Flow Cytometry-Based Cell Type-Specific Assessment of Target Regulation by Pulmonary siRNA Delivery.

Pulmonary siRNA delivery has attracted strong interest and has been reported to successfully mediate target gene knockdown in a number of disease models. However, the nature of the epithelial cells that eventually take up siRNA and the question if other lung cell types may also be transfected has so far been neglected. Therefore, we describe here a flow cytometry-based method using transgenic enhanced green fluorescence protein (EGFP) expressing mice for the differentiation of transfected lung cell populations based on their antigen expression.

Formulation and process considerations for the design of sildenafil-loaded polymeric microparticles by vibrational spray-drying.

CONTEXT AND OBJECTIVE: The current study reports the preparation and characterization of sildenafil-loaded poly(lactide-co-glycolide) (PLGA)-based microparticles (MPs) by means of vibrational spray-drying. Emphasis was placed on relevant formulation and process parameters with influence on the properties of obtained powders. Materials and methods, results and discussion: The solid state solubility of sildenafil in spray-dried PLGA-MPs amounted to 17 wt.%. Thus, a drug loading below and above the determined solubility limit resulted in solid solutions and phase separation (i.e. solid dispersions), respectively. Furthermore, interactions between sildenafil and the PLGA matrix were observed for the spray-dried MPs. Optimization of spray-drying conditions allowed for a fabrication of defined MPs (size range of ∼4-8 µm) displaying a high sildenafil encapsulation efficiency (>90%) and sustained sildenafil release (from ∼4 to >12 h). The individual drug release rates from the spray-dried formulations were mainly a function of the drug loading, applied polymer and MP size. Finally, a scale-up of the preparation process did not result in a relevant change of the physicochemical and in vitro drug release properties of the prepared powders. CONCLUSION: Identification of relevant formulation and spray-drying parameters enabled the fabrication of tailored sildenafil-loaded PLGA-based MPs, which meet the needs of the individual application (e.g. controlled drug delivery to the lungs).

Influence of amine-modified poly(vinyl alcohol)s on vibrating-membrane nebulizer performance and lung toxicity.

A suitable aerosol droplet size and formulation output rate is essential for the therapy of lung diseases under application of nebulizers. The current study investigated the potential of amine-modified poly(vinyl alcohol)s as excipients for inhalation delivery. A change of conductivity (effective at <0.1mg/ml) and viscosity (effective at >0.1mg/ml) of samples that were supplemented with charge-modified polymers had a significant influence on the generated droplet size (shift from ~8 to ~4 µm) and formulation throughput rate (shift from ~0.2 to ~1.0 g/min), where polymers with a higher amine density (and molecular weight) showed an elevated activity. Biocompatibility assessment of polymers in A549 cells and an isolated lung model resulted in cell lysis and lung edema formation dependent on the type (degree of amine substitution) and dose of polymer applied. Suitable compositions and concentrations of amine-modified poly(vinyl alcohol)s were identified with respect to an optimized nebulizer performance and acceptable biocompatibility. Charge-modified polymers represent novel excipients with potential to improve inhalation therapy.

Potential of the isolated lung technique for the examination of sildenafil absorption from lung-delivered poly(lactide-co-glycolide) microparticles.

Herein, we challenged the isolated lung (IL) technique to discriminate the performance of lung-delivered polymeric microparticles (MPs) having distinct drug release rates. For this purpose, sildenafil-loaded poly(lactide-co-glycolide) MPs were administered to the airspace of an IL model and the drug absorption profile was monitored. MPs (particle size of ~5µm) composed of PLGA of lower molecular weight (and glass transition temperature) manifested in the most rapid in vitro drug release (half-times ranging from <15 to ~200min). Moreover, microencapsulation resulted in a delayed sildenafil transfer over the air/perfusate barrier (half-times ranging from <5 to ~230min), where the actual ex vivo absorption profile depended on the release behavior of the utilized formulation. Finally, the obtained in vitro and ex vivo results were tested for level C, B and A correlations. The plotted data showed good agreement (R(2)>0.96) and the slopes of the resulting lines of regression (i.e., 0.80-0.85) indicated a slightly elevated in vitro drug release behavior. Overall, the IL model was able to differentiate between distinct microparticulate formulations and is, therefore, a valuable technique for early testing of potential inhalable controlled release medications.

Prolonged vasodilatory response to nanoencapsulated sildenafil in pulmonary hypertension.

