases, including cancer, infectious diseases, autoimmune and genetic disorders. These biotherapeutics pave the way for precision medicine with their unique and targeted capabilities. The production of high-quality biologics entails intricate manufacturing processes, including cell culture, fermentation, purification, and formulation, necessitating specialized facilities and expertise. These complex processes are subject to rigorous regulatory oversight to evaluate the safety, efficacy, and quality of biotherapeutics prior to clinical approval. Consequently, these drugs undergo extensive purification unit operations to achieve high purity by effectively removing impurities and contaminants. The field of personalized precision medicine necessitates the development of novel and highly efficient technologies. Microfluidic technology addresses unmet needs by enabling precise and compact separation, allowing rapid, integrated and continuous purification modules. Moreover, the integration of intelligent biomanufacturing systems with miniaturized devices presents an opportunity to significantly enhance the robustness of complex downstream processing of biopharmaceuticals, with the benefits of automation and advanced control. This allows seamless data exchange, real-time monitoring, and synchronization of purification steps, leading to improved process efficiency, data management, and decision-making. Integrating autonomous systems into biopharmaceutical purification ensures adherence to regulatory standards, such as good manufacturing practice (GMP), positioning the industry to effectively address emerging market demands for personalized precision nano-medicines. This perspective review will emphasize on the significance, challenges, and prospects associated with the adoption of continuous, integrated, and intelligent methodologies in small-scale downstream processing for various types of biologics. By utilizing microfluidic technology and intelligent systems, purification processes can be enhanced for increased efficiency, cost-effectiveness, and regulatory compliance, shaping the future of biopharmaceutical production and enabling the development of personalized and targeted therapies.

Continuous and autonomous-flow separation of laccase enzyme utilizing functionalized aqueous two-phase system with computer vision control.

Downstream processing of biomolecules, particularly therapeutic proteins and enzymes, presents a formidable challenge due to intricate unit operations and high costs. This study introduces a novel cysteine (cys) functionalized aqueous two-phase system (ATPS) utilizing polyethylene glycol (PEG) and potassium phosphate, referred as PEG-K3PO4/cys, for selective extraction of laccase from complex protein mixtures. A 3D-baffle micro-mixer and phase separator was meticulously designed and equipped with computer vision controller, to enable precise mixing and continuous phase separation under automated-flow. Microfluidic-assisted ATPS exhibits substantial increase in partition coefficient (Kflow = 16.3) and extraction efficiency (EEflow = 88 %) for laccase compared to conventional batch process. Integrated and continuous-flow process efficiently partitioned laccase, even in low concentrations and complex crude extracts. Circular dichroism spectra of laccase confirm structural stability of enzyme throughout the purification process. Eventually, continuous-flow microfluidic bioseparation is highly useful for seamless downstream processing of target biopharmaceuticals in integrated and autonomous manner.

Ultrafast Flow Synthesis of o-Functionalized Benzenesulfonyl Fluorides and Subsequent SuFEx Connections via Lithiated Chemistry.

Herein we present a flow-based, rapid, and straightforward approach to synthesize diverse functionalized sulfonyl fluorides by harnessing an aryllithium intermediate. The aryllithium intermediate was fully utilized under optimized conditions (0.016 s, -18 °C) to afford various functionalized sulfonyl fluorides and also intramolecular SuFEx cyclization products in high yields (27-94%). Furthermore, the integrated synthesis incorporating subsequent SuFEx connections with even unstable organolithium nucleophiles facilitated one-flow molecular assembly in high yields (42-72%).

Magnetic delivery and ultrasound-responsive release of chelating microcapsules for selective removal of urolithiasis.

A novel urolithiasis treatment in which a chelating solution encapsulated in poly(lactic-co-glycolic acid); PLGA-based microcapsules was delivered magnetically to specific urolithiasis sites and then subjected to ultrasound (US) to release the chelating solution and dissolve the stones. Using a double-droplet microfluidics method, a hexametaphosphate (HMP) chelating solution was encapsulated in an Fe3O4 nanoparticle (Fe3O4 NP)-loaded PLGA polymer shell with a thickness of <15 µm, forming homogenous microcapsules of 319 ± 14 µm in size. The obtained microcapsules (HMP/Fe3O4@PLGA) exhibited efficient magnetic mobility and US-responsive solution release. Moreover, in a Ψ-shaped flow chip, selective delivery of HMP from the microcapsules was achieved with high magnetic delivery efficiency (>90%), and an effective removal efficacy (>95%, 7 repeat cycles) of artificial calcium oxalate (5 mm in size) via a chelating effect. Eventually, the potential removal of urolithiasis in the body was verified using a PDMS-based kidney urinary flow-imitating chip with a human kidney stone (CaOx 100%, 5-7 mm in size) located in the minor calyx under an artificial urine counter flow (0.5 mL min-1). In the end, more than 50% of the stone, even in surgically tricky regions, was removed by 10 repeated treatments. Therefore, the selective approach of stone-dissolution capsules will help to develop alternative urolithiasis treatments to conventional surgical and systemic dissolution approaches.

