Horseshoe lamination mixer (HLM) sets new standards in the production of monodisperse lipid nanoparticles.

Microfluidic mixers promise unique conditions for the controlled and continuous preparation of nanoparticles by antisolvent precipitation. Nanoparticles may enable encapsulation of drug or mRNA molecules in the form of carrier nanoparticles or can provide higher bioavailability in the form of drug nanoparticles. The ultimate goal in microfluidic approaches is the production of nanoparticles with narrow size distributions while avoiding contaminations and achieving sufficiently high throughput. To achieve this, a novel microfluidic precipitation device was developed and realized by two-photon polymerization: mixing elements were designed in such a way that the liquids undergo a repeated Smale horseshoe transformation resulting in an increased interfacial area and mixing times of less than 10 ms. These elements and an additional 3D flow focusing ensure that no organic phase is exposed to the channel walls. The integration of a fluidic shield layer in the flow focusing proved to be useful to delay the precipitation process until reaching a sufficient distance to the injection nozzle. Lipid nanoparticle preparation with different concentrations of castor oil or the hard fat Softisan® 100 were performed at different flow rates and mixing ratios with and without a shield layer. Flow rates of up to 800 µl min-1 and organic phase mixing ratios of up to 20% resulted in particle sizes ranging from 42 nm to 166 nm with polydispersity indices from 0.04 to 0.11, indicating very narrowly distributed, and in most cases even monodisperse, nanoparticles. The occurrence of fouling can be completely suppressed with this new type of mixing elements, as long as Dean vortices are prevented. Moreover, this parameter range in the horseshoe lamination mixer provided a stable and continuous process, which enables a scalable production.

Goodbye fouling: a unique coaxial lamination mixer (CLM) enabled by two-photon polymerization for the stable production of monodisperse drug carrier nanoparticles.

Poorly soluble drugs can be incorporated in lipid carrier nanoparticles to achieve sufficient bioavailability and open up diverse routes of administration. Preparation by antisolvent precipitation in microfluidic systems enables excellent control of lipid nanoparticle size. However, particle-containing flows bear the risk of material deposition on microchannel surfaces, limiting reproducibility, prolonged continuous processing and scale-up by parallelization as required for practical use. The coaxial lamination mixer (CLM) introduced in this study can fully eliminate contact of the organic phase with the channel walls while efficiently mixing organic and aqueous phases. This unique micromixer, including a nozzle for coaxial injection, a sequence of stretch-and-fold elements and inlet filters, cannot be realized by conventional 2.5D microfabrication but only by 3D two-photon polymerization. Hydrodynamic focusing of the organic phase and fast coaxial lamination were studied in simulations and flow visualization experiments. Different concentrations of castor oil or a hard fat and polysorbate 80 dissolved in ethanol were injected and combined with purified water. Total flow rates of 100 and 200 µL min-1 and flow rate ratios of 15% or less resulted in particle sizes between 67 and 153 nm and polydispersity indices of 0.04 to 0.10. Extended preparation time revealed stable particle sizes and displayed no fouling, indicating that CLMs will even allow high throughput parallelization. Stable castor oil nanoemulsions loaded with the poorly soluble drugs fenofibrate or cannabidiol were prepared. In conclusion, the unique 3D design of the CLM enables prolonged, stable and scalable production of small as well as very narrowly distributed, in most cases even monodisperse drug-loaded lipid nanoparticles.

Antisolvent precipitation of lipid nanoparticles in microfluidic systems - A comparative study.

Microsystems offer promising possibilities to produce nanoparticles which can be used as carriers for poorly water-soluble active substances. The aim of the present study was to compare the preparation of lipid nanoparticles by precipitation in different microsystems: A segmented-flow micromixer, a high-pressure micromixer and the commercial NanoAssemblrTM platform with a staggered herringbone micromixer. A batch set-up served as reference experiment. Castor oil nanoemulsions prepared with polysorbate 80 as surfactant in the aqueous phase were in the size range of 36-160 nm. The particle sizes could be reduced to 43-93 nm when the surfactant was processed via the ethanolic phase. Furthermore, glycerol monooleate nanodispersions (65-141 nm) were manufactured with poloxamer 407 added as stabilizer via the aqueous phase. Deposition of lipid material in the segmented-flow micromixer could be reduced by a modification of the design. Preparation in the high-pressure mixer and in the herringbone mixer at high total flow rates resulted in the smallest particles for castor oil emulsions, but with bimodal distributions. The particle size of glycerol monooleate dispersions was smallest when prepared in the high-pressure micromixer and in the herringbone micromixer at a higher flow rate. In conclusion, microfluidic systems can be a useful tool to produce lipid nanoparticles.

Stabilized Production of Lipid Nanoparticles of Tunable Size in Taylor Flow Glass Devices with High-Surface-Quality 3D Microchannels.

Nanoparticles as an application platform for active ingredients offer the advantage of efficient absorption and rapid dissolution in the organism, even in cases of poor water solubility. Active substances can either be presented directly as nanoparticles or can be integrated in a colloidal carrier system (e.g., lipid nanoparticles). For bottom-up nanoparticle production minimizing particle contamination, precipitation processes provide an adequate approach. Microfluidic systems ensure a precise control of mixing for the precipitation, which enables a tunable particle size definition. In this work, a gas/liquid Taylor flow micromixer made of chemically inert glass is presented, in which the organic phases are injected through a symmetric inlet structure. The 3D structuring of the glass was performed by femtosecond laser ablation. Rough microchannel walls are typically obtained by laser ablation but were smoothed by a subsequent annealing process resulting in lower hydrophilicity and even rounder channel cross-sections. Only with such smooth channel walls can a substantial reduction of fouling be obtained, allowing for stable operation over longer periods. The ultrafast mixing of the solutions could be adjusted by simply changing the gas volume flow rate. Narrow particle size distributions are obtained for smaller gas bubbles with a low backflow and when the rate of liquid volume flow has a small influence on particle precipitation. Therefore, nanoparticles with adjustable sizes of down to 70 nm could be reliably produced in continuous mode. Particle size distributions could be narrowed to a polydispersity value of 0.12.

A Microfluidic Split-Flow Technology for Product Characterization in Continuous Low-Volume Nanoparticle Synthesis.

A key aspect of microfluidic processes is their ability to perform chemical reactions in small volumes under continuous flow. However, a continuous process requires stable reagent flow over a prolonged period. This can be challenging in microfluidic systems, as bubbles or particles easily block or alter the flow. Online analysis of the product stream can alleviate this problem by providing a feedback signal. When this signal exceeds a pre-defined range, the process can be re-adjusted or interrupted to prevent contamination. Here we demonstrate the feasibility of this concept by implementing a microfluidic detector downstream of a segmented-flow system for the synthesis of lipid nanoparticles. To match the flow rate through the detector to the measurement bandwidth independent of the synthesis requirements, a small stream is sidelined from the original product stream and routed through a measuring channel with 2 × 2 µm cross-section. The small size of the measuring channel prevents the entry of air plugs, which are inherent to our segmented flow synthesis device. Nanoparticles passing through the small channel were detected and characterized by quantitative fluorescence measurements. With this setup, we were able to count single nanoparticles. This way, we were able to detect changes in the particle synthesis affecting the size, concentration, or velocity of the particles in suspension. We envision that the flow-splitting scheme demonstrated here can be transferred to detection methods other than fluorescence for continuous monitoring and feedback control of microfluidic nanoparticle synthesis.