Nearly four million yearly deaths can be attributed to respiratory diseases, prompting a huge worldwide health emergency. Additionally, the COVID-19 pandemic’s death toll has surpassed six million, significantly increasing respiratory disease morbidity and mortality rates. Despite recent advances, it is still challenging for many drugs to be homogeneously distributed throughout the lungs, and specifically to reach the lower respiratory tract with an accurate sustained dose and minimal systemic side effects. Engineered nanocarriers can provide increased therapeutic efficacy while lessening potential biochemical adverse reactions. Poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer, has attracted significant interest as an inhalable drug delivery system. However, the influence of the nanocarrier surface charge and its intratracheal instillation has not been addressed so far. In this study, we fabricated red fluorescent PLGA nanocapsules (NCs)—Cy5/PLGA—with either positive (Cy5/PLGA+) or negative surface charge (Cy5/PLGA-). We report here on their excellent colloidal stability in culture and biological media, and after cryo-storage. Their lack of cytotoxicity in two relevant lung cell types, even for concentrations as high as 10 mg/mL, is also reported. More importantly, differences in the NCs’ cell uptake rates and internalization capacity were identified. The uptake of the anionic system was faster and in much higher amounts—10-fold and 2.5-fold in macrophages and epithelial alveolar cells, respectively. The in vivo study demonstrated that anionic PLGA NCs were retained in all lung lobules after 1 h of being intratracheally instilled, and were found to accumulate in lung macrophages after 24 h, making those nanocarriers especially suitable as a pulmonary immunomodulatory delivery system with a marked translational character.

Background: Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as an alternative to live-cell administration for Acute Respiratory Distress Syndrome (ARDS). MSC-EVs can be chiefly influenced by the environment to which the MSCs are exposed. Here, lipopolysaccharide (LPS) priming of MSCs was used as a strategy to boost the natural therapeutic potential of the EVs in acute lung injury (ALI). Methods: The regenerative and immunemodulatory effect of LPS-primed MSC-EVs (LPS-EVs) and non-primed MSC-EVs (C-EVs) were evaluated in vitro on alveolar epithelial cells and macrophage-like THP-1 cells. In vivo, ALI was induced in adult male rats by the intrapulmonary instillation of HCl and LPS. Rats (n = 8 to 22/group) were randomized to receive a single bolus (1 × 10⁸ particles) of LPS-EVs, C-EVs, or saline. Lung injury severity was assessed at 72 h in lung tissue and bronchoalveolar lavage. Results: In vitro, LPS-EVs improved wound regeneration and attenuated the inflammatory response triggered by the P. aeruginosa infection, enhancing the M2 macrophage phenotype. In in vivo studies, LPS-EVs, but not C-EVs, significantly decreased the neutrophilic infiltration and myeloperoxidase (MPO) activity in lung tissue. Alveolar macrophages from LPS-EVs-treated animals exhibited a reduced expression of CXCL-1, a key neutrophil chemoattractant. However, both C-EVs and LPS-EVs reduced alveolar epithelial and endothelial permeability, mitigating lung damage. Conclusions: EVs from LPS-primed MSCs resulted in a better resolution of ALI, achieving a greater balance in neutrophil infiltration and activation, while avoiding the complete disruption of the alveolar barrier. This opens new avenues, paving the way for the clinical implementation of cell-based therapies.

Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by acute hypoxemic respiratory failure. Pneumonia and sepsis are the most common causes, turning ARDS into a critical public health problem. Despite recent advances in pharmacological strategies, clinical trials have not demonstrated a reduction in ARDS‐associated mortality. This is in part connected to the singularity of the pulmonary physiological barrier, which hampers drug delivery, specifically at distal areas. To this aim, the use of polymeric nanocarriers as a platform for the efficient delivery of therapeutics to the lungs by nebulization is introduced. Herein, poly(lactic‐co‐glycolic acid) (PLGA) nanocapsules (NCs) loaded with human serum albumin, as an inhalable nanotherapeutic are prepared. The production of stable NCs aerosols in the inhalable range is achieved using a commercial device, while the nanocarrier's physicochemical parameters are only minimally altered after nebulization. Importantly, in vivo studies with healthy and acute lung injury animals show that after inhalation, the NCs are homogeneously distributed throughout the lungs, arriving at the distal areas. The NCs are internalized by alveolar type II cells, avoiding macrophage‐mediated lung clearance. These features make the PLGA NCs excellent vehicles for noninvasive pulmonary delivery, facilitating a ready‐to‐be‐used nanomedicine.

Sepsis is a syndromic response to infection and is frequently a final common pathway to death from many infectious diseases worldwide. The complexity and high heterogeneity of sepsis hinder the possibility to treat all patients with the same protocol, requiring personalized management. The versatility of extracellular vesicles (EVs) and their contribution to sepsis progression bring along promises for one-to-one tailoring sepsis treatment and diagnosis. In this article, we critically review the endogenous role of EVs in sepsis progression and how current advancements have improved EVs-based therapies toward their translational future clinical application, with innovative strategies to enhance EVs effect. More complex approaches, including hybrid and fully synthetic nanocarriers that mimic EVs, are also discussed. Several pre-clinical and clinical studies are examined through the review to offer a general outlook of the current and future perspectives of EV-based sepsis diagnosis and treatment.

Recent advances in super-resolution microscopy have enabled unprecedented visualization of cellular structures, tracking of nanomaterials in biological environments, or the elucidation of specific nano-bio interactions. Yet, dynamic quantification of cargo release from individual nanocarriers remains unexplored. Here, we leverage the high spatial resolution of direct stochastic optical reconstruction microscopy (dSTORM) to monitor protein release at the single-nanocarrier level. Poly(lactic-co-glycolic acid) (PLGA) nanocapsules labelled with Cyanine5 and loaded with bovine serum albumin (BSA) tagged with Alexa Fluor 488 are characterized using dSTORM alongside other characterization techniques. dSTORM allowed us to simultaneously observe changes in nanocarrier size and cargo localization over time. Our results demonstrate a time-dependent increase in nanocapsule diameter and a decrease in nanocarrier concentration. The quantitative analysis of individual nanocarriers reveals single-particle protein release profiles, characterized by an initial burst followed by sustained release, with complete release achieved after 30 days. This study represents the first application of super-resolution microscopy to spatially and temporally resolve protein release from nanocarriers, offering single-molecule sensitivity and nanometric resolution, and capturing heterogeneity that ensemble-averaged techniques overlook. Our approach complements other pharmacokinetic analyses and establishes a robust method to evaluate the cargo release from other nanocarriers by super-resolution microscopy.