Direct vasodilator delivery to the airways enables a selective therapy of pulmonary hypertension (PH). However, short-term effects of the applied medication require multiple daily inhalations. Controlled release formulations (polymeric nanomedicines) offer the potential of prolonging drug effects within the respiratory tract, thereby reducing the number of necessary inhalations. In the model of U46619-elicited PH, sildenafil and two sildenafil-loaded polymeric submicron particle formulations were evaluated for their pharmacodynamic and pharmacokinetic characteristics and acute tolerability. Lung-delivered sildenafil caused a selective dose-dependent decline of the pulmonary arterial pressure and vascular resistance. Compared to the transient pharmacodynamic effect observed for sildenafil, the same dose of nanoencapsulated sildenafil resulted in prolongation, but not augmentation, of the pulmonary vasodilatation. An extended pharmacokinetic profile was observed for nanoencapsulated sildenafil, and nanomedicines revealed no acute toxicity. The amplification of pulmonary vasodilatory response caused by nanoencapsulation of sildenafil offers an intriguing approach to ameliorate the therapy of PH. From the Clinical Editor: Pulmonary hypertension usually results in right heart failure long term. Current medical therapy includes the use of potent vasodilators such as sildenafil. In this article, the authors investigated the use of nanoencapsulated formulation for sustained delivery via inhalation route. An extended pharmacokinetic profile was seen for this nanoformulation with little side effects. It is hoped that clinical application of this would come to fruition soon.

Efficient and Tumor Targeted siRNA Delivery by Polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol).

Efficient delivery of functional nucleic acids into specific cells or tissues is still a challenge for gene therapy and largely depends on targeted delivery strategies. The folate receptor (FR) is known to be overexpressed extracellularly on a variety of human cancers and is therefore an outstanding gate for tumor-targeted Trojan horse-like delivery of therapeutics. In this study, an amphiphilic and biodegradable ternary copolymer conjugated with folate as ligand, polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol)-folate (PEI-PCL-PEG-Fol) was synthesized and evaluated for targeted siRNA delivery via folate-FR recognition. The amphiphilic character of similar polymers was shown previously to support endosomal release of endocytosed nanocarriers and to promote formation of long circulating micelles. The obtained PEI-PCL-PEG-Fol exhibited less cytotoxicity in comparison with the corresponding ternary copolymer without folate (PEI-PCL-PEG) and with unmodified PEI25kDa. Stable micelle-like polyplexes with hydrodynamic diameters about 100 nm were found to have a zeta potential of +8.6 mV, which was lower than that of micelleplexes without folate-conjugation (+13-16 mV). Nonetheless, increased cellular uptake and in vitro gene knockdown of PEI-PCL-PEG-Fol/siRNA micelleplexes were observed in SKOV-3 cells, an FR overexpressing cell line, in comparison with the nonfolate-conjugated ones. Moreover, PEI-PCL-PEG-Fol/siRNA micelleplexes exhibited excellent stability in vivo during the analysis of 120 min and a longer circulation half life than hyPEI25kDa/siRNA polyplexes. Most interestingly, the targeted delivery system yielded 17% deposition of the i.v. injected siRNA per gram in the tumor after 24 h due to the effective folate targeting and the prolonged circulation.

Modified vibrating-mesh nozzles for advanced spray-drying applications.

This work describes uniform polymer coatings allowing for an adjustment of the orifice dimension of vibrating-mesh nozzles and therefore, size of emerging formulation droplets and dried particles, which is of general interest for diverse spray-drying applications. Chemical vapor deposition of poly(p-xylylene) (PPXN) on aperture templates of the B-90 spray-dryer (orifice diameters: ∼4.0µm) caused a reduction of the opening cross-sections of ∼50%. Thus, a more efficient formulation atomization was observed (finer droplets). Likewise, application of PPXN-coated, rather than plain nozzles, resulted in significantly smaller (particle diameter: 1.3 vs. 3.6µm) and narrower distributed (span: ∼1.4 vs. ∼1.8) sildenafil-loaded poly(lactide-co-glycolide) microparticles. Prediction of the size of spray-dried microparticles using the size results of atomized droplets ("residual core method") was shown to be in agreement with the observed values. Formulations prepared with plain and PPXN-coated nozzles exhibited a sustained sildenafil release profile with mean dissolution times of ∼1.5 and ∼4.0h, respectively. Regardless of the starting aperture template, any desired orifice dimension and therefore, dried particle size could be achieved by generating adequate polymer deposits.

Biodegradable capsules as non-viral vectors for in vitro delivery of PEI/siRNA polyplexes for efficient gene silencing.

The efficiency of siRNA delivery is demonstrated to be improved by encapsulating the siRNA within a non-viral carrier based on layer-by-layer assembly of oppositely charged biodegradable and biocompatible polyelectrolytes. In comparison to other non-viral delivery vehicles such as polycation-based complexes, a smaller amount of siRNA was necessary to produce in vitro gene silencing as early as 20-30 h after incubation. Colloidal carriers based on assembled biodegradable polyelectrolytes offer several advantages, such as efficient intracellular delivery after endocytosis followed by release to the cytosol, as well as protection of the siRNA, which is crucial for its therapeutic activity.

siRNA delivery to the lung: what's new?

RNA interference (RNAi) has been thought of as the general answer to many unmet medical needs. After the first success stories, it soon became obvious that short interfering RNA (siRNA) is not suitable for systemic administration due to its poor pharmacokinetics. Therefore local administration routes have been adopted for more successful in vivo RNAi. This paper reviews nucleic acid modifications, nanocarrier chemistry, animal models used in successful pulmonary siRNA delivery, as well as clinical translation approaches. We summarize what has been published recently and conclude with the potential problems that may still hamper the efficient clinical application of RNAi in the lung.

