Lea Ann Dailey

Nebulization of biodegradable nanoparticles: impact of nebulizer technology and nanoparticle characteristics on aerosol features

Nanoparticles may be effective drug delivery systems for use in various pulmonary therapeutic schemes. This study investigated the effect of nebulization technology and nanoparticle characteristics on the features of aerosol generation. Suspensions of biodegradable nanoparticles consisting of commercially available poly(lactide-co-glycolide) and novel comb polymers were nebulized with a jet, ultrasonic, and piezo-electric crystal nebulizer. The effects of the nanoparticle suspensions on the aerosol droplet size, as well as the nanoparticle size before and after nebulization, were characterized via laser diffraction. While the individual nanoparticle suspensions showed no clinically relevant influence on aerosol droplet size, as compared to control experiments, an enhanced nanoparticle aggregation within the droplets was observed. This aggregation was further characterized by fluorescence and scanning electron microscopy. Dependency of aggregation on nebulizer technology and nanoparticle characteristics was noted. Nanoparticles exhibiting the highest surface hydrophobicity were particularly susceptible to aggregation when nebulized with a jet nebulizer. Aggregation was reduced with nanoparticles exhibiting a more hydrophilic surface or when using ultrasonic nebulizers. We conclude that the biodegradable nanoparticles contained in the suspensions did not affect the aerosol droplet size in a clinically relevant manner; however, both the nanoparticle characteristics and the technique of aerosol generation influence nanoparticle aggregation occurring during aerosolization.

Surfactant-Free, Biodegradable Nanoparticles for Aerosol Therapy Based on the Branched Polyesters, DEAPA-PVAL-g-PLGA

AbstractPurpose. This study describes the development of surfactant-free, biodegradable nanoparticle systems with varying physicochemical properties and their suitability for pulmonary application via nebulization. Methods. Nanoparticle suspensions were formulated from the branched polyester, diethylaminopropyl amine-poly(vinyl alcohol)-grafted-poly(lactide-co-glycolide) (DEAPA-PVAL-g-PLGA) alone, as well as with increasing amounts of carboxymethyl cellulose (CMC). Particle size, ζ potential, turbidity, and morphology (atomic force microscopy) were characterized. Three formulations were chosen for further study: Cationic nanoparticles without CMC, cationic nanoparticles with CMC, and anionic nanoparticles with an excess of CMC. Nanoparticle degradation was characterized, as well as stability during nebulization. Nanoparticle-cell interactions were investigated and quantified using confocal laser scanning microscopy and fluorescence spectrometry. Results. Nanoparticles ranged in size from 70-250 nm and displayed ζ potentials of +58.9 to −46.6 mV. Anionic nanoparticles showed the highest stability during nebulization. The degradation rate of each nanoparticle formulation decreased with increasing amounts of CMC. Cell association was highest among cationic nanoparticles (57% and 30%, respectively), although these were not internalized. Despite a lower rate of cell association (3%), anionic nanoparticles were internalized by A549 cells. Conclusions. Surfactant-free nanoparticles from DEAPA-PVAL-g-PLGA are versatile drug delivery systems; however, only the anionic formulations investigated were proven suitable for aerosol therapy.

Sparing methylation of β-cyclodextrin mitigates cytotoxicity and permeability induction in respiratory epithelial cell layers in vitro

Cyclodextrins (CDs) are promising solubility enhancers for inhaled drug delivery. However, they have dose-dependent effects on the respiratory epithelium, which may have advantages for permeability enhancement but also gives rise to safety concerns. In this study, the methyl thiazol tetrazolium (MTT) assay was used to compare a new sparingly methylated beta-CD, Kleptose Crysmebeta (Crysmeb) with the more established CD derivatives hydroxypropyl-gamma-cyclodextrin (HPgammaCD), randomly methylated beta-cyclodextrin (Rameb) and hydroxypropyl-beta-cyclodextrin (HPbetaCD). The betaCD derivatives affected cell metabolism in A549 cells in a concentration dependent manner with LD(50) of 56, 31 and 11 mM obtained for HPbetaCD, Crysmeb and Rameb, respectively. Calu-3 cells were less susceptible to betaCD with an LD(50) of 25 mM being obtained for Rameb only. Permeability increases in Calu-3 cell layers were observed with betaCD derivatives and a concentration dependency shown. The mechanism of permeability enhancement and its reversibility was investigated. Rameb produced an irreversible loss of cell layer barrier function at > or = 25 mM, but perturbations of epithelial integrity were moderate and reversible in the case of HPbetaCD and Crysmeb (25-50 mM). Given its high solubilisation capacity, the low toxicity and transient absorption promoting properties, this study identifies Crysmeb as a promising adjuvant in formulations for inhalation.

