Early-stage development of novel cyclodextrin-siRNA nanocomplexes allows for successful postnebulization transfection of bronchial epithelial cells.

BACKGROUND: Successful delivery of small interfering RNA (siRNA) to the lungs remains hampered by poor intracellular delivery, vector-mediated cytotoxicity, and an inability to withstand nebulization. Recently, a novel cyclodextrin (CD), SC12CDClickpropylamine, consisting of distinct lipophilic and cationic subunits, has been shown to transfect a number of cell types. However, the suitability of this vector for pulmonary siRNA delivery has not been assessed to date. To address this, a series of high-content analysis (HCA) and postnebulization assays were devised to determine the potential for CD-siRNA delivery to the lungs. METHODS: SC12CDClickpropylamine-siRNA mass ratios (MRs) were examined for size and zeta potential. In-depth analysis of nanocomplex uptake and toxicity in Calu-3 bronchial epithelial cells was examined using IN Cell(®) HCA assays. Nebulized SC12CDClickpropylamine nanocomplexes were assessed for volumetric median diameter (VMD) and fine particle fraction (FPF) and compared with saline controls. Finally, postnebulization stability was determined by comparing luciferase knockdown elicited by SC12CDClickpropylamine nanocomplexes before and after nebulization. RESULTS: SC12CDClickpropylamine-siRNA complexation formed cationic nanocomplexes of ≤200 nm in size depending on the medium and led to significantly higher levels of siRNA associated with Calu-3 cells compared with RNAiFect-siRNA-treated cells at all MRs (p CONCLUSIONS: SC12CDClickpropylamine nanocomplexes can be effectively nebulized for pulmonary delivery of siRNA using Aeroneb technology to mediate knockdown in airway cells. To the best of our knowledge, this is the first study examining the suitability of SC12CDClickpropylamine-siRNA nanocomplexes for pulmonary delivery. Furthermore, this work provides an integrated nanomedicine-device combination for future in vitro and in vivo preclinical and clinical studies of inhaled siRNA therapeutics

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Effective nebulization of interferon-γ using a novel vibrating mesh.

BACKGROUND: Interferon gamma (IFN-γ) is a clinically relevant immunomodulatory cytokine that has demonstrated significant potential in the treatment and management of respiratory diseases such as tuberculosis and pulmonary fibrosis. As with all large biomolecules, clinical translation is dependent on effective delivery to the disease site and delivery of IFN-γ as an aerosol offers a logical means of drug targeting. Effective localization is often hampered by instability and a lack of safe and efficient delivery systems. The present study sought to determine how effectively IFN-γ can be nebulized using two types of vibrating mesh nebulizer, each with differing mesh architectures, and to investigate the comparative efficiency of delivery of therapeutically active IFN-γ to the lungs. METHODS: Nebulization of IFN-γ was carried out using two different Aerogen vibrating mesh technologies with differing mesh architectures. These technologies represent both a standard commercially available mesh type (Aerogen Solo®) and a new iteration mesh (Photo-defined aperture plate (PDAP®). Extensive aerosol studies (aerosol output and droplet analysis, non-invasive and invasive aerosol therapy) were conducted in line with regulatory requirements and characterization of the stability and bioactivity of the IFN-γ post-nebulization was confirmed using SDS-PAGE and stimulation of Human C-X-C motif chemokine 10 (CXCL 10) also known as IFN-γ-induced protein 10KDa (IP 10) expression from THP-1 derived macrophages (THP-1 cells). RESULTS: Aerosol characterization studies indicated that a significant and reproducible dose of aerosolized IFN-γ can be delivered using both vibrating mesh technologies. Nebulization using both devices resulted in an emitted dose of at least 93% (100% dose minus residual volume) for IFN-γ. Characterization of aerosolized IFN-γ indicated that the PDAP was capable of generating droplets with a significantly lower mass median aerodynamic diameter (MMAD) with values of 2.79 ± 0.29 μm and 4.39 ± 0.25 μm for the PDAP and Solo respectively. The volume median diameters (VMD) of aerosolized IFN-γ corroborated this with VMDs of 2.33 ± 0.02 μm for the PDAP and 4.30 ± 0.02 μm for the Solo. SDS-PAGE gels indicated that IFN-γ remains stable after nebulization by both devices and this was confirmed by bioactivity studies using a THP-1 cell model in which an alveolar macrophage response to IFN-γ was determined. IFN-γ nebulized by the PDAP and Solo devices had no significant effect on the key inflammatory biomarker cytokine IP-10 release from this model in comparison to non-nebulized controls. Here we demonstrate that it is possible to combine IFN-γ with vibrating mesh nebulizer devices and facilitate effective aerosolisation with minimal impact on IFN-γ structure or bioactivity. CONCLUSIONS: It is possible to nebulize IFN-γ effectively with vibrating mesh nebulizer devices without compromising its stability. The PDAP allows for generation of IFN-γ aerosols with improved aerodynamic properties thereby increasing its potential efficiency for lower respiratory tract deposition over current technology, whilst maintaining the integrity and bioactivity of IFN-γ. This delivery modality therefore offers a rational means of facilitating the clinical translation of inhaled IFN-γ

