Electrophoretic Microfluidic Characterization of mRNA- and pDNA-Loaded Lipid Nanoparticles.

Lipid nanoparticles (LNPs) are clinically advanced nonviral gene delivery vehicles with a demonstrated ability to address viral, oncological, and genetic diseases. However, the further development of LNP therapies requires rapid analytical techniques to support their development and manufacturing. The method developed and described in this paper presents an approach to rapidly and accurately analyze LNPs for optimized therapeutic loading by utilizing an electrophoresis microfluidic platform to analyze the composition of LNPs with different clinical lipid compositions (Onpattro, Comirnaty, and Spikevax) and nucleic acid (plasmid DNA (pDNA) and messenger RNA (mRNA)) formulations. This method enables the high-throughput screening of LNPs using a 96- or 384-well plate with approximate times of 2-4 min per sample using a total volume of 11 µL. The lipid analysis requires concentrations approximately between 109 and 1010 particles/mL and has an average precision error of 10.4% and a prediction error of 19.1% when compared to using a NanoSight, while the nucleic acid analysis requires low concentrations of 1.17 ng/µL for pDNA and 0.17 ng/µL for mRNA and has an average precision error of 4.8% and a prediction error of 9.4% when compared to using a PicoGreen and RiboGreen assay. In addition, our method quantifies the relative concentration of nucleic acid per LNP. Utilizing this approach, we observed an average of 263 ± 62.2 mRNA per LNP and 126.3 ± 21.2 pDNA per LNP for the LNP formulations used in this study, where the accuracy of these estimations is dependent on reference standards. We foresee the utility of this technique in the high-throughput characterization of LNPs during manufacturing and formulation research and development.

On the potential of microscale electrokinetic cascade devices.

Phages used for phage therapy of multidrug resistant bacteria must be highly purified prior to use. There are limited purification approaches that are broadly applicable to many phage types. Electrokinetics has shown great potential to manipulate phages, but obstructions from the cell debris produced during phage propagation can severely diminish the capacity of an electrokinetic device to concentrate and purify phage samples. A multipart insulator-based electrokinetic device is proposed here to remove the larger, undesirable components of mixtures from phage preparations while transferring the freshly purified and concentrated sample to a second stage for downstream analysis. By combining the large debris prescreen and analysis stages in a streamlined system, this approach simultaneously reduces the impact of clogging and minimizes the sample loss observed during manual transferring of purified samples. Polystyrene particles were used to demonstrate a diminished sample loss of approximately one order of magnitude when using the cascade device as opposed to a manual transfer scheme. The purification and concentration of three different phage samples were demonstrated using the first stage of the cascade device as a prescreen. This design provides a simple method of purifying and concentrating valuable samples from a complex mixture that might impede separation capacity in a single channel.

Simultaneous Determination of Linear and Nonlinear Electrophoretic Mobilities of Cells and Microparticles.

Direct-current insulator-based electrokinetics (DC-iEK) is a branch of microfluidics that has demonstrated to be an attractive and efficient technique for manipulating micro- and nano- particles, including microorganisms. A unique feature of DC-iEK devices is that nonlinear EK effects are enhanced by the presence of regions of higher field intensity between the insulating structures. Accurate computational models, describing particle and cell behavior, are crucial to optimize the design and improve the performance of DC-iEK devices. The electrokinetic equilibrium condition (EEEC) is a recently introduced fundamental concept that has radically shifted the perspective behind the analysis of particle manipulation in these microfluidic devices. The EEEC takes into consideration previously neglected nonlinear effects on particle migration and indicates that these effects are central to control particle motion in DC-iEK devices. In this study, we present a simultaneous experimental characterization of linear and nonlinear electrokinetic (EK) parameters, that is, the electrophoretic mobility (µEP(1)), the particle zeta potential (ζP), the EEEC, and the electrophoretic mobility of the second kind (µEP(3)), for four types of polystyrene microparticles and four cell strains. For this, we studied the electromigration of polystyrene microparticles ranging in size from 2 to 6.8 µm, three bacteria strains (B. cereus, E. coli, and S. enterica) and a yeast cell (S. cerevisiae), ranging in size from 1 to 6.3 µm, in a polydimethylsiloxane (PDMS) microfluidic channel with a rectangular cross-section. The results illustrated that electrokinetic particle trapping can occur by linear and nonlinear electrophoresis and electroosmosis reaching an equilibrium, without the presence of insulating posts. The experimentally measured parameters reported herein will allow optimizing the design of future DC-iEK devices for a wide range of applications (e.g., to separate multiple kinds of particles and microorganisms) and for developing computational models that better represent reality.