Evolution of the structure of lipid nanoparticles for nucleic acid delivery: From in situ studies of formulation to colloidal stability.

The development of lipid nanoparticle (LNP) based therapeutics for delivery of RNA has triggered the advance of new strategies for formulation, such as high throughput microfluidics for precise mixing of components into well-defined particles. In this study, we have characterised the structure of LNPs throughout the formulation process using in situ small angle x-ray scattering in the microfluidic chip, then by sampling in the subsequent dialysis process. The final formulation was investigated with small angle x-ray (SAXS) and neutron (SANS) scattering, dynamic light scattering (DLS) and cryo-TEM. The effect on structure was investigated for LNPs with a benchmark lipid composition and containing different cargos: calf thymus DNA (DNA) and two model mRNAs, polyadenylic acid (polyA) and polyuridylic acid (polyU). The LNP structure evolved during mixing in the microfluidic channel, however was only fully developed during the dialysis. The colloidal stability of the final formulation was affected by the type of incorporated nucleic acids (NAs) and decreased with the degree of base-pairing, as polyU induced extensive particle aggregation. The main NA LNP peak in the SAXS data for the final formulation were similar, with the repeat distance increasing from polyU

A droplet acoustofluidic platform for time-controlled microbead-based reactions.

Droplet microfluidics is a powerful method used to characterize chemical reactions at high throughput. Often detection is performed via in-line optical readout, which puts high demands on the detection system or makes detection of low concentration substrates challenging. Here, we have developed a droplet acoustofluidic chip for time-controlled reactions that can be combined with off-line optical readout. The principle of the platform is demonstrated by the enzymatic conversion of fluorescein diphosphate to fluorescein by alkaline phosphatase. The novelty of this work is that the time of the enzymatic reaction is controlled by physically removing the enzymes from the droplets instead of using chemical inhibitors. This is advantageous as inhibitors could potentially interact with the readout. Droplets containing substrate were generated on the chip, and enzyme-coupled microbeads were added into the droplets via pico-injection. The reaction starts as soon as the enzyme/bead complexes are added, and the reaction is stopped when the microbeads are removed from the droplets at a channel bifurcation. The encapsulated microbeads were focused in the droplets by acoustophoresis during the split, leaving the product in the side daughter droplet to be collected for the analysis (without beads). The time of the reaction was controlled by using different outlets, positioned at different lengths from the pico-injector. The enzymatic conversion could be measured with fluorescence readout in a separate PDMS based assay chip. We show the ability to perform time-controlled enzymatic assays in droplet microfluidics coupled to an off-line optical readout, without the need of enzyme inhibitors.

Acoustic focusing of beads and cells in hydrogel droplets.

The generation of hydrogel droplets using droplet microfluidics has emerged as a powerful tool with many applications in biology and medicine. Here, a microfluidic system to control the position of particles (beads or astrocyte cells) in hydrogel droplets using bulk acoustic standing waves is presented. The chip consisted of a droplet generator and a 380 µm wide acoustic focusing channel. Droplets comprising hydrogel precursor solution (polyethylene glycol tetraacrylate or a combination of polyethylene glycol tetraacrylate and gelatine methacrylate), photoinitiator and particles were generated. The droplets passed along the acoustic focusing channel where a half wavelength acoustic standing wave field was generated, and the particles were focused to the centre line of the droplets (i.e. the pressure nodal line) by the acoustic force. The droplets were cross-linked by exposure to UV-light, freezing the particles in their positions. With the acoustics applied, 89 ± 19% of the particles (polystyrene beads, 10 µm diameter) were positioned in an area ± 10% from the centre line. As proof-of-principle for biological particles, astrocytes were focused in hydrogel droplets using the same principle. The viability of the astrocytes after 7 days in culture was 72 ± 22% when exposed to the acoustic focusing compared with 70 ± 19% for samples not exposed to the acoustic focusing. This technology provides a platform to control the spatial position of bioparticles in hydrogel droplets, and opens up for the generation of more complex biological hydrogel structures.

Fabrication of Silicon Microfluidic Chips for Acoustic Particle Focusing Using Direct Laser Writing.