Evaluation of polymer choice on immunogenicity of chitosan coated PLGA NPs with surface-adsorbed pneumococcal protein antigen PspA4Pro

Polymeric nanoparticles (NPs) are recognized as potential delivery vehicles for vaccines. PLGA is a biocompatible polymer synonymous with polymeric NPs, which can be coated with other polymers such as chitosan that has intrinsic adjuvant properties as well as mucoadhesive properties. Numerous modifications and variations exist for PLGA and chitosan, which can influence the NP characteristics and the resulting immunogenicity. The current study investigated variations for making chitosan coated PLGA NPs incorporating recombinant pneumococcal surface protein A from family 2, clade 4 (PspA4Pro) antigen as a vaccine targeting the vast majority of pneumococcal strains and determine the effect of the polymers on particle size, surface charge, and surface marker upregulation on a dendritic cell (DC) line in vitro. PLGA variations tested with the ester-terminal group had the greatest detriment for prospective vaccine use, due to the lowest PspA4Pro adsorption and induction of CD40 and CD86 cell surface markers on DCs. The negatively charged chitosans exhibited the lowest surface marker expressions, similar to the uncoated NP, supporting the commonly accepted notion that positive surface charge augments immunogenic effects of the NPs. However, the study indicated that NPs made from PLGA with an acid terminated group, and chitosan HCl salt, exhibit particle characteristics, antigen adsorption efficiency and immunogenicity, which could be most suitable as a vaccine formulation

Estudio del glicoconjugado glucosa/manosa en mucosa gástrica de cerdos diagnosticados con Helicobacter pylori

Helicobacter pylori, a Gram negative bacillus, is associated with gastritis, ulcer, and gastric cancer. The worldwide prevalence of H. pylori infection is greater than 50%, constituting a public health problem. Different species of Helicobacter were described, which affect both animals and humans, and may be responsible for zoonoses. The colonization process of this bacterium depends largely on its binding to glycoconjugates present in the gastric mucosa of the host. The pig represents the animal model of choice for the study of infection by this bacterium. The objective of the present work was to analyze the behavior of the glucose/mannose glycoconjugate in the gastric mucosa of pigs affected by gastritis caused by Helicobacter sp. Samples from the antral region of the stomach of crossbred pigs were used, obtained from slaughterhouse in the Río Cuarto (Argentina). The samples were classified into different study groups according to the type of gastritis and the presence or absence of Helicobacter sp. A lectin histochemistry study for glucose/mannose glycoconjugate was carried out, the evaluated under microscope and statistical analysis performed. The results obtained indicate that there were no significant differences between the groups in the glucose/mannose expression in epithelium and gastric glands, but there were significant differences in the expression of this glycoconjugate in lamina propria between Helicobacter sp positive acute gastritis with respect to the Helicobacter sp negative acute gastritis groups and with normal mucosa group. This results reveal that there is relationship between the increase in glucose/mannose expression and the presence of Helicobacter sp in the lamina propria of pig gastric mucosa.Helicobacter sp, bacilo Gram negativo, se encuentra asociado a gastritis, úlcera y cáncer gástrico.La prevalencia mundial de la infección por H. pylori es mayor al 50%, constituyendo un problema de salud pública. Diferentes especies de Helicobacter afectan tanto a animales como seres humanos, pudiendo ser responsables de zoonosis. El proceso de colonización de esta bacteria depende en gran medida de su unión a glicoconjugados presentes en la mucosa gástrica del hospedero. El cerdo representa el modelo animal de elección para el estudio de la infección por esta bacteria. El objetivo del presente trabajo fue analizar el comportamiento del glicoconjugado glucosa/manosa en la mucosa gástrica de cerdos afectados por gastritis producida por Helicobacter sp. Se utilizaron muestras de la región antral del estómago de cerdos mestizos, obtenidas de frigoríficos de Río Cuarto (Argentina). Dichas muestras fueron clasificadas en distintos grupos de estudio de acuerdo al tipo de gastritis y a la presencia y/o ausencia de Helicobacter sp. Las muestras fueron luego sometidas a lectin-histoquímica para la marcación del glicoconjugado glucosa/manosa y posteriormente evaluadas mediante microscopía óptica y análisis estadístico. Los resultados obtenidos indican que no hay diferencias entre los grupos en cuanto a la expresión de glucosa/manosa en epitelio y glándulas gástricas, pero sí se presentan diferencias significativas en la expresión de este glicoconjugado en lamina propia entre el grupo gastritis aguda positivo a Helicobacter sp con respecto a los grupos gastritis aguda negativo a Helicobacter sp. y grupo con mucosa normal. Los resultados estadísticos revelan que existe relación entre el aumento en la marcación de glucosa/manosa y la presencia de Helicobacter sp en lamina propia de mucosa gástrica de cerdos

