Liposomes for Inhalation.

Inhalation of liposomes formulated with phospholipids similar to endogenous lung surfactants and lipids offers biocompatibility and versatility within the pulmonary medicine field to treat a range of diseases such as lung cancer, cystic fibrosis and lung infections. Manipulation of the physicochemical properties of liposomes enables innovative design of the carrier to meet specific delivery, release and targeting requirements. This delivery system offers several benefits: improved pharmacokinetics with reduced toxicity, enhanced therapeutic efficacy, increased delivery of poorly soluble drugs, taste masking, biopharmaceutics degradation protection and targeted cellular therapy. This section provides an overview of liposomal formulation and delivery, together with their applications for different disease states in the lung.

An overview of in vitro and in vivo techniques for characterization of intranasal protein and peptide formulations for brain targeting.

The surge in neurological disorders necessitates innovative strategies for delivering active pharmaceutical ingredients to the brain. The non-invasive intranasal route has emerged as a promising approach to optimize drug delivery to the central nervous system by circumventing the blood-brain barrier. While the intranasal approach offers numerous advantages, the lack of a standardized protocol for drug testing poses challenges to both in vitro and in vivo studies, limiting the accurate interpretation of nasal drug delivery and pharmacokinetic data. This review explores the in vitro experimental assays employed by the pharmaceutical industry to test intranasal formulation. The focus lies on understanding the diverse techniques used to characterize the intranasal delivery of drugs targeting the brain. Parameters such as drug release, droplet size measurement, plume geometry, deposition in the nasal cavity, aerodynamic performance and mucoadhesiveness are scrutinized for their role in evaluating the performance of nasal drug products. The review further discusses the methodology for in vivo characterization in detail, which is essential in evaluating and refining drug efficacy through the nose-to-brain pathway. Animal models are indispensable for pre-clinical drug testing, offering valuable insights into absorption efficacy and potential variables affecting formulation safety. The insights presented aim to guide future research in intranasal drug delivery for neurological disorders, ensuring more accurate predictions of therapeutic efficacy in clinical contexts.

The development of a 3D-printed in vitro integrated oro-pharyngeal air-liquid interface cellular throat model for drug transport.

To simulate the deposition of drugs in the oro-pharynx region, several in vitro models are available such as the United States Pharmacopeia-Induction Port (USP-IP) throat and the Virginia Commonwealth University (VCU) models. However, currently, there is no such in vitro model that incorporates a biological barrier to elucidate drug transport across the pharyngeal cells. Cellular models such as in vitro air-liquid interface (ALI) models of human respiratory epithelial cell lines are extensively used to study drug transport. To date, no studies have yet been performed to optimise the ALI culture conditions of the human pharyngeal cell line Detroit 562 and determine whether it could be used for drug transport. Therefore, this study aimed to develop a novel 3D-printed throat model integrated with an ALI cellular model of Detroit 562 cells and optimise the culture conditions to investigate whether the combined model could be used to study drug transport, using Lidocaine as a model drug. Differentiating characteristics specific to airway epithelia were assessed using 3 seeding densities (30,000, 60,000, and 80,000 cells/well (c/w), respectively) over 21 days. The results showed that Detroit 562 cells completely differentiates on day 18 of ALI for both 60,000 and 80,000 c/w with significant mucus production, showing response to bacterial and viral stimuli and development of functional tight junctions and Lidocaine transport with no significant differences observed between the ALI models with the 2 cell seeding densities. Results showed the suitability of the Low density (60,000 c/w or 1.8 × 105 cells/cm2) ALI model to study drug transport. Importantly, the developed novel 3D-printed throat model integrated with our optimised in vitro Detroit 562 ALI model showed transport of Lidocaine throat spray. Overall, the study highlights the potential of the novel 3D-printed bio-throat integrated model as a promising in vitro system to investigate the transport of inhalable drug therapies targeted at the oro-pharyngeal region.

In vitro and in vivo applications of a universal and synthetic thermo-responsive drug delivery hydrogel platform.

