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.

Quantification and Stability Impact Assessment of Drop Stresses in Biologic Drug Products.

Product can experience a combination of cavitation and agitation stresses as a result of dropping post-manufacture. We optimized terephthalic acid (TA) dosimetry, hydroxyphenyl fluorescein fluorimetry, and p-nitrophenol calorimetry as tools to detect and quantify the levels of hydroxyl radicals generated in solution. Using TA dosimetry, we determined the level of hydroxyl radicals generated from a vial drop and found that it is a function of drop height and fill volume and that protein and excipients may serve to mitigate but not completely quench the radicals. Additionally, we optimized sonication and friability as scale-down models to simulate dropping stresses and applied them to assess the impact on the stability of biologics. Our results suggest that chemical degradation dominates when a protein is subjected to cavitation stress alone, and that physical degradation induced by air-liquid and solid-liquid interfaces is the dominant degradation mode when there is a combination of cavitation and agitation stress. Taken together, this work provides a quick and simplistic approach that can be applied during drug product process development to evaluate the impact of drop stresses on the stability of biologic drug product.

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

Enrichment of Placental Trophoblast Cells from Clinical Cervical Samples Using Differences in Surface Adhesion on an Inclined Plane.

Placental trophoblast cells present in cervical samples have great potential towards non-invasive prenatal testing. However, cervical samples are highly heterogeneous, largely comprised of maternal cervical cells with only a small quantity of trophoblast cells. In order to use these rare cells for diagnostic applications, there is a need to enrich and isolate them from the heterogeneous maternal sample. Our goal was to investigate the use of gravitational flow on an inclined surface and optimize parameters including angle of incline, surface material, incubation time on the surface, solution volume, and device channel width in order to identify a design allowing label-free enrichment of trophoblast cells. In this work we detail the development of a new method and device for controlling cell adhesion to a surface vs. rolling into a collection area. The enrichment device design was developed for ease of use by non-specialized personal and on a slide surface for the ability to be directly integrated onto an automatic cell picker instrument, which can be used for downstream single cell isolation. JEG-3 trophoblast cells were used with clinical cervical samples to present the effect of the different optimization parameters on enrichment. We further provide an assessment of the impact shear stress and thickness of the liquid layer has on cell enrichment. We found that this method provides a maximum JEG-3 enrichment using polystyrene surfaces at a 50° incline with a 5 min incubation period prior to inclined flow. This resulted in a 396 ± 52% increase in purity of the trophoblast cells from the clinical cervical samples as confirmed using human leukocyte antigen G (HLA-G) antibody staining with fluorescence imaging to identify JEG-3 cells. Ultimately, this method is inexpensive, quick, and has the potential for direct integration into fetal cell isolation platforms.

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Placental Trophoblast-Inspired Lipid Bilayers for Cell-Free Investigation of Molecular Interactions.

The placenta plays a key role in regulating the maternal-fetal transport but it is a difficult organ to study due to a lack of existing in vitro models. Lipid bilayers inspired by the placenta can provide a facile new in vitro tool with promise for screening molecular transport across this important organ. Here we developed lipid bilayers that mimic the composition of human placental trophoblast cells at different times during the course of pregnancy. Mass spectrometry identified five major lipid classes (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin) present at varying concentrations in trophoblasts representative of the first and third trimesters and full-term placenta. We successfully developed supported and suspended lipid bilayers mimicking these trophoblast lipid compositions and then demonstrated the utility of these synthetic placenta models for investigating molecular interactions. Specifically, we investigated the interactions with di(2-ethylhexyl) phthalate (DEHP), a common plasticizer and environmental toxicant, and amphotericin B, a common yet toxic, antifungal therapeutic. Overall, we observed that DEHP adsorbs and potentially embeds itself within all placental lipid bilayers, with varying levels of interaction. For both amphotericin B and a liposomal formulation of amphotericin B, AmBisome, we noted lower levels of permeation in transport studies with bilayers and trophoblast cells compared with DEHP, likely driven by differences in size. AmBisome interacted less with both the supported and suspended placental lipid bilayers in comparison to amphotericin B, suggesting that drug delivery carriers can vary the impact of a pharmaceutical agent on these lipid structures. We found that the apparent permeability observed in suspended bilayers was approximately an order of magnitude less than those observed for trophoblast monolayers, which is typical of lipid bilayers. Ultimately, these placenta mimetic lipid bilayers can serve as a platform for the rapid initial screening of molecular interactions with the maternal-fetal interface to better inform future testing.

