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

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

Pneumococcal Surface Protein A-Hybrid Nanoparticles Protect Mice from Lethal Challenge after Mucosal Immunization Targeting the Lungs

Pneumococcal disease remains a global burden, with current conjugated vaccines offering protection against the common serotype strains. However, there are over 100 serotype strains, and serotype replacement is now being observed, which reduces the effectiveness of the current vaccines. Pneumococcal surface protein A (PspA) has been investigated as a candidate for new serotype-independent pneumococcal vaccines, but requires adjuvants and/or delivery systems to improve protection. Polymeric nanoparticles (NPs) are biocompatible and, besides the antigen, can incorporate mucoadhesive and adjuvant substances such as chitosans, which improve antigen presentation at mucosal surfaces. This work aimed to define the optimal NP formulation to deliver PspA into the lungs and protect mice against lethal challenge. We prepared poly(glycerol-adipate-co-ω-pentadecalactone) (PGA-co-PDL) and poly(lactic-co-glycolic acid) (PLGA) NPs using an emulsion/solvent evaporation method, incorporating chitosan hydrochloride (HCl-CS) or carboxymethyl chitosan (CM-CS) as hybrid NPs with encapsulated or adsorbed PspA. We investigated the physicochemical properties of NPs, together with the PspA integrity and biological activity. Furthermore, their ability to activate dendritic cells in vitro was evaluated, followed by mucosal immunization targeting mouse lungs. PGA-co-PDL/HCl-CS (291 nm) or CM-CS (281 nm) NPs produced smaller sizes compared to PLGA/HCl-CS (310 nm) or CM-CS (299 nm) NPs. Moreover, NPs formulated with HCl-CS possessed a positive charge (PGA-co-PDL +17 mV, PLGA + 13 mV) compared to those formulated with CM-CS (PGA-co-PDL −20 mV, PLGA −40 mV). PspA released from NPs formulated with HCl-CS preserved the integrity and biological activity, but CM-CS affected PspA binding to lactoferrin and antibody recognition. PspA adsorbed in PGA-co-PDL/HCl-CS NPs stimulated CD80+ and CD86+ cells, but this was lower compared to when PspA was encapsulated in PLGA/HCl-CS NPs, which also stimulated CD40+ and MHC II (I-A/I-E)+ cells. Despite no differences in IgG being observed between immunized animals, PGA-co-PDL/HCl-CS/adsorbed-PspA protected 83% of mice after lethal pneumococcal challenge, while 100% of mice immunized with PLGA/HCl-CS/encapsulated-PspA were protected. Therefore, this formulation is a promising vaccine strategy, which has beneficial properties for mucosal immunization and could potentially provide serotype-independent protection

Attenuation of Cigarette-Smoke-Induced Oxidative Stress, Senescence, and Inflammation by Berberine-Loaded Liquid Crystalline Nanoparticles: In Vitro Study in 16HBE and RAW264.7 Cells

Cigarette smoke is considered a primary risk factor for chronic obstructive pulmonary disease. Numerous toxicants present in cigarette smoke are known to induce oxidative stress and airway inflammation that further exacerbate disease progression. Generally, the broncho-epithelial cells and alveolar macrophages exposed to cigarette smoke release massive amounts of oxidative stress and inflammation mediators. Chronic exposure of cigarette smoke leads to premature senescence of airway epithelial cells. This impairs cellular function and ultimately leads to the progression of chronic lung diseases. Therefore, an ideal therapeutic candidate should prevent disease progression by controlling oxidative stress, inflammation, and senescence during the initial stage of damage. In our study, we explored if berberine (an alkaloid)-loaded liquid crystalline nanoparticles (berberine-LCNs)-based treatment to human broncho-epithelial cells and macrophage inhibits oxidative stress, inflammation, and senescence induced by cigarette-smoke extract. The developed berberine-LCNs were found to have favourable physiochemical parameters, such as high entrapment efficiency and sustained in vitro release. The cellular-assay observations revealed that berberine-LCNs showed potent antioxidant activity by suppressing the generation of reactive oxygen species in both broncho-epithelial cells (16HBE) and macrophages (RAW264.7), and modulating the genes involved in inflammation and oxidative stress. Similarly, in 16HBE cells, berberine-LCNs inhibited the cigarette smoke-induced senescence as revealed by X-gal staining, gene expression of CDKN1A (p21), and immunofluorescent staining of p21. Further in-depth mechanistic investigations into antioxidative, anti-inflammatory, and antisenescence research will diversify the current findings of berberine as a promising therapeutic approach for inflammatory lung diseases caused by cigarette smoking.</jats:p

Berberine-Loaded Liquid Crystalline Nanoparticles Inhibit Non-Small Cell Lung Cancer Proliferation and Migration in Vitro

Abstract Non-small cell lung cancer (NSCLC) is reported to have a high incidence rate and is one of the most prevalent types of lung cancer contributing towards 85 percent of all incidences of lung cancer. Berberine is a potent isoquinoline alkaloid which offers a broad range of therapeutical and pharmacological actions against cancer. However, extremely low water solubility and poor oral bioavailability have largely restricted its therapeutic applications. To overcome these limitations, we synthesized Berberine-loaded liquid crystalline nanoparticles (LCNs) and investigated them in vitro for their antiproliferative and antimigratory properties in human lung epithelial cancer cell line (A549). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Trypan blue staining, and colony forming assays were used to evaluate the anti-proliferative activity, while scratch wound healing assay and a modified Boyden chamber assay were employed to determine the anti-migratory activity. We also investigated critical proteins associated with lung cancer. The developed nanoparticles (NPs) were found to have an average particle size of 181.3 nm with spherical shape, high entrapment efficiency (75.35 %) and have shown sustained release behaviour. The most remarkable findings reported with Berberine-loaded LCNs were significant suppression of proliferation, inhibition of colony formation, inhibition of invasion or migration via Epithelial Mesenchymal Transition (EMT) related proteins associated with cancer progression. Further in-depth investigations into anti-cancer mechanistic research will expand and strengthen the current findings of Berberine-LCNs as a potential NSCLC treatment option.</jats:p