A synthetic and thermo-responsive polymer, poly(N-isopropylacrylamide)-co-(polylactide/2-hydroxy methacrylate)-co-(oligo (ethylene glycol)), is used to formulate a universal carrier platform for sustained drug release. The enabling carrier, denoted as TP, is prepared by dissolving the polymer in an aqueous solution at a relatively neutral pH. A wide range of therapeutic moieties can be incorporated without the need for the addition of surfactants, organic solvents, and other reagents to the carrier system. The resulting solution is flowable through fine gauge needle, allowing accurate administration of TP to the target site. After injection, TP carrier undergoes a coil to globe phase transition to form a hydrogel matrix at the site. The benign nature of the polymer carrier and its physical gelation process are essential to preserve the biological activity of the encapsulated compounds while the adhesive hydrogel nature of the matrix allows sustained elusion and controlled delivery of the incorporated therapeutics. The TP carrier system has been shown to be non-toxic and elicits a minimal inflammatory response in multiple in vitro studies. These findings suggest the suitability of TP as an enabling carrier of therapeutics for localized and sustained drug delivery. To confirm this hypothesis, the capabilities of TP to encapsulate and effectively deliver multiple therapeutics of different physicochemical characteristics was evaluated. Specifically, a broad range of compounds were tested, including ciprofloxacin HCl, tumor necrosis factor-alpha (TNF-α), transforming growth factor beta 1 (TGF-ß1), and recombinant human bone morphogenetic protein 2 (BMP2). In vitro studies confirmed that TP carrier is able to control the release of the encapsulated drugs over an extended period of time and mitigate their burst release regardless of the compounds' physiochemical properties for the majority of the loaded therapeutics. Importantly, in vitro and in vivo animal studies showed that the released drugs from the TP hydrogel matrix remained potent and bioactive, confirming the high potential of the TP polymer system as an enabling carrier.

Development of excipients free inhalable co-spray-dried tobramycin and diclofenac formulations for cystic fibrosis using two and three fluid nozzles.

This study aims to investigate the effect of physicochemical properties and aerosol performance of two (2FN) and three-fluid nozzles (3FN) on the inhalable co-formulation of tobramycin and diclofenac dry powders. Combination formulations of tobramycin and diclofenac at 2:1 and 4:1 w/w ratios were prepared at a laboratory scale using a spray dryer in conjunction with a 2FN or 3FN. Powder size, morphology, solid-state characteristics, and aerodynamic and dissolution properties were characterised. The nozzle types and the formulation composition influenced the yield, particle size, solid-state properties, aerosolization behaviour and dissolution of the co-spray dried formulations. In particular, using the 2FN the co-spray dried formulation of tobramycin and diclofenac at 2:1 w/w showed smaller particle size (D50, 3.01 ± 0.06 µm), high fine particle fractions (FPF) (61.1 ± 3.6% for tobramycin and 65.92 ± 3 for diclofenac) and faster dissolution with approx. 70% diclofenac released within 3 h and approx. 90% tobramycin was released within 45 min. However, the 3FN for the co-spray dried formulation of tobramycin and diclofenac at a 2:1 w/w ratio showed a larger particle size (D50, 3.42 ± 0.02 µm), lower FPF (40.6 ± 3.4% for tobramycin and 36.9 ± 0.84 for diclofenac) and comparative slower dissolution with approx. 60% diclofenac was released within 3 h and 80% tobramycin was released within 45 min. A similar trend was observed when the tobramycin to diclofenac ratio was increased to 4:1 w/w. Overall results suggest that spray drying with 2FN showed a superior and viable approach to producing excipients-free inhalable co-spray dried formulations of tobramycin and diclofenac. However, the formulation produced using the 3FN showed higher enrichment of hydrophobic diclofenac and an ability to control the tobramycin drug release in vitro.

The application of in vitro cellular assays for analysis of electronic cigarettes impact on the airway.

Electronic (e)-cigarettes have been marketed for more than a decade as an alternative to conventional cigarettes. Their popularity and use among adolescents have grown significantly during recent years. While e-cigarettes do not release carcinogenic aromatic hydrocarbons, they can generate reactive carbonyls and radicals during the heating process in vitro. Emphasis has been placed in recent studies to introduce more rigorous and physiologically relevant in vitro models to characterise the toxicological profile of e-cigarettes. However, significant challenges are present due to difficulties for the developed systems to fully represent the in vivo inhalation settings. Furthermore, research protocols that fail to simulate the characteristics of e-cigarettes can affect the findings of in vitro studies. This review will illustrate the status quo of e-cigarette assays in vitro, discussing the various cellular assays used for evaluating the safety profile of e-cigarettes. Future directions will also be provided to better assist the scientific community in interpreting the health risks of e-cigarettes.