Flow Synthesis of Gigantic Porphyrinic Cages: Facile Synthesis of P12 L24 and Discovery of Kinetic Product P9 L18.

A continuous flow methodology for the facile and high-yielding synthesis of the porphyrin-based self-assembled organic cage, P12 L24 is reported, along with the serendipitous discovery of a kinetic product, P9 L18 cage, which has been characterized by MALDI-TOF MS, NMR, and AFM analysis. A theoretical study suggests a tricapped trigonal prismatic geometry for P9 L18 . Unlike P12 L24 , P9 L18 is unstable and readily decomposes into monomers and small oligomers. While the batch synthesis produces only the thermodynamic product P12 L24 , the continuous flow process generates not only the thermodynamic product but also kinetic products, such as P9 L18 , illustrating the advantages of the continuous flow process for the synthesis of self-assembled cages and the exploration of new non-equilibrium assemblies.

Ex-situ generation and synthetic utilization of bare trifluoromethyl anion in flow via rapid biphasic mixing.

Fluoroform (CF3H) is the simplest reagent for nucleophilic trifluoromethylation intermediated by trifluoromethyl anion (CF3-). However, it has been well-known that CF3- should be generated in presence of a stabilizer or reaction partner (in-situ method) due to its short lifetime, which results in the fundamental limitation on its synthetic utilization. We herein report a bare CF3- can be ex-situ generated and directly used for the synthesis of diverse trifluoromethylated compounds in a devised flow dissolver for rapid biphasic mixing of gaseous CF3H and liquid reagents that was designed and structurally optimized by computational fluid dynamics (CFD). In flow, various substrates including multi-functional compounds were chemoselectively reacted with CF3-, extending to the multi-gram-scale synthesis of valuable compounds by 1-hour operation of the integrated flow system.

Meta optimization based on real-time benchmarking of multiple surrogate models for autonomous flow synthesis.

Optimizing a wide range of reaction parameters, steps, and pathways is currently considered one of the most complex and challenging problems in microflow-based organic synthesis. As a novel solution, Bayesian optimization (BO) has been utilized to efficiently guide the optimized conditions of flow reactors; however, the benchmarking process for selecting the optimal model among various surrogate models remains inefficient. In this work, we report meta optimization (MO) by benchmarking multiple surrogate models in real-time without any pre-work, which is realized by evaluating the expected values obtained by the regressor used to build each surrogate model, enabling efficient optimization of reaction conditions. By the comparison of the performance of MO with that of various BOs on four datasets of different flow syntheses, it was verified that MO consistently performs the best-in-class for all emulators developed through machine learning, while the conventional BOs based on surrogate models such as the Gaussian process, random forest, neural network ensemble, and gradient boosting demonstrated varying performances from each emulator, which implies that benchmarking is required.

Microalgae-Based Biohybrid Microrobot for Accelerated Diabetic Wound Healing.

A variety of wound healing platforms have been proposed to alleviate the hypoxic condition and/or to modulate the immune responses for the treatment of chronic wounds in diabetes. However, these platforms with the passive diffusion of therapeutic agents through the blood clot result in the relatively low delivery efficiency into the deep wound site. Here, a microalgae-based biohybrid microrobot for accelerated diabetic wound healing is developed. The biohybrid microrobot autonomously moves at velocity of 33.3 µm s-1 and generates oxygen for the alleviation of hypoxic condition. In addition, the microrobot efficiently bound with inflammatory chemokines of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) for modulating the immune responses. The enhanced penetration of microrobot is corroborated by measuring fibrin clots in biomimetic wound using microfluidic devices and the enhanced retention of microrobot is confirmed in the real wounded mouse skin tissue. After deposition on the chronic wound in diabetic mice without wound dressing, the wounds treated with microrobots are completely healed after 9 days with the significant decrease of inflammatory cytokines below 31% of the control level and the upregulated angiogenesis above 20 times of CD31+ cells. These results confirm the feasibility of microrobots as a next-generation platform for diabetic wound healing.

Modular Flow Reactors for Valorization of Kraft Lignin and Low-Voltage Hydrogen Production.

