Nanomaterials fusing with the skin: Alpha-tocopherol phosphate delivery into the viable epidermis to protect against ultraviolet radiation damage.

Vitamin E (alpha tocopherol, α-T) is an important skin antioxidant, but its penetration into the viable epidermis, where it acts, is very limited. This study investigated if phosphorylating α-tocopherol (α-TP) to form a provitamin, improved its interactions with skin, its passage into the tissue, and thus its ability to protect the skin from ultraviolet radiation (UVR) damage. At pH 7.4, when the α-TPO4-1 microspecies predominated in solution, dynamic light scattering measurements showed that α-TP formed nanoaggregates with a median hydrodynamic diameter of 9 nm (Critical aggregation constant, CAC, - 4.2 mM). At 9.0 when the α-TPO4-2 microspecies predominated there was no aggregation. The passage of α-TP nanoaggregates through regenerated cellulose membranes was significantly slower than the α-TP monomers (at pH 9) suggesting that aggregation slowed diffusion. However, a lotion formulation containing the nanoaggregates delivered more α-TP into the skin compared to the formulation containing the monomers. In addition, the nanosized α-TP aggregates delivered 8-fold more active into the stratum corneum (SC) (252.2 µg/cm2 vs 29.5 µg/cm2) and 4 fold more active into the epidermis (85.1 µg/cm2 vs 19 µg/cm2, respectively, p < 0.05) compared to α-T. Langmuir subphase injection studies at pH 7.4 (surface pressure 10 mN m-1) showed that the α-TP nanoaggregates more readily fused with the SC compared to the monomers and the membrane compression studies demonstrated that α-TP fluidised the SC lipids. Together the fusion with the SC and its fluidisation were proposed as the causes of the better α-TP penetration into the skin, which enhanced potential of α-TP to protect from UVR-induced skin damage compared to α-T.

In vitro and in vivo antitubercular activity of benzothiazinone-loaded human serum albumin nanocarriers designed for inhalation.

The aim of this study was to investigate the potential of human serum albumin (HSA) as a solubilising agent/drug delivery vehicle for pulmonary administration of antimycobacterial benzothiazinone (BTZ) compounds. The solubility of four novel BTZ compounds (IR 20, IF 274, FG 2, AR 112) was enhanced 2 to 140-fold by incubation with albumin (0.38-134 µg/mL). Tryptophan 213 residue quenching studies indicated moderate binding strength to Sudlow's site I. Nanoparticle manufacture achieved 37-60% encapsulation efficiency in HSA particles (169 nm, zeta potential -31 mV). Drug release was triggered by proteases with >50% released in 4 h. The antimycobacterial activity of IR 20 and FG 2 loaded in HSA nanoparticles was enhanced compared to DMSO/phosphate buffered saline (PBS) or albumin/PBS solutions in an in vitro M. tuberculosis-infected macrophage model. Intranasal instillation was used to achieve pulmonary delivery daily over 10 days to M. tuberculosis infected mice for FG2 HSA nanoparticles (0.4 mg/kg), FG 2 DMSO/saline (0.4 and 8 mg/kg) and a reference compound, BTZ043, DMSO/saline (0.4 and 8 mg/kg). A lower lung M. tuberculosis burden was apparent for all BTZ cohorts, but only significant for BTZ043 at both doses. In conclusion, mechanisms of HSA nanoparticle loading and release of BTZ compounds were demonstrated, enhanced antimycobacterial activity of the nanoparticle formulations was demonstrated in a biorelevant in vitro bioassay and the effectiveness of BTZ by pulmonary delivery in vivo was established with pilot evidence for effectiveness when delivered by HSA nanoparticles. Finally, the feasibility of developing an inhaled nanoparticle-in-microparticle powder formulation was ascertained.

Antibiotic-in-Cyclodextrin-in-Liposomes: Formulation Development and Interactions with Model Bacterial Membranes.

Gram-negative bacteria possess numerous defenses against antibiotics, due to the intrinsic permeability barrier of their outer membrane (OM), explaining the recalcitrance of some common and life-threatening infections. We report the formulation of a new drug, PPA148, which shows promising activity against all Gram-negative bacteria included in the ESKAPEE pathogens. PPA148 was solubilized by inclusion complexation with cyclodextrin followed by encapsulation in liposomes. The complex and liposomal formulation presented increased activity against E. coli compared to the pure drug when assessed with the Kirby Bauer assay. The novel formulation containing 1 µg PPA148 reached similar efficacy levels equivalent to those of 30 µg of pure rifampicin. A range of biophysical techniques was used to explore the mechanism of drug uptake. Langmuir trough (LT) and neutron reflectivity (NR) techniques were employed to monitor the interactions between the drug and the formulation with model membranes. We found evidence for liposome fusion with the model Gram-negative outer membrane and for cyclodextrins acting as inner membrane (IM) permeation enhancers without presenting intrinsic antimicrobial activity. An antibiotic-in-cyclodextrin-in-liposomes (ACL) formulation was developed, which targets both the bacterial OM and IM, and offers promise as a means to breach the Gram-negative cell envelope.