We have developed a fast and simple method for fabricating microfluidic channels in silicon using direct laser writing. The laser microfabrication process was optimised to generate microfluidic channels with vertical walls suitable for acoustic particle focusing by bulk acoustic waves. The width of the acoustic resonance channel was designed to be 380 µm, branching into a trifurcation with 127 µm wide side outlet channels. The optimised settings used to make the microfluidic channels were 50% laser radiation power, 10 kHz pulse frequency and 35 passes. With these settings, six chips could be ablated in 5 h. The microfluidic channels were sealed with a glass wafer using adhesive bonding, diced into individual chips, and a piezoelectric transducer was glued to each chip. With acoustic actuation at 2.03 MHz a half wavelength resonance mode was generated in the microfluidic channel, and polystyrene microparticles (10 µm diameter) were focused along the centre-line of the channel. The presented fabrication process is especially interesting for research purposes as it opens up for rapid prototyping of silicon-glass microfluidic chips for acoustofluidic applications.

On-chip background dilution in droplets with high particle recovery using acoustophoresis.

Droplet microfluidics has shown great potential for on-chip biological and chemical assays. However, fluid exchange in droplet microfluidics with high particle recovery is still a major bottleneck. Here, using acoustophoresis, we present for the first time a label-free method to achieve continuous background dilution in droplets containing cells with high sample recovery. The system comprises droplet generation, acoustic focusing, droplet splitting, picoinjection, and serpentine mixing on the same chip. The capacities of the picoinjection and the droplet split to dilute the background fluorescent signal in the droplets have been characterized. The sample recovery at different droplet split ratios has also been characterized. The results show a maximum of 4.3-fold background dilution with 87.7% particle recovery. We also demonstrated that the system can be used to dilute background fluorescent signal in droplets containing either polystyrene particles or endothelial cells.

An acoustofluidic platform for non-contact trapping of cell-laden hydrogel droplets compatible with optical microscopy.

Production of cell-laden hydrogel droplets as miniaturized niches for 3D cell culture provides a new route for cell-based assays. Such production can be enabled by droplet microfluidics and here we present a droplet trapping system based on bulk acoustic waves for handling hydrogel droplets in a continuous flow format. The droplet trapping system consists of a glass capillary equipped with a small piezoelectric transducer. By applying ultrasound (4 MHz), a localized acoustic standing wave field is generated in the capillary, trapping the droplets in a well-defined cluster above the transducer area. The results show that the droplet cluster can be retained at flow rates of up to 76 µl/min, corresponding to an average flow speed of 3.2 mm/s. The system allows for important operations such as continuous perfusion and/or addition of chemical reagents to the encapsulated cells with in situ optical access. This feature is demonstrated by performing on-chip staining of the cell nuclei. The key advantages of this trapping method are that it is label-free and gentle and thus well-suited for biological applications. Moreover, the droplets can easily be released on-demand, which facilitates downstream analysis. It is envisioned that the presented droplet trapping system will be a valuable tool for a wide range of multistep assays as well as long-term monitoring of cells encapsulated in gel-based droplets.

Particle Manipulation Methods in Droplet Microfluidics.

This Feature describes the different particle manipulation techniques available in the droplet microfluidics toolbox to handle particles encapsulated inside droplets and to manipulate whole droplets. We address the advantages and disadvantages of the different techniques to guide new users.

An intra-droplet particle switch for droplet microfluidics using bulk acoustic waves.

To transfer cell- and bead-assays into droplet-based platforms typically requires the use of complex microfluidic circuits, which calls for methods to switch the direction of the encapsulated particles. We present a microfluidic chip where the combination of acoustic manipulation at two different harmonics and a trident-shaped droplet-splitter enables direction-switching of microbeads and yeast cells in droplet microfluidic circuits. At the first harmonic, the encapsulated particles exit the splitter in the center daughter droplets, while at the second harmonic, the particles exit in the side daughter droplets. This method holds promises for droplet-based assays where particle-positioning needs to be selectively controlled.

Controlled Lateral Positioning of Microparticles Inside Droplets Using Acoustophoresis.

In this paper, we utilize bulk acoustic waves to control the position of microparticles inside droplets in two-phase microfluidic systems and demonstrate a method to enrich the microparticles. In droplet microfluidics, different unit operations are combined and integrated on-chip to miniaturize complex biochemical assays. We present a droplet unit operation capable of controlling the position of microparticles during a trident shaped droplet split. An acoustic standing wave field is generated in the microchannel, and the acoustic forces direct the encapsulated microparticles to the center of the droplets. The method is generic, requires no labeling of the microparticles, and is operated in a noncontact fashion. It was possible to achieve 2+-fold enrichment of polystyrene beads (5 µm in diameter) in the center daughter droplet with an average recovery of 89% of the beads. Red blood cells were also successfully manipulated inside droplets. These results show the possibility to use acoustophoresis in two-phase systems to enrich microparticles and open up the possibility for new droplet-based assays that are not performed today.