Prospective nanoparticle treatments for lymphangioleiomyomatosis.

INTRODUCTION: Lymphangioleiomyomatosis (LAM) is a rare lung disease that is characterized by smooth muscle-like cell growth in the lungs. The current available oral treatment rapamycin slows down the disease progression but does not result in a cure. Rapamycin is also limited by its low bioavailability and dose-related adverse side effects. New treatments are, therefore, underway to investigate alternative targets and combination therapies for LAM. In recent years, much focus has been on the development of therapies based on inhaled nanotechnology using carriers to deliver drugs, as it is shown to improve drug solubility, local targeted treatment, and bioavailability. AREAS COVERED: This review, therefore, focuses on future prospective treatments for LAM using nanoparticles and lipid-based nanocarriers, including liposomes, solid lipid nanoparticles, micelles, and polymeric nanoparticles. It also investigates how nanoparticles' physicochemical factors such as size and charge can affect the treatment of both pulmonary and extrapulmonary LAM. EXPERT OPINION: Advanced clinical research is still needed to demonstrate the full potential and drive future commercialization of LAM treatments delivered via inhaled lipid nanobased formulations. If successful, the resultant effects will be seen in the improvement in the life expectancy and life quality of LAM patients.

How Do Mechanics Guide Fibroblast Activity? Complex Disruptions during Emphysema Shape Cellular Responses and Limit Research.

The emphysema death toll has steadily risen over recent decades, causing the disease to become the third most common cause of death worldwide in 2019. Emphysema is currently incurable and could be due to a genetic condition (Alpha-1 antitrypsin deficiency) or exposure to pollutants/irritants, such as cigarette smoke or poorly ventilated cooking fires. Despite the growing burden of emphysema, the mechanisms behind emphysematous pathogenesis and progression are not fully understood by the scientific literature. A key aspect of emphysematous progression is the destruction of the lung parenchyma extracellular matrix (ECM), causing a drastic shift in the mechanical properties of the lung (known as mechanobiology). The mechanical properties of the lung such as the stiffness of the parenchyma (measured as the elastic modulus) and the stretch forces required for inhalation and exhalation are both reduced in emphysema. Fibroblasts function to maintain the structural and mechanical integrity of the lung parenchyma, yet, in the context of emphysema, these fibroblasts appear incapable of repairing the ECM, allowing emphysema to progress. This relationship between the disturbances in the mechanical cues experienced by an emphysematous lung and fibroblast behaviour is constantly overlooked and consequently understudied, thus warranting further research. Interestingly, the failure of current research models to integrate the altered mechanical environment of an emphysematous lung may be limiting our understanding of emphysematous pathogenesis and progression, potentially disrupting the development of novel treatments. This review will focus on the significance of emphysematous lung mechanobiology to fibroblast activity and current research limitations by examining: (1) the impact of mechanical cues on fibroblast activity and the cell cycle, (2) the potential role of mechanical cues in the diminished activity of emphysematous fibroblasts and, finally, (3) the limitations of current emphysematous lung research models and treatments as a result of the overlooked emphysematous mechanical environment.

Tobramycin and Colistin display anti-inflammatory properties in CuFi-1 cystic fibrosis cell line.

Current cystic fibrosis (CF) treatment strategies are primarily focused on oral/inhaled anti-inflammatories and antibiotics, resulting in a considerable treatment burden for CF patients. Therefore, combination treatments consisting of anti-inflammatories with antibiotics could reduce the CF treatment burden. However, there is an imperative need to understand the potential drug-drug interactions of these combination treatments to determine their efficacy. Thus, this study aimed to determine the interactions of the anti-inflammatory agent Ibuprofen with each of the CF-approved inhaled antibiotics (Tobramycin, Colistin and its prodrug colistimethate sodium/Tadim) and anti-bacterial and anti-inflammatory efficacy. Chemical interactions of the Ibuprofen:antibiotic combinations were elucidated using High-Resolution Mass-Spectrometry (HRMS) and 1H NMR. HRMS showed pairing of Ibuprofen and Tobramycin, further confirmed by 1H NMR whilst no pairing was observed for either Ibuprofen:Colistin or Ibuprofen:Tadim combinations. The anti-bacterial activity of the combinations against Pseudomonas aeruginosa showed that neither paired nor non-paired Ibuprofen:antibiotic therapies altered the anti-bacterial activity. The anti-inflammatory efficacy of the combination therapies was next determined at two different concentrations (Low and High) using in vitro models of NuLi-1 (healthy) and CuFi-1 (CF) cell lines. Differential response in the anti-inflammatory efficacy of Ibuprofen:Tobramycin combination was observed between the two concentrations due to changes in the structural conformation of the paired Ibuprofen:Tobramycin complex at High concentration, confirmed by 1H NMR. In contrast, the non-pairing of the Ibuprofen:Colistin and Ibuprofen:Tadim combinations showed a significant decrease in IL-8 secretion at both the concentrations. Importantly, all antibiotics alone showed anti-inflammatory properties, highlighting the inherent anti-inflammatory properties of these antibiotics.