The delivered dose: Applying particokinetics to in vitro investigations of nanoparticle internalization by macrophages

PURPOSE The aim of this study was to investigate the impact of nanoparticle dosimetry on the interpretation of results from in vitro experiments involving particle-cell interactions. Three different dose metrics were evaluated: 1) The administered dose (particle mass, number or surface area administered per volume media at the onset of an experiment), 2) the delivered dose (particle mass, number or surface area to reach the cell monolayer via diffusion and sedimentation over the duration of an experiment) and 3) the cellular dose (particle mass, number or surface area internalized by the cells during the experiment). The In Vitro Sedimentation and Diffusion and Dosimetry model (ISDD) was used to calculate particle sedimentation and diffusion in cell culture media to predict delivered dose values. These were compared with administered doses and experimentally determined cellular dose values. METHODS Dosing conditions and predicted delivered dose values were computed in silico using ISDD. In vitro cell association experiments were performed by exposing fluorescently labelled polystyrene beads of 50, 100, 200, 700 and 1000nm diameter to J774A.1 macrophage-like cells and determining the internalized particle content (cellular dose) via fluorescence spectroscopy. Experiments were repeated using lipopolysachharide (LPS) to activate and cytochalasin D to inhibit phagocytosis. RESULTS Only a small fraction (0.03-0.33%) of the administered dose was able to interact with the cells for all particle sizes tested. Measured cellular doses in non-activated J774A.1 cells corresponded well with computed delivered dose values for all particle sizes tested under six different exposure conditions. When cellular doses were averaged and normalized to their corresponding delivered doses, the percentage values of cell-associated particles were: 36 ± 10%(50 nm), 15 ± 3%(100 nm), 22 ± 6%(200 nm), 18 ± 4%(700 nm), and 42 ± 19%(1000 nm). Activation of J774A.1 cells with LPS significantly increased the cellular dose (normalized to the delivered dose) in all particle sizes except 50 nm, while cytochalasin D treatment significantly reduced the cellular dose of 100, 200 and 1000 nm particles. CONCLUSIONS This study demonstrates that dose correction using the ISDD model (i.e. normalization of cellular dose values to the delivered dose) is essential for accurate interpretation of results derived from in vitro particle-cell interaction studies (e.g. particle uptake, cytotoxicity, mechanisms of action, pharmacodynamic studies, etc.). It is of particular relevance to the field of particulate drug delivery systems, because the low density nature of most biomaterials used as drug carriers will result in very low fractions of the administered particle dose reaching the cell monolayer under most commonly used experimental conditions.

Quantitative assessment of nanoparticle surface hydrophobicity and its influence on pulmonary biocompatibility.

To date, the role of nanoparticle surface hydrophobicity has not been investigated quantitatively in relation to pulmonary biocompatibility. A panel of nanoparticles spanning three different biomaterial types, pegylated lipid nanocapsules, polyvinyl acetate (PVAc) and polystyrene nanoparticles, were characterized for size, surface charge, and stability in biofluids. Surface hydrophobicity of five nanoparticles (50-150nm) was quantified using hydrophobic interaction chromatography (HIC) and classified using a purpose-developed hydrophobicity scale: the HIC index, range from 0.00 (hydrophilic) to 1.00 (hydrophobic). This enabled the relationship between the nanomaterial HIC index value and acute lung inflammation after pulmonary administration to mice to be investigated. The nanomaterials with low HIC index values (between 0.50 and 0.64) elicited little or no inflammation at low (22cm(2)) or high (220cm(2)) nanoparticle surface area doses per animal, whereas equivalent surface area doses of the two nanoparticles with high HIC index values (0.88-0.96) induced neutrophil infiltration, elevation of pro-inflammatory cytokines and adverse histopathology findings. In summary, a HIC index is reported that provides a versatile, discriminatory, and widely available measure of nanoparticle surface hydrophobicity. The avoidance of high (HIC index>~0.8) surface hydrophobicity appears to be important for the design of safe nanomedicines for inhalation therapy.

