PEGylation of Tobramycin Improves Mucus Penetration and Antimicrobial Activity against Pseudomonas aeruginosa Biofilms in Vitro.

Pseudomonas aeruginosa is the predominant pathogen in the persistent lung infections of cystic fibrosis (CF) patients among other diseases. One of the mechanisms of resistance of P. aeruginosa infections is the formation and presence of biofilms. Previously, we demonstrated that PEGylated-tobramycin (Tob-PEG) had superior antimicrobial activity against P. aeruginosa biofilms compared to tobramycin (Tob). The goal of this study was to optimize the method of PEGylation of Tob and assess its activity in an in vitro CF-like mucus barrier biofilm model. Tob was PEGylated using three separate chemical conjugation methods and analyzed by 1H NMR. A comparison of the Tob-PEG products from the different conjugation methods showed significant differences in the reduction of biofilm proliferation after 24 h of treatment. In the CF-like mucus barrier model, Tob-PEG was significantly better than Tob in reducing P. aeruginosa proliferation after only 5 h of treatment ( p < 0.01). Finally, Tob-PEG caused a reduction in the number of surviving P. aeruginosa biofilm colonies higher than that of Tob ( p < 0.0001). We demonstrate the significantly improved antimicrobial activity of Tob-PEG against P. aeruginosa biofilms compared to Tob using two PEGylation methods. Tob-PEG had better in vitro activity compared to that of Tob against P. aeruginosa biofilms growing in a CF-like mucus barrier model.

In vitro and in vivo characterisation of PEG-lipid-based micellar complexes of salmon calcitonin for pulmonary delivery.

PURPOSE: To investigate DSPE-PEG(2000)-based micellar formulations of salmon calcitonin (sCT) for their ability to improve pulmonary delivery. METHODS: Micelles were characterised by DLS and (31)P-NMR spectroscopy. Stability against sCT degrading peptidases, trypsin, α-chymotrypsin and neutrophil elastase as well as their influence on transepithelial absorption was investigated in vitro. In vivo performance of sCT micelles was studied in an experimental model of intratracheal aerosolisation into rats. RESULTS: Micelles with a mean hydrodynamic diameter of 12 nm spontaneously assembled, when a total concentration of 0.02 mM of PEG-lipid and sCT (at 1:1 molar ratio) was exceeded. Nuclear magnetic resonance confirmed the presence of small micellar structures. The micellar formulation showed increased stability against enzymatic digestion. In vitro studies also showed that sCT micelles were able to enhance transepithelial absorption. Data obtained from in vivo experiments provided evidence of significantly (P < 0.05) higher mean plasma concentrations of sCT, after inhalation of micelles compared to sCT solution, at 60 and 90 min, a significantly higher AUC (inf) and a relative bioavailability of 160 ± 55% when compared to plain sCT solution. CONCLUSIONS: The herein described PEG-lipid micelles are promising carriers for enhanced pulmonary delivery of sCT.

Evaluation of HPßCD-PEG microparticles for salmon calcitonin administration via pulmonary delivery.

For therapeutic peptides, the lung represents an attractive, noninvasive route into the bloodstream. To achieve optimal bioavailability and control their fast rate of absorption, peptides can be protected by coprocessing with polymers such as polyethylene glycol (PEG). Here, we formulated and characterized salmon calcitonin (sCT)-loaded microparticles using linear or branched PEG (L-PEG or B-PEG) and hydroxypropyl-beta-cyclodextrin (HPßCD) for pulmonary administration. Mixtures of sCT, L-PEG or B-PEG and HPßCD were co-spray dried. Based on the particle properties, the best PEG:HPßCD ratio was 1:1 w:w for both PEGs. In the sCT-loaded particles, the L-PEG was more crystalline than B-PEG. Thus, L-PEG-based particles had lower surface free energy and better aerodynamic behavior than B-PEG-based particles. However, B-PEG-based particles provided better protection against chemical degradation of sCT. A decrease in sCT permeability, measured across Calu-3 bronchial epithelial monolayers, occurred when the PEG and HPßCD concentrations were both 1.6 wt %. This was attributed to an increase in buffer viscosity, caused by the two excipients. sCT pharmacokinetic profiles in Wistar rats were evaluated using a 2-compartment model after iv injection or lung insufflation. The maximal sCT plasma concentration was reached within 3 min following nebulization of sCT solution. L-PEG and B-PEG-based microparticles were able to increase T(max) to 20 ± 1 min and 18 ± 8 min, respectively. Furthermore, sCT absolute bioavailability after L-PEG-based microparticle aerosolization at 100 µg/kg was 2.3 times greater than for the nebulized sCT solution.

Dynamical and rheological properties of fluorinated surfactant films adsorbed at the pressurized CO2-H2O interface.

The dynamics of adsorption, interfacial tension, and rheological properties of two phosphocholine-derived partially fluorinated surfactants FnHmPC, designed to compensate for the weak CO(2)-surfactant tail interactions, were determined at the pressurized CO(2)-H(2)O interface. The two surfactants differ only by the length of the hydrocarbon spacer (5 CH(2) in F8H5PC and 11 CH(2) in F8H11PC) located between the terminal perfluoroalkyl chain and the polar head. The length of this spacer was found to have a critical impact on the adsorption kinetics and elasticity of the interfacial surfactant film. F8H5PC is soluble in both water and CO(2) phases and presents several distinct successive interfacial behaviors when bulk water concentration (C(W)) increases and displays a nonclassical isotherm shape. The isotherms of F8H5PC are similar for the three CO(2) pressures investigated and comprise four regimes. In the first regime, at low C(W), the interfacial tension is controlled by the organization that occurs between H(2)O and CO(2). The second regime corresponds to the adsorption of the surfactant as a monolayer until the CO(2) phase is saturated with F8H5PC, resulting in a first inflection point. In this regime, F8H5PC molecules reach maximal compaction and display the highest apparent interfacial elasticity. In the third regime, a second inflection is observed that corresponds to the critical micelle concentration of the surfactant in water. At the highest concentrations (fourth regime), the interfacial films are purely viscous and highly flexible, suggesting the capacity for this surfactant to produce water-in-CO(2) microemulsion. In this regime, surfactant adsorption is very fast and equilibrium is reached in less than 100 s. The behavior of F8H11PC is drastically different: it forms micelles only in the water phase, resulting in a classical Gibbs interface. This surfactant decreases the interfacial tension down to 1 mN/m and forms a strongly elastic interface. As this surfactant forms a very cohesive interface, it should be suitable for formulating stable water-in-CO(2) emulsions. The finding that the length of the hydrocarbon spacer in partially fluorinated surfactants can drastically influence film properties at the CO(2)-H(2)O interface should help control the formation of microemulsions versus emulsions and help elaborate a rationale for the design of surfactants specifically adapted to pressurized CO(2).

Moisture diffusion and permeability characteristics of hydroxypropylmethylcellulose and hard gelatin capsules.

The primary objective of this paper is to compare the sorption characteristics of hydroxypropylmethylcellulose (HPMC) and hard gelatin (HG) capsules and their ability to protect capsule contents. Moisture sorption and desorption isotherms for empty HPMC and HG capsules have been investigated using dynamic vapour sorption (DVS) at 25°C. All sorption studies were analysed using the Young-Nelson model equations which distinguishes three moisture sorption types: monolayer adsorption moisture, condensation and absorption. Water vapour diffusion coefficients (D), solubility (S) and permeability (P) parameters of the capsule shells were calculated. ANOVA was performed with the Tukey comparison test to analyse the effect of %RH and capsule type on S, P, and D parameters. The moisture uptake of HG capsules were higher than HPMC capsules at all %RH conditions studied. It was found that values of D and P across HPMC capsules were greater than for HG capsules at 0-40 %RH; whereas over the same %RH range S values were higher for HG than for HPMC capsules. S values decreased gradually as the %RH was increased up to 60% RH. To probe the effect of moisture ingress, spray dried lactose was loaded into capsules. Phase evolution was characterised by scanning electron microscopy (SEM), X-ray powder diffraction (XRD), and differential scanning calorimetry (DSC). The capsules under investigation are not capable of protecting spray dried lactose from induced solid state changes as a result of moisture uptake. For somewhat less moisture sensitive formulations, HPMC would appear to be a better choice than HG in terms of protection of moisture induced deterioration.

Encapsulation into PEG-liposomes does not improve the bioavailability of pulmonary delivered salmon calcitonin.

BACKGROUND: Salmon calcitonin (sCT) is approved for the short-term treatment of Paget's disease and hypercalcemia. As pulmonary delivery might improve the drug's efficacy, a variety of liposomal sCT formulations for inhalation were prepared and characterized with the intention of developing a controlled release formulation. METHODS: The influence of pH of the loading buffer, charge of the vesicular surface, and membrane rigidity on particle size, ζ-potential, and sCT encapsulation efficiency of formulations was studied. The most promising systems were investigated for their ability to withstand nebulization stresses using an Aeroneb(®) vibrating mesh device. In vitro studies were carried out to determine sCT release from the vesicles and the bioactivity of the peptide post nebulization. Lastly, pharmacokinetics of sCT liposomes following intratracheal aerosolization in an experimental rat model were investigated and compared with intravenous injection. RESULTS: Liposomes prepared with acidic loading buffer and comprising rigid lipid membranes showed an optimal compromise between small particle size, high encapsulation efficiency, and sCT stability. Polyethylene glycol (PEG) liposomes showed the highest encapsulation efficiency overall, regardless of the ζ-potential of the vesicles. Positive surface charge, however, yielded higher entrapment in non-PEGylated liposomes. All liposomes tested were stable during nebulization. The bioactivity of sCT after formulation into liposomes was 52-55%. Intratracheal nebulization in rats revealed that the bioavailability and other pharmacokinetic parameters were not enhanced by liposomes, when compared with sCT solution. Following intravenous administration, however, liposomes showed significantly higher bioavailability and AUCinf (area under the curve to the infinity time point) than controls. CONCLUSIONS: The developed liposomal formulations were not optimal carriers for pulmonary delivery of sCT. Due to the low amounts of peptide released from the vesicles, enzymatic digestion by peptidases in the airspace reduced the bioavailability significantly. Liposomal encapsulation of sCT, nevertheless, resulted in improved pharmacokinetics following injection.