Bloom, E. | bioRxiv Science

bioRxiv2024

Microfluidic platform enables shear-less aerosolization of lipid nanoparticles for messenger RNA inhalation

Leveraging the extensive surface area of the lungs for gene therapy, inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a novel microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient mRNA delivery to the respiratory system. Our results demonstrated the superiority of the novel MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful, lung-specific mRNA transfection without observable signs of toxicity. This pioneering MAP represents a significant advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.

bioengineering↗

bioRxiv2022

Destabilization of F-actin by Mechanical Stress Deprivation or Tpm3.1 Inhibition Promotes a Pathological Phenotype in Tendon Cells

The actin cytoskeleton is a central mediator between mechanical force and cellular phenotype. In tendon, it is speculated that mechanical stress deprivation regulates gene expression by filamentous (F-) actin destabilization. However, the molecular mechanisms that stabilize tenocyte F-actin networks remain unclear. Tropomyosins (Tpms) are master regulators of F-actin networks. There are over 40 mammalian Tpm isoforms, with each isoform having the unique capability to stabilize F-actin sub-populations. Thus, the specific Tpm(s) expressed by a cell defines overall F-actin organization. Here, we investigated F-actin destabilization by stress deprivation of tendon and tested the hypothesis that stress fiber-associated Tpm(s) stabilize tenocyte F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon fascicles downregulated tenocyte genes (collagen-I, tenascin-C, scleraxis, -smooth muscle actin) and upregulated matrix metalloproteinase-3. Concomitant with mRNA modulation were increases in DNAse-I/Phallodin (G/F-actin) staining, confirming F-actin destabilization by tendon stress deprivation. To investigate the molecular regulation of F-actin stabilization, we first identified the Tpms expressed by mouse tendons. Tendon cells from different origins (tail, Achilles, plantaris) express three isoforms in common: Tpm1.6, 3.1, and 4.2. We examined the function of Tpm3.1 since we previously determined that it stabilizes F-actin stress fibers in lens epithelial cells. Tpm3.1 associated with F-actin stress fibers in native and primary tendon cells. Inhibition of Tpm3.1 depolymerized F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin stability in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

cell biology↗