Recent studies have found that green hydrogen production and biomass utilization technologies can be combined to efficiently produce both hydrogen and value-added chemicals using biomass as an electron and proton source. However, the majority of them have been limited to proof-of-concept demonstrations based on batch systems. Here the authors report the design of modular flow systems for the continuous depolymerization and valorization of lignin and low-voltage hydrogen production. A redox-active phosphomolybdic acid is used as a catalyst to depolymerize lignin with the production of aromatic compounds and extraction of electrons for hydrogen production. Individual processes for lignin depolymerization, byproduct separation, and hydrogen production with catalyst reactivation are modularized and integrated to perform the entire process in the serial flow. Consequently, this work enabled a one-flow process from biomass conversion to hydrogen gas generation under a cyclic loop. In addition, the unique advantages of the fluidic system (i.e., effective mass and heat transfer) substantially improved the yield and efficiency, leading to hydrogen production at a higher current density (20.5 mA cm-2 ) at a lower voltage (1.5 V) without oxygen evolution. This sustainable eco-chemical platform envisages scalable co-production of valuable chemicals and green hydrogen for industrial purposes in an energy-saving and safe manner.

Microfluidic-driven ultrafast self-assembly of a dipeptide into stimuli-responsive 0D, 1D, and 2D nanostructures and as hydrolase mimic.

Numerous peptides have been utilized to explore the efficacy of their self-assembly to produce nanostructures to mimic the self-organization capability of biomolecules in nature. Self-assembled nanostructures have significant applicability for a range of diverse applications. While the ability to create self-assembled functional materials has greatly improved, the self-assembly process, which results in ordered 0D, 1D, and 2D nanostructures, is still time-consuming. Moreover, in situ structural transformation from one self-assembled structure to another with different dimensions presents an additional challenge. Therefore, in this report, we demonstrate self-assembly in an ultrafast fashion to access four different nanostructures, namely, twisted bundle (TB), nanoparticle (NP), nanofiber (NF), and nanosheet (NS), from a simple dipeptide with the aid of simple microfluidic reactors by applying different stimuli. Additionally, an integrated microfluidic system enabled rapid structural switchover between two types in an ultrashort period of time. It is interesting to note that the formation of the twisted bundle (TB) morphology enabled the formation of an extended entangled network, which resulted in the formation of a hydrogel (1 w/v%). In addition, the nanostructures obtained using the ultrafast self-assembly process were investigated to study their hydrolase enzyme activity mimicking performance against a model substrate (p-NPA) reaction. Intriguingly, we found that our nanostructures were suitably well ordered, and when taking molecular mass into consideration, showed improved catalytic efficiency as compared to the native enzymes.

Production of H2-Free Carbon Monoxide from Formic Acid Dehydration: The Catalytic Role of Acid Sites in Sulfated Zirconia.

The formic acid (CH2O2) decomposition over sulfated zirconia (SZ) catalysts prepared under different synthesis conditions, such as calcination temperature (500-650 °C) and sulfate loading (0-20 wt.%), was investigated. Three sulfate species (tridentate, bridging bidentate, and pyrosulfate) on the SZ catalysts were characterized by using temperature-programmed decomposition (TPDE), Fourier-transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). The acidic properties of the SZ catalysts were investigated by the temperature-programmed desorption of iso-propanol (IPA-TPD) and pyridine-adsorbed infrared (Py-IR) spectroscopy and correlated with their catalytic properties in formic acid decomposition. The relative contributions of Brønsted and Lewis acid sites to the formic acid dehydration were compared, and optimal synthetic conditions, such as calcination temperature and sulfate loading, were proposed.

A Perfluoropolyether Microfluidic Device for Cell-Based Drug Screening with Accurate Quantitative Analysis.

Microfluidic drug screening technologies have been extensively explored to evaluate the pharmacology and therapeutic implications of promising chemical compounds in multiplexed physiological microenvironments in vivo. However, conventional poly(dimethylsiloxane) microchips are susceptible to adsorption by hydrophobic molecules on channel surfaces and permeation in the matrix. These can significantly compromise the drug availability and accuracy of dose-dependent quantitative analyses. Here, we prepared a perfluorinated polyether (PFPE) microchip via digital light processing 3D printing as a quantitative drug screening platform for precise concentration-dependent pharmaceutical assays. Cells cultured on PFPE microchips exhibited excellent viability with a spread morphology as well as superior proliferative capability. Importantly, PFPE constructions with a low surface energy significantly prevented the nonspecific molecular adsorption into their surfaces or permeation into the matrix. In particular, the PFPE multibranched channel preserved the concentration of the pharmaceutical drug during the perfusion process and generated a linear concentration gradient, resulting in a dose-dependent chemotherapeutic effect. We suggest that the biocompatible and nonadsorbing PFPE microchannel can provide a cell-based drug screening platform for concentration-dependent quantitative analyses.