Development and in vitro characterization of a novel pMDI diclofenac formulation as an inhalable anti-inflammatory therapy for cystic fibrosis.

Anti-inflammatory treatment options for cystic fibrosis (CF) patients are currently limited and as such, there is an imperative need to develop new anti-inflammatory agents to reduce the persistent inflammation present within CF lungs. This study explored the potential of Diclofenac (DICLO) as a novel inhaled anti-inflammatory drug for CF treatment. The anti-inflammatory activity of DICLO on an air-liquid interface (ALI) cell culture model of healthy (NuLi-1) and CF (CuFi-1) airways showed a significant reduction in the secretion of pro-inflammatory cytokines, IL-6 and IL-8. Therefore, pressurized metered dose inhaler (pMDI) DICLO formulations were developed to allow targeted DICLO delivery to CF airways. As such, two pMDI DICLO formulations with varying ethanol concentrations: 5% (w/w) equating to 150 µg of DICLO per dose (Low dose), and 15% (w/w) equating to 430 µg of DICLO per dose (High dose) were developed and characterized to determine the optimum formulation. The Low dose pMDI DICLO formulation showed a significantly smaller particle diameter with uniform distribution resulting in a greater aerosol performance when compared to High dose formulation. Consequently, the Low dose pMDI DICLO formulation was further evaluated in terms of in vitro transport characteristics and anti-inflammatory activity. Importantly, the DICLO pMDI displayed anti-inflammatory activity in both healthy and CF in vitro models, highlighting the potential of an aerosolized low-dose DICLO formulation as a promising inhaled anti-inflammatory therapy for CF treatment.

Real-time quantitative monitoring of in vitro nasal drug delivery by a nasal epithelial mucosa-on-a-chip model.

OBJECTIVES: A human nasal epithelial mucosa (NEM) on-a-chip is developed integrated with a novel carbon nanofibers-modified carbon electrode for real-time quantitative monitoring of in vitro nasal drug delivery. The integration of platinum electrodes in the chip also enables real-time measurement of transepithelial electrical resistance (TEER). METHODS: The air-liquid interface culture of nasal epithelial RPMI 2650 cells in the NEM-on-a-chip was optimized to mimic the key functional characteristics of the human nasal mucosa. The epithelial transport of ibuprofen in the NEM-on-a-chip was electrochemically monitored in real-time under static and physiologically realistic dynamic flow conditions. RESULTS: The NEM-on-a-chip mimics the mucus production and nasal epithelial barrier function of the human nasal mucosa. The real-time drug quantification by the NEM-on-a-chip was validated versus the high-performance liquid chromatography method. The drug transport rate monitored in the NEM-on-a-chip was influenced by the flow in the bottom compartment of the chip, highlighting the importance of emulating the dynamic in vivo condition for nasal drug transport studies. CONCLUSION: This novel NEM-on-a-chip can be a low-cost and time-efficient alternative to the costly laborious conventional techniques for in vitro nasal drug transport assays. Importantly, its dynamic microenvironment enables conducting nasal drug transport tests under physiologically relevant dynamic conditions.

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.

Development and Evaluation of Paclitaxel and Curcumin Dry Powder for Inhalation Lung Cancer Treatment.

