Moritz Beck-Broichsitter

Characterization of novel spray-dried polymeric particles for controlled pulmonary drug delivery.

Numerous studies have addressed the controlled pulmonary drug delivery properties of colloidal particles. However, only scant information on the potential of spray-drying for submicron particle preparation is available. By exploiting the advantages of spray-drying, the characteristics of submicron particles can be optimized to meet the requirements necessary for lung application. Submicron particles were prepared from organic poly(d,l-lactide-co-glycolide) (PLGA) solutions, and composite particles were spray-dried from aqueous PLGA nanosuspensions. The feed concentration, as well as the spray-mesh diameter influenced the resulting particle sizes. Nanoparticles were virtually unaffected after spray-drying. The aerodynamic characteristics of both particle species revealed aerosol particle sizes suitable for deposition in the deep lungs (≤4μm). While the entrapped drug was released within ~90min from the composite particles, extensive drug retardation (~480min) was observed for PLGA particles spray-dried from organic solution. These results suggest that nanospray-drying is a convenient method to prepare submicron, controlled drug delivery vehicles useful for pulmonary application potentially allowing access to alveolar tissue.

Pulmonary drug delivery with aerosolizable nanoparticles in an ex vivo lung model

The use of colloidal carrier systems for pulmonary drug delivery is an emerging field of interest in nanomedicine. The objective of this study was to compare the pulmonary absorption and distribution characteristics of the hydrophilic model drug 5(6)-carboxyfluorescein (CF) after aerosolization as solution or entrapped into nanoparticles in an isolated rabbit lung model (IPL). CF-nanoparticles were prepared from a new class of biocompatible, fast degrading, branched polyesters by a modified solvent displacement method. Physicochemical properties, morphology, encapsulation efficiency, in vitro drug release, stability of nanoparticles to nebulization, aerosol characteristics as well as pulmonary dye absorption and distribution profiles after nebulization in an IPL were investigated. CF-nanoparticles were spherical in shape with a mean particle size of 195.3+/-7.1nm, a polydispersity index of 0.225+/-0.017 and a zeta-potential of -28.3+/-0.3mV. Encapsulation efficiencies of CF were as high as about 60% (drug loading of 3% (w/w)); 90% of the entrapped CF were released during the first 50min in vitro. Nanoparticle characteristics were not significantly affected by the aerosolization process utilizing a vibrating mesh nebulizer. After deposition of equal amounts of CF in the IPL, less CF was detected in the perfusate for CF-nanoparticles (plateau concentration 9.2+/-2.4ng/ml) when compared to CF aerosolized from solution (17.7+/-0.8ng/ml). In conclusion, the data suggest that inhalative delivery of biodegradable nanoparticles may be a viable approach for pulmonary drug delivery. Moreover, a targeting effect to the lung tissue is claimed.

Amphiphilic biodegradable PEG-PCL-PEI triblock copolymers for FRET-capable in vitro and in vivo delivery of siRNA and quantum dots.

Amphiphilic triblock copolymers represent a versatile delivery platform capable of co-delivery of nucleic acids, drugs, and/or dyes. Multifunctional cationic triblock copolymers based on poly(ethylene glycol), poly-ε-caprolactone, and polyethylene imine, designed for the delivery of siRNA, were evaluated in vitro and in vivo. Moreover, a nucleic acid-unpacking-sensitive imaging technique based on quantum dot-mediated fluorescence resonance energy transfer (QD-FRET) was established. Cell uptake in vitro was measured by flow cytometry, whereas transfection efficiencies of nanocarriers with different hydrophilic block lengths were determined in vitro and in vivo by quantitative real-time PCR. Furthermore, after the proof of concept was demonstrated by fluorescence spectroscopy/microscopy, a prototype FRET pair was established by co-loading QDs and fluorescently labeled siRNA. The hydrophobic copolymer mediated a 5-fold higher cellular uptake and good knockdown efficiency (61 ± 5% in vitro, 55 ± 18% in vivo) compared to its hydrophilic counterpart (13 ± 6% in vitro, 30 ± 17% in vivo), which exhibited poor performance. FRET was demonstrated by UV-induced emission of the acceptor dye. Upon complex dissociation, which was simulated by the addition of heparin, a dose-dependent decrease in FRET efficiency was observed. We believe that in vitro/in vivo correlation of the structure and function of polymeric nanocarriers as well as sensitive imaging functionality for mechanistic investigations are prerequisites for a more rational design of amphiphilic gene carriers.