Laminar flow-assisted synthesis of amorphous ZIF-8-based nano-motor with enhanced transmigration for photothermal cancer therapy.

Because of their biocompatibility, there are promising applications in various fields for enzyme-powered nano-motors. However, enzymes can undergo denaturation under harsh conditions. Here, we report the flow-assisted synthesis of an enzyme-based amorphous ZIF-8 nano-motor (A-motor; Pdop@urease@aZIF-8) for enhanced movement and protection of polydopamine and enzymes. Multiple laminar flow types with varied input ratios effectively entrapped enzymes into amorphous ZIF-8 shells in a serial flow with a momentary difference. The obtained A-motor exhibited superior enzymatic activity and photothermal ablation properties with excellent durability due to the protection the amorphous shell offers from the external environment. Furthermore, in the bio-mimic 2D membrane model, the enhanced mobility of the A-motor afforded high transmigration (>80%), which had a powerful effect on bladder cancer cell ablation via photothermal therapy. This work envisages that the rapid flow approach will facilitate scalable manufacturing of the nano-motors under low stress to vulnerable biomolecules, which would be extended to nano-biomedical applications in various body environments.

Rapid Flow Synthesis of a Biomimetic Carbonate Apatite as an Effective Drug Carrier.

A facile synthesis of apatite nanocrystals analogous to bioapatites with increased biocompatibility and biodegradability can remedy the shortcomings of the widely applied synthetic hydroxyapatite (HAp) for bone defect treatment. Here, we propose an expeditious synthesis method to develop a biomimetic B-type carbonate apatite (CAp) with a simple capillary microfluidic device at room temperature. The process not only eliminates fluctuations with the addition of carbonate but also produces safe CAp drug carriers through simultaneous alendronate incorporation to the CAp structure. CAp displayed superior mineralization on osteoblast-like MG-63 cells when compared with HAp and HAp drug carriers that were produced using identical methods. Furthermore, alendronate-incorporated CAp drug carriers potentially displayed higher cancer cell suppression when applied to breast cancer cells attached to the bone tissue model, which signifies enhanced cancer metastasis to bone suppression due to the likelihood of increased alendronate release of CAp owing to its faster dissolution. Overall, our results may provide promising opportunities for enhanced clinical CAp application for bone defect treatment, particularly for bone loss and cancer to bone metastasis.

Cytocompatible asymmetrical coating for Janus carrier synthesis through capillary wetting and ascending.

Despite the successful implementation of elegant strategies for the fabrication of Janus microstructures, two critical factors have limited the applicability of most techniques for the partial modification of living cell surfaces: harsh conditions that could disintegrate cells, and the lack of an effective route to accomplish a mild modification for living cells. In this study, an expeditious synthesis, named lower-half occupation by capillary ascended liquids (LOCAL), is proposed for the fabrication of asymmetrical structures surrounding not only microbeads but also both living adherent and buoyant mammalian cells. The microbeads or living cells are safely supported and trapped on the apical sides of a micropillar array, which prevents them from contacting the bottom substrate. As the coating agents further transfer and contact the trapped particles through interpillar capillary flow, the autonomous capillary ascending coats the free bottom surfaces of the target particles within 2 min, with significantly small quantities of coating agents. The self-assembled architectures of the cells demonstrate thoroughly maintained cell viability, highlighting the potential of the LOCAL method as a desirable alternative to the widely applied state-of-art methods for developing Janus beads and Janus cells.

Scalable Subsecond Synthesis of Drug Scaffolds via Aryllithium Intermediates by Numbered-up 3D-Printed Metal Microreactors.

Continuous-flow microreactors enable ultrafast chemistry; however, their small capacity restricts industrial-level productivity of pharmaceutical compounds. In this work, scale-up subsecond synthesis of drug scaffolds was achieved via a 16 numbered-up printed metal microreactor (16N-PMR) assembly to render high productivity up to 20 g for 10 min operation. Initially, ultrafast synthetic chemistry of unstable lithiated intermediates in the halogen-lithium exchange reactions of three aryl halides and subsequent reactions with diverse electrophiles were carried out using a single microreactor (SMR). Larger production of the ultrafast synthesis was achieved by devising a monolithic module of 4 numbered-up 3D-printed metal microreactor (4N-PMR) that was integrated by laminating four SMRs and four bifurcation flow distributors in a compact manner. Eventually, the 16N-PMR system for the scalable subsecond synthesis of three drug scaffolds was assembled by stacking four monolithic modules of 4N-PMRs.