Aerosol and intranasal delivery of monoclonal antibodies to prevent transmission in pig influenza infection models

There is an urgent need for robust animal models to assess novel therapies that prevent the transmission of respiratory pathogens. We developed two complementary pig influenza models, direct and contact challenge, to evaluate the ability of monoclonal antibodies to block transmission. Using the strongly neutralizing 2–12 C mAb targeting H1N1pdm09 haemagglutinin, we established a benchmark for comparing mAb delivery routes and platforms. Intravenous administration of 2–12 C consistently showed the highest efficacy in the direct challenge model. The contact influenza challenge model, which best mimics natural exposure, was further optimized by evaluating key parameters, including timing of co-housing, infectious dose, and delivery routes. Aerosol and intravenous delivery of 2–12 C were equally potent, preventing infection in contact animals, while intranasal delivery prevented infection in some but not all animals. The pig direct and contact influenza challenge models provide powerful platforms for the evaluation therapeutic strategies to prevent influenza disease and transmission in humans

Cellular senescence in chronic obstructive pulmonary disease: Molecular mechanisms and therapeutic interventionsa.

Chronic obstructive pulmonary disease (COPD) is the world's fourth highest reason for mortality, accounting for 3.5 million deaths in 2021, and about 5% of total global deaths. Emphysema and chronic bronchitis are the two major pathologies of COPD. Tobacco smoke, dust, vapors, and fumes, outdoor air pollutants, genetic factors, ageing, infections, and asthma are the risk factors of COPD. On the other hand, senescence is permanent halt in cell cycle accompanied by phenotypic alterations due to ageing, oxidative stress like; irreparable DNA damage, telomere shortening, oncogene activation or inactivation of tumor suppressors. COPD is often considered an accelerated ageing process of the lungs, with senescent cells impairing tissue repair and regeneration, causing progressive lung function decline. Although, cellular senescence is seen as powerful defense against risk of carcinogenesis in COPD as it arrests cell proliferation irreversibly, excessive collection of senescent cells releases senescence-associated secretory phenotype (SASP) that increase oxidative stress to lungs and leads to long-term inflammation, tissue damage, and hindered lung recovery. This review will address the accelerated ageing process and cellular senescence in COPD, therapeutic approaches targeting senescence regulation in COPD; clinical research and trial studies demonstrating the use of therapies aimed at senescence in COPD along with current obstacles and potential solutions

Unravelling the role of nanomedicine in attenuating inflammation, oxidative stress and cellular ageing in chronic obstructive pulmonary disease

Abstract Chronic obstructive pulmonary disease is a chronic lung disease which causes obstruction and inflammation in the airways or other parts of the lung. It is often associated with structural changes in the lung due to persistent inflammation caused by prolong exposure to cigarette smoke. Other factors such as oxidative stress, chronic inflammation and cellular senescence also play a major role in the progression of the disease. Chronic inflammation is responsible for cell cycle dysfunction. Cellular senescence is associated in the pathogenesis of COPD, which can accelerate the lung aging process. Cellular senescence can elevate the level inflammatory mediators, which can comprise lung function and structure. This review explores various pathologic mechanisms which are involved in the progression of COPD. It also explores the application of nanostructure-based drug delivery systems such as solid lipid nanoparticles, polymeric nanoparticles, liposomes, nanoemulsions, dendrimers and other miscellaneous nanostructures in overcoming challenges associated with current conventional treatments for COPD. This review explores recent advancements in the field of nanostructures-based drug delivery systems for COPD treatment.</jats:p