Using Polar Ion-Pairs to Control Drug Delivery to the Airways of the Lungs.

The rapid absorptive clearance of drugs delivered to the airways of the lungs means that many inhaled medicines have a short duration of action. The aim of this study was to investigate whether forming polar ion-pairs can modify drug absorption to slow down clearance from the airways. Salbutamol was used as a model drug and was formulated as ion-pairs in an aqueous solution with three negatively charged hydrophilic counterions: sulfate (molecular weight (MW) 142), gluconate (MW 218), and phytate (MW 736) (association constants of 1.57, 2.27, and 4.15, respectively) and one negatively charged hydrophobic counterion, octanoate (MW 166) (association constant, 2.56). All of the counterions were well tolerated by Calu-3 human bronchial epithelial cells when screened for toxicity in vitro using conditions that in silico simulations suggested maintain >80% drug-counterion association. The transport of salbutamol ion-pairs with higher polar surface area (PSA), i.e., the sulfate (PSA 52%), gluconate (PSA 50%), and phytate (PSA 79%) ion-pairs, was significantly lower compared to that of the drug alone (PSA 30%, p < 0.05). In contrast, the octanoate ion-pair (PSA 23%) did not significantly alter the salbutamol transport. The transport data for the gluconate ion-pair suggested that the pulmonary absorption half-life of the ion-paired drug would be double that of salbutamol base, and this illustrates the promise of increasing drug polarity using noncovalent complexation as an approach to control drug delivery to the airways of the lungs.

Development of new in vitro models of lung protease activity for investigating stability of inhaled biological therapies and drug delivery systems.

Proteases play a vital role in lung health and are critically important to the metabolic clearance of inhaled protein-based therapeutics after inhalation. Surprisingly little is known about lung fluid protease composition and there is a consequent lack of biorelevant experimental models, which limits research and development in the burgeoning field of inhaled biologics. The aim of this study was to quantify proteases in human lung fluid and to use this data to design novel in vitro experimental models of lung lining fluid possessing biorelevant lung protease activity for use in biopharmaceutical stability studies. As a proof of concept, these novel models were used to investigate the effect of proteolytic activity on the stability of albumin nanoparticles, a biologic nanoparticle formulation widely investigated as a pulmonary drug delivery system. Bronchoalveolar lavage fluid was collected from healthy human volunteers and proteomic analysis was used to quantify the predominant proteases. Based on these data, four new lung protease models were constructed based on: (i) trypsin as a sole protease, (ii) dipeptidyl peptidase IV, cathepsin D, cathepsin H, and angiotensin converting enzyme in ratio and concentration to mimic the protease concentration in healthy lungs. Neutrophil elastase was used to model protease activity in inflammation. Albumin nanoparticles of 100 nm diameter remained intact over 48 h in phosphate buffered saline, but were degraded more rapidly in trypsin (50% reduction in 10 min) compared to the healthy lung protease model (50% reduction in 150 min). The addition of neutrophil elastase to the healthy lung protease model resulted in a similar, but more variable degradation profile. Nanoparticle degradation was associated with concomitant appearance of small fragments and aggregates. In conclusion, we have characterised the protease concentration in the lungs of healthy humans, designed models of lung protease activity and demonstrated their utility in studying albumin nanoparticle degradation. These methods and models have wide application to study the influence of proteases in lung disease, expression of proteases in respiratory cell culture models, stability of peptide and protein-based drugs and inhaled drug delivery systems.

Mucus penetrating properties of soft, distensible lipid nanocapsules.

Designing nanomaterials to release their drug pay-load upon exposure to an exogenous trigger can help to direct drug delivery, but how the triggered release, which often modifies the nanomaterial properties, influences the biological fate of these systems is currently unknown. The aim of this study was to investigate how the triggered drug release from PEG coated, soft, 50 nm distensible lipid nanocapsules (LNC) influenced their diffusion across a mucus barrier. The translocation speed of the non-triggered LNC across a 35 µm thick purified gastric mucin (PGM) barrier was 3 times faster (30.08 ±â€¯2.49 × 10-10 cm2 s-1) compared to equivalent-sized negatively charged polystyrene particles (9.87 ±â€¯0.61 × 10-10 cm2 s-1, p < 0.05). In cystic fibrosis mucus (CFM), harvested from patient primary cells, the non-triggered LNC translocation speed was similar to the PGM, but the polystyrene particle diffusion was so slow it could not be measured. The trigger induced LNC distension process had no effect on the particle diffusion rate in both PGM and CFM (p > 0.05) in a static mucus barrier, but when shear was applied to the barrier the distended LNCs diffused more slowly (3.97 ±â€¯1.38 × 10-8 cm2 s-1, p < 0.05) compared to the non-distended materials (4.94 ±â€¯0.04 × 10-8 cm2 s-1). This data suggested the rapid mucus penetration of the distended LNCs, despite their increased size, was a consequence of their capacity to take a less tortuous path through the barrier, i.e., they experienced less steric hinderance, compared to the non-distended LNC.