Characterisation of the decomposition behaviour of S-nitrosoglutathione and a new class of analogues: S-Nitrosophytochelatins

S-Nitrosoglutathione (GSNO) is one of the most abundant S-nitrosothiols present in the body, playing an important role in many important physiological functions. Depletion of GSNO in some pathophysiological conditions makes GSNO a potentially interesting therapeutic molecule. Phytochelatins are glutathione analogues with the following structure: (gamma-glutamyl-cysteine)(n)-glycine. S-Nitroso derivatives of phytochelatins (SNOPCs) carry a greater number of S-nitrosothiol groups per molecule than GSNO and might therefore be very useful as therapeutic agents. The aim of this study was to investigate the in vitro decomposition behaviour of SNOPCs under various physicochemical stress conditions and compare it to the decomposition behaviour of GSNO. SNOPCs were generally less stable than GSNO under all experimental conditions tested, which included exposure to light, variation of pH and temperature as well as exposure to different concentrations of exogenous free thiol in the form of reduced glutathione (GSH). Even under light exclusion at ambient temperature the SNOPCs retained only 40% of their intact SNO groups after a 48h incubation time compared to 90% for GSNO. SNOPCs were also shown to readily take part in transnitrosation reactions when incubated with free glutathione. These properties suggest that SNOPCs could be employed as an investigation tool or possibly as therapeutic agents.

Enrichment of immunoregulatory proteins in the biomolecular corona of nanoparticles within human respiratory tract lining fluid.

UNLABELLED When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate semi-quantitative profiles of corona proteins formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles. FROM THE CLINICAL EDITOR Inhaled nanoparticles often acquire a layer of protein corona while they go through the respiratory tract. Here, the authors investigated the identity of these proteins. The proper identification would improve the understanding of the use of inhaled nanoparticles in future therapeutics.

New poly(lactic-co-glycolic acid) derivatives: Modular polymers with tailored properties.

Poly(lactic-co-glycolic acid) (PLGA) is one of the most widely used polymers in drug delivery, despite several well-characterized shortcomings. Polymer modification is one approach to improve PLGA-based formulation properties, such as drug stability, drug release profiles, mechanism of polymer degradation and the possibility of drug targeting. A brief summary of recent reports on PLGA modifications is provided and a new class of branched polyester derivatives is introduced. In vitro and in vivo applications of the new branched polyesters as protein carriers, gene delivery vehicles, vaccine adjuvants and pulmonary drug delivery vehicles are described.:

In vivo biocompatibility, clearance, and biodistribution of albumin vehicles for pulmonary drug delivery.

The development of clinically acceptable albumin-based nanoparticle formulations for use in pulmonary drug delivery has been hindered by concerns about the toxicity of nanomaterials in the lungs combined with a lack of information on albumin nanoparticle clearance kinetics and biodistribution. In this study, the in vivo biocompatibility of albumin nanoparticles was investigated following a single administration of 2, 20, and 390 μg/mouse, showing no inflammatory response (TNF-α and IL-6, cellular infiltration and protein concentration) compared to vehicle controls at the two lower doses, but elevated mononucleocytes and a mild inflammatory effect at the highest dose tested. The biodistribution and clearance of 111In labelled albumin solution and nanoparticles over 48 h following a single pulmonary administration to mice was investigated by single photon emission computed tomography and X-ray computed tomography imaging and terminal biodistribution studies. 111In labelled albumin nanoparticles were cleared more slowly from the mouse lung than 111In albumin solution (64.1 ± 8.5% vs 40.6 ± 3.3% at t = 48 h, respectively), with significantly higher (P < 0.001) levels of albumin nanoparticle-associated radioactivity located within the lung tissue (23.3 ± 4.7%) compared to the lung fluid (16.1 ± 4.4%). Low amounts of 111In activity were detected in the liver, kidneys, and intestine at time points > 24 h indicating that small amounts of activity were cleared from the lungs both by translocation across the lung mucosal barrier, as well as mucociliary clearance. This study provides important information on the fate of albumin vehicles in the lungs, which may be used to direct future formulation design of inhaled nanomedicines.

Enhanced gene expression and reduced toxicity in mice using polyplexes of low-molecular-weight poly(ethylene imine) for pulmonary gene delivery

The aim of this study was to elicit improved gene expression and decreased cytotoxicity for pulmonary gene therapy by replacing the commonly used carrier 25 kDa branched poly(ethylene imine) (BPEI) by two PEI derivatives, low-molecular-weight PEI (LMWPEI) and polyethylene glycol−grafted PEI (PEGPEI). All polymers were shown to condense DNA to spherical particles of approximately 100 nm. Biocompatibility was investigated in vitro and in vivo. Although transfection was less efficient with LMWPEI-DNA in vitro, this polyplex caused the highest luciferase expression in the mouse lung after intratracheal instillation. While PEGPEI luciferase expression in vitro was approximately three times higher when compared to BPEI, a transfection rate at the level of naked DNA was observed in vivo. LMWPEI polyplexes were located in both the bronchial and alveolar cells, whereas BPEI polyplexes were mainly detected in bronchial cells. LMWPEI combines low cytotoxicity with high transfection efficiency in the mouse lung in vivo, rendering it a promising strategy for pulmonary gene delivery.