Despite the effort to develop efficient targeted drug delivery for lung cancer treatment, the outcome remains unsatisfactory with a survival rate of 15% after 5 years of diagnosis. Inhalation formulation is an ideal alternative that could ensure the direct deposition of chemotherapeutics to the lungs. However, the design of an inhalable formulation that could simultaneously achieve a high local chemotherapeutic dose to the solid tumor and exert low pulmonary toxicities is a challenge, as the presence of 10-30% of chemotherapeutics in the lung is sufficient to induce toxicity. Therefore, this study aimed to develop a simple dry powder inhalation (DPI) formulation containing a model chemotherapeutic agent (paclitaxel, PTX) and a natural antioxidant (curcumin, CUR) that acts to protect healthy lung cells from injury during direct lung delivery. The co-jet-milling of CUR and PTX resulted in formulations with suitable aerosol performance, as indicated in the high fine particle fractions (FPF) (>60%) and adequate mass median aerodynamic diameter (MMAD). The CUR/PTX combination showed a more potent cytotoxic effect against lung cancer cells. This is evident from the induction of apoptosis/necrotic cell death and G2/M cell cycle arrests in both A549 and Calu-3 cells. The increased intracellular ROS, mitochondrial depolarization and reduced ATP content in A549 and Calu-3 cells indicated that the actions of CUR and PTX were associated with mitochondrial oxidative stress. Interestingly, the presence of CUR is crucial to neutralize the cytotoxic effects of PTX against healthy cells (Beas-2B), and this is dose-dependent. This study presents a simple approach to formulating an effective DPI formulation with preferential cytotoxicity towards lung cancer.

Using individualized three-dimensional printed airway models to guide airway stent implantation.

Airway stents are used to manage central airway obstructions by restoring airway patency. Current manufactured stents are limited in shape and size, which pose issues in stent fenestrations needed to be manually created to allow collateral ventilation to airway branches. The precise location to place these fenestrations can be difficult to predict based on 2-dimensional computed tomography images. Inspiratory computed tomography scans were obtained from 3 patients and analysed using 3D-Slicer™, Blender™ and AutoDesk® Meshmixer™ programmes to obtain working 3D-airway models, which were 3D printed. Stent customizations were made based on 3D-model dimensions, and fenestrations into the stent were cut. The modified stents were then inserted as per usual technique. Two patients reported improved airway performance; however, stents were later removed due to symptoms related to in-stent sputum retention. In a third patient, the stent was removed a few weeks later due to the persistence of fistula leakage. The use of a 3D-printed personalized airway model allowed for more precise stent customization, optimizing stent fit and allowing for cross-ventilation of branching airways. We determine that an airway model is a beneficial tool for stent optimization but does not prevent the development of some stent-related complications such as airway secretions.

An in vitro model for assessing drug transport in cystic fibrosis treatment: Characterisation of the CuFi-1 cell line.

Cystic fibrosis (CF) is a disease that most commonly affects the lungs and is characterized by mucus retention and a continuous cycle of bacterial infection and inflammation. Current CF treatment strategies are focused on targeted drug delivery to the lungs. Novel inhalable drug therapies require an in vitro CF model that appropriately mimics the in vivo CF lung environment to better understand drug delivery and transport across the CF epithelium, and predict drug therapeutic efficacy. Therefore, the aim of this research was to determine the appropriate air-liquid interface (ALI) culture method of the CuFi-1 (CF cell line) compared to the NuLi-1 (healthy cell line) cells to be used as in vitro models of CF airway epithelia. Furthermore, drug transport on both CuFi-1 and NuLi-1 was investigated to determine whether these cell lines could be used to study transport of drugs used in CF treatment using Ibuprofen (the only anti-inflammatory drug currently approved for CF) as a model drug. Differentiating characteristics specific to airway epithelia such as mucus production, inflammatory response and tight junction formation at two seeding densities (Low and High) were assessed throughout an 8-week ALI culture period. This study demonstrated that both the NuLi-1 and CuFi-1 cell lines fully differentiate in ALI culture with significant mucus secretion, IL-6 and IL-8 production, and functional tight junctions at week 8. Additionally, the High seeding density was found to alter the phenotype of the NuLi-1 cell line. For the first time, this study identifies that ibuprofen is transported via the paracellular pathway in ALI models of NuLi-1 and CuFi-1 cell lines. Overall, these findings highlight that NuLi-1 and CuFi-1 as promising in vitro ALI models to investigate the transport properties of novel inhalable drug therapies for CF treatment.

Properties of rapamycin solid lipid nanoparticles for lymphatic access through the lungs & part I: the effect of size.