Biophysical investigation of pulmonary surfactant surface properties upon contact with polymeric nanoparticles in vitro

UNLABELLED Nanoparticulate drug carriers have been proposed for the targeted and controlled release of pharmaceuticals to the lung. However, inhaled particles may adversely affect the biophysical properties of pulmonary surfactant. This study examines the influence of polymeric nanoparticles with distinct physicochemical properties on the adsorption and dynamic surface tension lowering properties of pulmonary surfactant. Nanoparticles had a mean size of 100 nm with narrow size distributions. Although poly(styrene) and poly(D,L-lactide-co-glycolide) nanoparticles revealed a dose-dependent influence on biophysics of pulmonary surfactant, positively-charged nanoparticles made from poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate) showed no effect. This behavior is attributed to the differences in ζ-potential and surface hydrophobicity, which in turn involves an altered adsorption pattern of the positively charged surfactant proteins to the nanoparticles. This study suggests that polymeric nanoparticles do not substantially affect the biophysical properties of pulmonary surfactant and may be a viable drug-delivery vehicle for the inhalative treatment of respiratory diseases. FROM THE CLINICAL EDITOR Inhaled nanoparticulate drug carriers may adversely affect the biophysical properties of pulmonary surfactant. In this study the influence of polymeric nanoparticles was characterized from this standpoint, with the conclusion that polymeric nanoparticles do not substantially affect the biophysical properties of pulmonary surfactant and may be viable drug-delivery vehicles for inhalational treatment.

Pulmonary targeting with biodegradable salbutamol-loaded nanoparticles.

BACKGROUND Aerosol therapy using particulate drug carriers has become an increasingly attractive method to deliver therapeutic or diagnostic compounds to the lung. Polymeric nanoparticles are widely investigated carriers in nanomedicine. The targeted and controlled release of drugs from nanoparticles for pulmonary delivery, however, is a research field that has been so far rather unexploited. Therefore, the objective of this study was to compare the pulmonary absorption and distribution characteristics of salbutamol after aerosolization as solution or entrapped into novel polymeric nanoparticles in an isolated rabbit lung model (IPL). METHODS Physicochemical properties, morphology, encapsulation efficiency, in vitro drug release, stability of nanoparticles to nebulization, as well as pulmonary drug absorption and distribution after nebulization in the IPL were investigated. RESULTS Salbutamol-loaded poly(D,L-lactide-co-glycolide) (PLGA) and poly(vinyl sulfonate-co-vinyl alcohol)-graft-poly(D,L-lactide-co-glycolide) (VS(72)-10) nanoparticles were prepared by a modified solvent displacement technique with a mean particle size of approximately 120 nm and a polydispersity index below 0.150. VS(72)-10 nanoparticles showed a more negative zeta-potential of -54.2 +/- 3.3 mV compared to PLGA nanoparticles (-36.5 +/- 2.6 mV). Salbutamol encapsulation efficiency was 25.2 +/- 4.9% and 63.4 +/- 3.5% for PLGA and VS(72)-10 nanoparticles, respectively. After nebulization utilizing the MicroSprayer physicochemical properties of salbutamol-loaded VS(72)-10 nanoparticles were virtually unchanged, whereas nebulized salbutamol-loaded PLGA nanoparticles showed a significant increase in mean particle size and polydispersity. In vitro release studies demonstrated a sustained release of the encapsulated salbutamol from VS(72)-10 nanoparticles. In parallel, a sustained salbutamol release profile was observed after aerosol delivery of these particles to the IPL as reflected by a lower salbutamol recovery in the perfusate (40.2 +/- 5.8%) when compared to PLGA nanoparticles (55.2 +/- 9.1%) and salbutamol solution (62.8 +/- 7.1%). CONCLUSIONS The current study suggests that inhalative delivery of biodegradable nanoparticles may be a viable approach for the treatment of respiratory diseases.