Antimicrobial molecules in the lung: formulation challenges and future directions for innovation.

Inhaled antimicrobials have been extremely beneficial in treating respiratory infections, particularly chronic infections in a lung with cystic fibrosis. The pulmonary delivery of antibiotics has been demonstrated to improve treatment efficacy, reduce systemic side effects and, critically, reduce drug exposure to commensal bacteria compared with systemic administration, reducing selective pressure for antimicrobial resistance. This review will explore the specific challenges of pulmonary delivery of a number of differing antimicrobial molecules, and the formulation and technological approaches that have been used to overcome these difficulties. It will also explore the future challenges being faced in the development of inhaled products and respiratory infection treatment, and identify future directions of innovation, with a particular focus on respiratory infections caused by multiple drug-resistant pathogens.

Controlled drug release from lung-targeted nanocarriers via chemically mediated shell permeabilisation.

Nanocarriers can aid therapeutic agent administration to the lung, but controlling drug delivery from these systems after deposition in the airways can be problematic. The aim of this study was to evaluate if chemically mediated shell permeabilisation could help manipulate the rate and extent of nanocarrier drug release. Rifampicin was loaded into lipid shell (loading efficiency 41.0±11.4%, size 50nm) and polymer shell nanocarriers (loading efficiency 25.9±2.3%, size 250nm). The drug release at pH 7.4 (lung epithelial pH) and 4.2 (macrophage endosomal pH) with and without the chemical permeabilisers (Pluronic L62D - lipid nanocarriers; H(+)- polymer nanocarriers) was then tested. At pH 7.4 the presence of the permeabilisers increased nanocarrier drug release rate (from 3.2µg/h to 6.8µg/h for lipid shell nanocarriers, 2.3µg/h to 3.4µg/h for polymer shell nanocarriers) and drug release extent (from 50% to 80% for lipid shell nanocarriers, from 45% to 76% for polymer shell nanocarriers). These effects were accompanied by lipid nanocarrier distension (from 50 to 240nm) and polymer shell hydrolysis. At pH 4.2 the polymer nanocarriers did not respond to the permeabiliser, but the lipid nanocarrier maintained a robust drug release enhancement response and hence they demonstrated that the manipulation of controlled drug release from lung-targeted nanocarriers was possible through chemically mediated shell permeabilisation.

Surface chemistry of photoluminescent F8BT conjugated polymer nanoparticles determines protein corona formation and internalization by phagocytic cells.

Conjugated polymer nanoparticles are being developed for a variety of diagnostic and theranostic applications. The conjugated polymer, F8BT, a polyfluorene derivative, was used as a model system to examine the biological behavior of conjugated polymer nanoparticle formulations stabilized with ionic (sodium dodecyl sulfate; F8BT-SDS; ∼207 nm; -31 mV) and nonionic (pegylated 12-hydroxystearate; F8BT-PEG; ∼175 nm; -5 mV) surfactants, and compared with polystyrene nanoparticles of a similar size (PS200; ∼217 nm; -40 mV). F8BT nanoparticles were as hydrophobic as PS200 (hydrophobic interaction chromatography index value: 0.96) and showed evidence of protein corona formation after incubation with serum-containing medium; however, unlike polystyrene, F8BT nanoparticles did not enrich specific proteins onto the nanoparticle surface. J774A.1 macrophage cells internalized approximately ∼20% and ∼60% of the F8BT-SDS and PS200 delivered dose (calculated by the ISDD model) in serum-supplemented and serum-free conditions, respectively, while cell association of F8BT-PEG was minimal (<5% of the delivered dose). F8BT-PEG, however, was more cytotoxic (IC50 4.5 µg cm(-2)) than F8BT-SDS or PS200. The study results highlight that F8BT surface chemistry influences the composition of the protein corona, while the properties of the conjugated polymer nanoparticle surfactant stabilizer used determine particle internalization and biocompatibility profile.

Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity.

ArdA antirestriction proteins are encoded by genes present in many conjugative plasmids and transposons within bacterial genomes. Antirestriction is the ability to prevent cleavage of foreign incoming DNA by restriction-modification (RM) systems. Antimodification, the ability to inhibit modification by the RM system, can also be observed with some antirestriction proteins. As these mobile genetic elements can transfer antibiotic resistance genes, the ArdA proteins assist their spread. The consequence of antirestriction is therefore the enhanced dissemination of mobile genetic elements. ArdA proteins cause antirestriction by mimicking the DNA structure bound by Type I RM enzymes. The crystal structure of ArdA showed it to be a dimeric protein with a highly elongated curved cylindrical shape [McMahon SA et al. (2009) Nucleic Acids Res 37, 4887-4897]. Each monomer has three domains covered with negatively charged side chains and a very small interface with the other monomer. We investigated the role of the domain forming the dimer interface for ArdA activity via site-directed mutagenesis. The antirestriction activity of ArdA was maintained when up to seven mutations per monomer were made or the interface was disrupted such that the protein could only exist as a monomer. The antimodification activity of ArdA was lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests, first, that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme, and second, that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction.