Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids.

Lipid-based nanocarriers have emerged as helpful tools to deliver sensible biomolecules such as proteins and oligonucleotides. To have a fast and robust microfluidic-based nanoparticle synthesis method, the setup of versatile equipment should allow for the rapid transfer to scale cost-effectively while ensuring tunable, precise and reproducible nanoparticle attributes. The present work aims to assess the effect of different micromixer geometries on the manufacturing of lipid nanocarriers taking into account the influence on the mixing efficiency by changing the fluid-fluid interface and indeed the mass transfer. Since the geometry of the adopted micromixer varies from those already published, a Design of Experiment (DoE) was necessary to identify the operating (total flow, flow rate ratio) and formulation (lipid concentration, lipid molar ratios) parameters affecting the nanocarrier quality. The suitable application of the platform was investigated by producing neutral, stealth and cationic liposomes, using DaunoXome®, Myocet®, Onivyde® and Onpattro® as the benchmark. The effect of condensing lipid (DOTAP, 3-10-20 mol%), coating lipids (DSPE-PEG550 and DSPE-PEG2000), as well as structural lipids (DSPC, eggPC) was pointed out. A very satisfactory encapsulation efficiency, always higher than 70%, was successfully obtained for model biomolecules (myoglobin, short and long nucleic acids).

Development and optimization of microfluidic assisted manufacturing process to produce PLGA nanoparticles.

Nanomedicine consists in the application of nanotechnology in medicine to revolutionize the healthcare sector through transformative new diagnostic and therapeutic tools. In this field, nanostructures or nanocarriers (i.e., nanoparticles) are extensively used as a drug delivery system. Despite the well-defined profits offered by nanomedicines based on poly (lactic-co-glycolic acid) (PLGA), the major barriers hampering the launch of a nanoparticles-based product on the market are batch-to-batch variations and its lack of reproducibility from the benchtop to an industrial scale production. Currently, microfluidics technology has emerged as potential tool to achieve a continuous manufacturing with a precise control over fluids mixing and particles quality attributes. This work aims at defining a tailored strategy to produce PLGA NPs, exploiting a new microfluidic device. Moreover, Design of Experiments (DoE) and computational fluid dynamics approaches were exploited to understand the main process parameters and material attributes affecting the quality of the final product as well as the NPs manufacturing process. Finally, the ability to incorporate a drug into the PLGA nanoparticles was investigated by using Curcumin as model payload reaching encapsulation efficiency in the rank 28-44%. This paper is proposed as useful guide for the preparation of PLGA NPs by microfluidic technique.

Comparison among the Quantification of Bacterial Pathogens by qPCR, dPCR, and Cultural Methods.

The demand for rapid methods for the quantification of pathogens is increasing. Among these methods, those based on nucleic acids amplification (quantitative PCRs) are the most widespread worldwide. Together with the qPCR, a new approach named digital PCR (dPCR), has rapidly gained importance. The aim of our study was to compare the results obtained using two different dPCR systems and one qPCR in the quantification of three different bacterial pathogens: Listeria monocytogenes, Francisella tularensis, and Mycobacterium avium subsp. paratuberculosis. For this purpose, three pre-existing qPCRs were used, while the same primers and probes, as well as PCR conditions, were transferred to two different dPCR systems: the QX200 (Bio-Rad) and the Quant Studio 3D (Applied Biosystems). The limits of detection and limits of quantification for all pathogens, and all PCR approaches applied, were determined using genomic pure DNAs. The quantification of unknown decimal suspensions of the three bacteria obtained by the three different PCR approaches was compared through the Linear Regression and Bland and Altman analyses. Our results suggest that, both dPCRs are able to quantify the same amount of bacteria, while the comparison among dPCRs and qPCRs, showed both over and under-estimation of the bacteria present in the unknown suspensions. Our results showed qPCR over-estimated the amount of M. avium subsp. paratuberculosis and F. tularensis cells. On the contrary, qPCR, compared to QX200 dPCR, under-estimated the amount of L. monocytogenes cells. However, the maximum difference among PCRs approaches was <0.5 Log10, while cultural methods underestimated the number of bacteria by one to two Log10 for Francisella tularensis and Mycobacterium avium subsp. paratuberculosis. On the other hand, cultural and PCRs methods quantified the same amount of bacteria for L. monocytogenes, suggesting for this last pathogen, PCRs approaches can be considered as a valid alternative to the cultural ones.