Nebulization performance of biodegradable sildenafil-loaded nanoparticles using the Aeroneb® Pro: Formulation aspects and nanoparticle stability to nebulization

Polymeric nanoparticles meet the increasing interest for drug delivery applications and hold great promise to improve controlled drug delivery to the lung. Here, we present a series of investigations that were carried out to understand the impact of formulation variables on the nebulization performance of novel biodegradable sildenafil-loaded nanoparticles designed for targeted aerosol therapy of life-threatening pulmonary arterial hypertension. Narrowly distributed poly(D,L-lactide-co-glycolide) nanoparticles (size: ∼200 nm) were prepared by a solvent evaporation technique using poly(vinyl alcohol) (PVA) as stabilizer. The aerodynamic and output characteristics using the Aeroneb Pro nebulizer correlated well with the dynamic viscosity of the employed fluids for nebulization. The nebulization performance was mainly affected by the amount of employed stabilizer, rather than by the applied nanoparticle concentration. Nanoparticles revealed physical stability against forces generated during aerosolization, what is attributed to the adsorbed PVA layer around the nanoparticles. Sildenafil was successfully encapsulated into nanoparticles (encapsulation efficiency: ∼80%). Size, size distribution and sildenafil content of nanoparticles were not affected by nebulization and the in vitro drug release profile demonstrated a sustained sildenafil release over ∼120 min. The current study suggests that the prepared sildenafil-loaded nanoparticles are a promising pharmaceutical for the therapy of pulmonary arterial hypertension.

Development of a biodegradable nanoparticle platform for sildenafil: Formulation optimization by factorial design analysis combined with application of charge-modified branched polyesters

Biodegradable nanoparticles have gained tremendous attraction as carriers for controlled drug delivery to the lung. Despite numerous advances in the field, e.g. development of suitable methods for pulmonary administration of polymeric nanoparticles, a sufficient association of the therapeutic agent with the carrier system as well as drug release in a controlled fashion remain considerable challenges. Hence, this study examines the optimization of biodegradable sildenafil-loaded nanoparticle formulations intended for aerosol treatment of pulmonary hypertension. A factorial design analysis was employed to identify the important experimental factors involved in the preparation of nanoparticles by the solvent evaporation technique. The effect of tailored charge-modified branched polyesters on drug loading and in vitro drug release from nanoparticles was also evaluated. Moreover, colloidal stability of obtained nanoparticles was assessed, and stabilization of nanoparticles by lyophilization was accomplished without additional excipients. Essential experimental factors were identified and optimized to allow the preparation of nanoparticles composed of linear polyesters with a sildenafil content of ~5 wt.%. The in vitro drug release profile from these nanoparticles demonstrated a sustained release of sildenafil over ~90 min. Application of charge-modified branched polyesters enhanced the drug content in nanoparticles and drug release profile, according to the charge-density present in the employed polymer. Accordingly an increase in drug loading by a factor of ~1.4, a prolonged drug release profile from nanoparticles over ~240 min was achieved. Sildenafil release from nanoparticles made of linear and charge-modified branched polyesters was governed by a diffusion process. The obtained drug diffusion coefficients were decreased as the charge-density present in the applied polymer was increased, which promotes the strategy to improve drug loading and release rates by electrostatic interactions between polymer and drug. In addition, nanoparticles showed high colloidal stability in different media of importance for pulmonary application and were successfully stabilized by lyophilization. In conclusion, optimization of the nanoparticle preparation process together with the application of tailored polymeric materials facilitated the synthesis of promising drug carriers for sildenafil that permit a novel treatment modality for severe pulmonary hypertension.

Polymeric nanocarriers for drug delivery to the lung

Successful delivery of nano-particles to the lung has to take account of the potential biological and pathophysiological barriers of the target organ. Hence this review attempts to provide an overview of the anatomy, physiology, and pathophysiology of the lung. In particular, pathological changes may have crucial impact on successful nano-carrier delivery. The latest approaches in nano-encapsulation and pulmonary delivery will be discussed for small molecular drugs, usually having a fast release due to short diffusion distances. In peptide and protein delivery, the stability of organic solvents and prevention from denaturation are main aspects which are addressed. Gene delivery may be divided into DNA, mRNA and siRNA delivery. They have in common that most of the polymers used still have toxic effects and are only poorly degradable. Polymers with reduced toxic potential and biodegradable polyamides will be presented in this review. The attractiveness of pulmonary drug delivery both for loco-regional and systemic application is likely to increase in the future as nano-carriers may allow more efficient and selective drug deposition in the lung.

Optimising the self-assembly of siRNA loaded PEG-PCL-lPEI nano-carriers employing different preparation techniques

Amphiphilic cationic block copolymers consisting of poly(ethylene glycol), poly(ε-caprolactone) and poly(ethylene imine) spontaneously assemble to nano-sized particulate carriers, which can be utilised for complexation of nucleic acids (small-interfering RNA), representing a multifunctional vector system, designed for drug and gene delivery. Apart from polymer design and charge ratio, a more homogeneous complexation could lead to a more uniform charge distribution, subsequently increasing colloidal stability, RNA protection and consequently transfection efficiency. Microfluidic mixing techniques, bringing cationic polymer and nucleic acid together at a constant ratio during the entire mixing process, have the potential for a gentler complexation. In the present study carriers were prepared by a solvent displacement technique. In a first step complex size for addition of RNA during (addition to the aqueous or the organic phase) or after (classical pipetting or microfluidic mixing) carrier assembly was determined by dynamic light scattering. Suitable N/P ratios have previously been selected by measuring size and ζ-potential as a function of N/P. Subsequently, for the most promising techniques (loading after assembly), colloidal stability, the ability to protect RNA as well as transfection efficiency in vitro were compared. Finally, parameters for the superior microfluidic mixing process were optimised with the help of a central composite design. Generally, gentler loading leads to more homogeneous complexes. Hence, possibly due to a more consistent surface coating, loading after carrier assembly resulted in less aggregation. In comparison to bulk mixing, microfluidic assembly exhibited smaller diameters (179±11 vs. 230±97nm), less heterogeneity (PDI=0.205±0.028 vs. 0.353±0.161), enhanced RNA protection (RNA recovery=30.6±1.0 vs. 15.4±1.4%) as well as increased transfection performance (34.8±1.5 vs. 24.5±2.2% knockdown). Therefore, microfluidic complexation represents a reproducible alternative for formulating gene delivery carriers with superior colloidal stability, RNA protection and transfection efficiency.

Biophysical inhibition of synthetic vs. naturally-derived pulmonary surfactant preparations by polymeric nanoparticles.

Reasonable suspicion has accumulated that inhaled nano-scale particulate matter influences the biophysical function of the pulmonary surfactant system. Hence, it is evident to provide novel insights into the extent and mechanisms of nanoparticle-surfactant interactions in order to facilitate the fabrication of safe nanomedicines suitable for pulmonary applications. Negatively- and positively-charged poly(styrene) nanoparticles (diameters of ~100nm) served as model carriers. Nanoparticles were incubated with several synthetic and naturally-derived pulmonary surfactants to characterize the sensitivity of each preparation to biophysical inactivation. Changes in surface properties (i.e. adsorption and dynamic surface tension behavior) were monitored in a pulsating bubble surfactometer. Both nanoparticle formulations revealed a dose-dependent influence on the biophysical behavior of all investigated pulmonary surfactants. However, the surfactant sensitivity towards inhibition depended on both the carrier type, where negatively-charged nanoparticles showed increased inactivation potency compared to their positively-charged counterparts, and surfactant composition. Among the surfactants tested, synthetic mixtures (i.e. phospholipids, phospholipids supplemented with surfactant protein B, and Venticute®) were more susceptible to surface-activity inhibition as the more complex naturally-derived preparations (i.e. Alveofact® and large surfactant aggregates isolated from rabbit bronchoalveolar lavage fluid). Overall, nanoparticle characteristics and surfactant constitution both influence the extent of biophysical inhibition of pulmonary surfactants.