Anidulafungin liposome nanoparticles exhibit antifungal activity against planktonic and biofilm Candida albicans.

Fungal infections can cause significant patient morbidity and mortality. Nanoparticle therapeutics have the potential to improve treatment of these infections. Here we report the development of liposomal nanoparticles incorporating anidulafungin, a potent antifungal, with the goal of increasing its solubility and aiding in localization to fungi. Liposomes were fabricated with three concentrations of anidulafungin yielding monodisperse ~100 nm unilamellar vesicles. All three formulations inhibited planktonic Candida albicans growth at a minimum inhibitory concentration equivalent to free drug. All three formulations also disrupted preformed C. albicans biofilms, reducing fungal burden by as much as 99%, exhibiting superior biofilm disruption compared with free drug. Liposome formulations tested in vivo in C. albicans infected Galleria mellonella wax moth larvae demonstrated increased survival compared to free drug equivalents, leading to a survival of 33 to 67% of larvae over 7 days depending on the liposome utilized compared with only 25% survival of larvae administered free drug. Liposomal formulations along with free anidulafungin did not cause red blood cell lysis. Ultimately, the liposome formulations reported here increased anidulafungin solubility, displayed promising efficacy against planktonic and biofilm C. albicans, and improved the survival of C. albicans-infected G. mellonella compared to free anidulafungin.

Investigating interactions of phthalate environmental toxicants with lipid structures.

Di(2-ethylhexyl) phthalate (DEHP) is one of the most abundant plasticizers in common household products. It leaches from materials, resulting in exposure associated with detrimental health effects. The main objective of this study was to investigate how DEHP and its metabolite, mono(2-ethylhexyl) phthalate (MEHP), interact with and permeate lipid structures, namely vesicles and planar bilayers. Using dynamic light scattering, we observed significant changes in the size and polydispersity of L-α-phosphatidylcholine (egg PC) vesicles when incubated with DEHP but not MEHP at the same concentrations (100 and 200 µM). We demonstrated that these effects are mitigated by pre-treatment with chitosan nanoparticles which adsorb the phthalates. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we observed a concentration dependence on the interaction of DEHP with egg PC supported lipid bilayers (SLBs). QCM-D results suggested lipid removal for 5 and 100 µM DEHP, and adsorption and potential embedment in the bilayer at 50 and 200 µM DEHP. SLB mass decrease was observed for all concentrations of MEHP (5, 50, 100, and 200 µM), suggesting lipid removal. We also investigated the permeability of DEHP and MEHP as well as several small molecules across a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) suspended lipid bilayer. We found that DEHP and MEHP both had low permeabilities, but only DEHP remained associated with the bilayer. Exposure to DEHP and MEHP influenced how several common small molecules interacted with DOPC bilayers. Ultimately, this work provides insight into mechanisms of phthalate interactions with lipid structures, having implications for human health.

A Rapid Method for Label-Free Enrichment of Rare Trophoblast Cells from Cervical Samples.

Extravillous trophoblasts (EVTs) have the potential to provide the entire fetal genome for prenatal testing. Previous studies have demonstrated the presence of EVTs in the cervical canal and the ability to retrieve a small quantity of these cells by cervical sampling. However, these small quantities of trophoblasts are far outnumbered by the population of cervical cells in the sample, making isolation of the trophoblasts challenging. We have developed a method to enrich trophoblast cells from a cervical sample using differential settling of the cells in polystyrene wells. We tested the addition of small quantities of JEG-3 trophoblast cell line cells into clinical samples from standard Pap tests taken at 5 to 20 weeks of gestation to determine the optimal work flow. We observed that a 4 min incubation in the capture wells led to a maximum in JEG-3 cell settling on the surface (71 ± 10% of the initial amount added) with the removal of 91 ± 3% of the cervical cell population, leading to a 700% enrichment in JEG-3 cells. We hypothesized that settling of mucus in the cervical sample affects the separation. Finally, we performed a proof-of-concept study using our work flow and CyteFinder cell picking to verify enrichment and pick individual JEG-3 and trophoblast cells free of cervical cells. Ultimately, this work provides a rapid, facile, and cost-effective method for enriching native trophoblasts from cervical samples for use in subsequent non-invasive prenatal testing using methods including single cell picking.