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.

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.

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.

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.

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.

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.

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.