Investigating potential TRPV1 positive feedback to explain TRPV1 upregulation in airway disease states.

OBJECTIVE: The airway epithelium is a potential source of pathophysiology through activation of transient potential receptor vallinoid type 1 (TRPV1) channel. A positive feedback cycle caused by TRPV1 activity is hypothesized to induce upregulation and production of inflammatory cytokines, leading to exacerbations of chronic airway diseases. These cytokine and protein regulation effects were investigated in this study. METHODS: Healthy (BEAS-2B) and cancer-derived (Calu-3) airway epithelial cell lines were assessed for changes to TRPV1 protein expression and mRNA expression following exposure to capsaicin (5-50 µM), and TRPV1 modulators including heat (43 °C), and hydrochloric acid (pH 3.4 to pH 6.4). Cytotoxicity was measured to determine the working concentration ranges of treatment. Subsequent bronchoconstriction by TRPV1 activation with capsaicin was measured on guinea pig airway tissue to confirm locally mediated activity without the action of known neuronal inputs. RESULTS: TRPV1 protein expression was not different for all capsaicin, acidity, and heat exposures (p > 0.05), and was replicated in mRNA protein expression (p > 0.05). IL-6 and IL-8 expression were lower in BEAS-2B and Calu-3 cell lines exposed with acidity and heat (p < 0.05), but not consistently with capsaicin exposure, with potential cytotoxic effects possible. CONCLUSIONS: TRPV1 expression was present in airway epithelial cells but its expression was not changed after activation by TRPV1 activators. Thus, it was not apparent the reason for reported TRPV1 upregulation in patients with airway disease states. More complex mechanisms are likely involved and will require further investigation.

Delivery of pDNA to lung epithelial cells using PLGA nanoparticles formulated with a cell-penetrating peptide: understanding the intracellular fate.

The combination of nanoparticles (NPs) and cell-penetrating peptide (CPP) represents a new opportunity to develop plasmid DNA (pDNA) delivery systems with desirable properties for lung delivery. In this study, poly(lactide-co-glycolide) (PLGA) NPs containing pDNA were formulated with and without CPP using a double-emulsion technique. NPs were characterized in regards of size, surface charge, release profile, pDNA encapsulation efficiency and pDNA integrity. Cellular uptake, intracellular trafficking, uptake mechanism and pDNA expression were assessed in both A549 and Beas-2B cells. Manufactured PLGA-NPs efficiently encapsulated pDNA with approximately 50% released in the first 24 h of incubation. Addition of CPP was essential to promote NP internalization in both cell lines, with 83.85 ± 1.2% and 96.76 ± 1.7% of Beas-2B and A549 cells, respectively, with internalized NP-DNA-CPP after 3 h of incubation. Internalization appears to occur mainly via clathrin-mediated endocytosis, with other pathways also being used by the different cell lines. An endosomal-escape mechanism seems to happen in both cell lines, and eGFP expression was observed in Beas-2B after 96 h of incubation. In summary, the NP-DNA-CPP delivery system efficiently encapsulated and protected pDNA structure and is being investigated as a promising tool for gene delivery to the lungs.

Nanotoxicologic Effects of PLGA Nanoparticles Formulated with a Cell-Penetrating Peptide: Searching for a Safe pDNA Delivery System for the Lungs.

The use of cell-penetrating peptides (CPPs) in combination with nanoparticles (NPs) shows great potential for intracellular delivery of DNA. Currently, its application is limited due to the potential toxicity and unknown long-term side effects. In this study NPs prepared using a biodegradable polymer, poly(lactic⁻co⁻glycolic acid (PLGA) in association with a CPP, was assessed on two lung epithelial cell lines (adenocarcinomic human alveolar basal epithelial cells (A549) and normal bronchial epithelial cells (Beas-2B cells)). Addition of CPP was essential for intracellular internalization. No effects were observed on the mitochondrial activity and membrane integrity. Cells exposed to the NPs⁻DNA⁻CPP showed low inflammatory response, low levels of apoptosis and no activation of caspase-3. Increase in necrotic cells (between 10%⁻15%) after 24 h of incubation and increase in autophagy, induced by NPs⁻DNA⁻CPP, are likely to be related to the lysosomal escape mechanism. Although oxidative stress is one of the main toxic mechanisms of NPs, NPs⁻DNA⁻CPP showed decreased reactive oxygen species (ROS) production on Beas-2B cells, with potential antioxidant effect of CPP and no effect on A549 cells. This NP system appears to be safe for intracellular delivery of plasmid DNA to the lung epithelial cells. Further investigations should be conducted in other lung-related systems to better understand its potential effects on the lungs.

Delivery of pDNA Polyplexes to Bronchial and Alveolar Epithelial Cells Using a Mesh Nebulizer.

PURPOSE: In this study, a cell penetrating peptide was used as an uptake enhancer for pDNA delivery to the lungs. METHODS: Polyplexes were prepared between pDNA and CPP. Intracellular delivery of pDNA was assessed in both alveolar (A549) and bronchial (Calu-3) epithelial cells. Aerosol delivery was investigated using a mesh nebulizer. RESULTS: Efficient intracellular delivery of pDNA occurs in both A549 and Calu-3 cells when delivered as polyplexes. Protection against nucleases and endosomal escape mechanism occurs when pDNA is formulated within the polyplexes. For aerosol delivery, 1% (w/v) mannitol was able to protect naked DNA structure during nebulization with a significant increase in fine particle fraction (particles <5 µm). The structure of polyplexes when delivered via a mesh nebulizer using 1% (w/v) mannitol could partially withstand the shear forces involved in aerosolization. Although some loss in functionality occurred after nebulization, membrane-associated fluorescence was observed in A549 cells. In Calu-3 cells mucus entrapment was a limiting factor for polyplex delivery. CONCLUSIONS: The presence of CPP is essential for efficient intracellular delivery of pDNA. The polyplexes can be delivered to lung epithelial cells using mesh nebulizer. The use of different excipients is essential for further optimization of these delivery systems.

Inhaled gene delivery: a formulation and delivery approach.

INTRODUCTION: Gene therapy is a potential alternative to treat a number of diseases. Different hurdles are associated with aerosol gene delivery due to the susceptibility of plasmid DNA (pDNA) structure to be degraded during the aerosolization process. Different strategies have been investigated in order to protect and efficiently deliver pDNA to the lungs using non-viral vectors. To date, no successful therapy involving non-viral vectors has been marketed, highlighting the need for further investigation in this field. Areas covered: This review is focused on the formulation and delivery of DNA to the lungs, using non-viral vectors. Aerosol gene formulations are divided according to the current delivery systems for the lung: nebulizers, dry powder inhalers and pressurized metered dose inhalers; highlighting its benefits, challenges and potential application. Expert opinion: Successful aerosol delivery is achieved when the supercoiled DNA structure is protected during aerosolization. A formulation strategy or compounds that can protect, stabilize and efficiently transfect DNA into the cells is desired in order to produce an effective, low-cost and safe formulation. Nebulizers and dry powder inhalers are the most promising approaches to be used for aerosol delivery, due to the lower shear forces involved. In this context it is also important to highlight the importance of considering the 'pDNA-formulation-device system' as an integral part of the formulation development for a successful nucleic acid delivery.