Background: Lymphangioleiomyomatosis (LAM) is characterized by growth of smooth muscle-like cells in the lungs that spread to other organs via lymphatic vessels. Current oral rapamycin treatment is limited by low bioavailability of approximately 15%. Aim: The effect of inhaled rapamycin solid lipid nanoparticles (Rapa-SLNs) size on its penetration through the lymphatics. Method: Three Rapa-SLN formulations (200-1000 nm) were produced and assessed for particle characteristics and further for toxicity and performance in vitro. Results: Rapa-SLNs of 200 nm inhibited proliferation in TSC2-negative mouse embryonic fibroblast cells and penetrated the respiratory epithelium and lymphatic endothelium significantly faster compared with free rapamycin and larger Rapa-SLNs. Conclusion: Rapa-SLN approximately 200 nm allows efficient entry of rapamycin into the lymphatic system and is therefore a promising treatment for LAM patients.

Paclitaxel-eluting silicone airway stent for preventing granulation tissue growth and lung cancer relapse in central airway pathologies.

BACKGROUND: Airway stents are used to treat obstructive central airway pathologies including palliation of lung cancer, but face challenges with granulation tissue growth. Paclitaxel is a chemotherapy drug that also suppresses growth of granulation tissue. Yet, side effects arise from administration with toxic solubilizers. By incorporating paclitaxel in silicone stents, delivery of paclitaxel can be localized, and side effects minimized. METHODS: Paclitaxel was incorporated into Liquid Silicone Rubber (LSR) containing polydimethylsiloxane, either as a powder or solution, prior to curing. Drug release study was compared in vitro at 37°C over 10 days. Drug release was quantified using HPLC, and bronchial cell lines were grown on LSR to investigate drug cytotoxicity, and expression of inflammatory markers, specifically interleukin-6 and interleukin-8. RESULTS: Release rate of paclitaxel incorporated into silicone rubber was consistent with the Korsmeyer and Weibull models (R2 > 0.96). Paclitaxel exposure reduced IL-8 levels in cancer cell lines, whilst no cytotoxic effect was observed in all cell lines at treatment concentration levels (≤ 0.1% (w/v) paclitaxel in silicone). CONCLUSIONS: Incorporating paclitaxel into a silicone matrix for future use in a tracheobronchial stent was investigated. Drug release from silicone was observed and is a promising avenue for future treatments of central airway pathologies.

Properties of rapamycin solid lipid nanoparticles for lymphatic access through the lungs & part II: the effect of nanoparticle charge.

Aim: Lymphangioleiomyomatosis is characterized by smooth muscle-like cells in the lungs that spread to other organs via lymphatic vessels. Oral rapamycin is restricted by low bioavailability approximately 15%. The aim of the present study is to systematically investigate the effect of inhaled rapamycin solid lipid nanoparticles (Rapa-SLN) surface charge on efficacy and penetration into the lymphatics. Materials & methods: Rapa-SLN formulations with different charge: neutral, positive and negative, were produced and assessed for their physicochemical particle characteristics and efficacy in vitro. Results: Negative Rapa-SLNs were significantly faster at entering the lymphatic endothelium and more potent at inhibiting lymphanigiogenesis compared with neutral and positive Rapa-SLNs. Conclusion: Negative Rapa-SLNs showed efficient lymphatic access and should therefore be investigated further as a treatment for targeting extrapulmonary lymphangioleiomyomatosis.

Advances in the use of cell penetrating peptides for respiratory drug delivery.

Introduction: Respiratory diseases are leading causes of death in the world, still inhalation therapies are the largest fail in drug development. There is an evident need to develop new therapies. Biomolecules represent apotential therapeutic agent in this regard, however their translation to the clinic is hindered by the lack of tools to efficiently deliver molecules. Cell penetrating peptides (CPPs) have arisen as apotential strategy for intracellular delivery that could theoretically enable the translation of new therapies.Areas covered: In this review, the use of CPPs as astrategy to deliver different molecules (cargoes) to treat lung-relateddiseases will be the focus. Abrief description of these molecules and the innovative methods in designing new CPPs is presented. The delivery of different cargoes (proteins, peptides, poorly soluble drugs and nucleic acids) using CPPs is discussed, focusing on benefits to treat different respiratory diseases like inflammatory disorders, cystic fibrosis and lung cancer.Expert opinion: The advantages of using CPPs to deliver biomolecules and poorly soluble drugs to the lungs is evident. This field has advanced in the past few years toward targeted intracellular delivery, although further studies are needed to fully understand its potential and limitations in vitro and in vivo.

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.