Quo vadis polyplex?

Polymeric siRNA delivery has been an important field for the Journal of Controlled Release since the early 2000s. With currently about 1600 publications on this topic that can be found in Scopus, we have to ask ourselves why the clinical translation has been so slow. In this review we address the importance of in vivo studies, the problem of biocompatibility of the carrier systems, intracellular entrapment and endosomal release, as well as stability issues in the presence of serum. Most of all, we ask the question: Quo vadis? Where do polymeric carriers go inside the cell and inside the body and how does this affect repeated administration? We show that the gap of knowledge on polymer biodistribution and excretion has not been closed yet and needs to be addressed to develop safe RNAi therapeutics.

Functionalized polyglycerol amine nanogels as nanocarriers for DNA.

Polyglycerol based nanogels (nPG) can function as cellular delivery systems. These nPGs are synthesized with different amine densities (nPG amines) by acid-catalyzed epoxide-opening polymerization using a mini-emulsion approach and surface modification. All the synthesized nanogels are characterized by NMR, dynamic light scattering, and ζ-potential, showing slightly positive surface charge and a homogeneous size of ≈100 nm. The use of these systems for delivery applications is demonstrated with regard to polyplex formation, cytotoxicity, and cellular uptake studies. It is depicted that the CE50 value of the high loaded nPG amines is eight times higher than the low loaded ones. The influence of the amine loading percentage on the nanogel and the effects of polyvalency in these architecture is discussed.

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.

Polymer nanoparticle-based controlled pulmonary drug delivery.

The development of novel formulations for controlled pulmonary drug delivery purposes has gained remarkable interest in medicine. Although nanomedicine represents attractive concepts for the treatment of numerous systemic diseases, scant information is available on the controlled drug release characteristics of colloidal formulations following lung administration, which might be attributed to the lack of methods to follow their absorption and distribution behavior in the pulmonary environment.In this chapter, we describe the methods of preparation and characterization of drug-loaded polymeric nanoparticles prepared from biodegradable charge-modified branched polyesters, aerosolization of the nanosuspensions using a vibrating-mesh nebulizer, and evaluation of the pulmonary pharmacokinetics (i.e., absorption and distribution characteristics) of the nanoscale drug delivery vehicles following aerosol delivery to the airspace of an isolated lung model. The disclosed methodology may contribute to the design of advanced colloids for the treatment of respiratory disorders.

Feasibility of macrophage mediated on-demand drug release from surface eroding poly(ethylene carbonate).

Macrophage induced surface degradation of poly(ethylene carbonate) (PEC) was investigated under in vitro conditions. Degradation of PEC with the MW of 41 kDa (PEC41) was slower than that of PEC with the MW of 200 kDa (PEC200). In terms of macrophage mediated drug release from PEC matrix, in cell-free medium, less than 1% of levofloxacin was released from both PEC samples in 10 days, while more than 60 and 20% of the drug, levofloxacin, can be detected in medium with macrophages from PEC200 and PEC41 films, respectively. This work indicated that on-demand drug delivery induced by macrophages can be achieved with PEC polymer.

Reflectance infrared spectroscopy for in-line monitoring of nicotine during a coating process for an oral thin film.

A process analytical method using reflectance infrared spectrometry was developed for the in-line monitoring of the amount of the active pharmaceutical ingredient (API) nicotine during a coating process for an oral thin film (OTF). In-line measurements were made using a reflectance infrared (RI) sensor positioned after the last drying zone of the coating line. Real-time spectra from the coating process were used for modelling the nicotine content. Partial least squares (PLS1) calibration models with different data pre-treatments were generated. The calibration model with the most comparable standard error of calibration (SEC) and the standard error of cross validation (SECV) was selected for an external validation run on the production coating line with an independent laminate. Good correlations could be obtained between values estimated from the reflectance infrared data and the reference HPLC test method, respectively. With in-line measurements it was possible to allow real-time adjustments during the production process to keep product specifications within predefined limits hence avoiding loss of material and batch.

In situ forming parenteral depot systems based on poly(ethylene carbonate): effect of polymer molecular weight on model protein release.

The objective of this study was to investigate the effect of molecular weight (MW) on the drug release from poly(ethylene carbonate) (PEC) based surface-eroding in situ forming depots (ISFD). In phosphate buffered saline (PBS) pH 7.4, 63.7% of bovine serum albumin BSA was released from high MW PEC of 200 kDa (PEC200) in DMSO (15%, w/w) in 2 days, while during the same time period, the release of BSA from PEC41 samples was only 22.5%. At higher concentrations of PEC41 (25%, w/w), the initial burst was further reduced, and even after 6 days, only 16.3% was released. Compared to depots based on PEC200, there was lower rate of solvent release, slower phase inversion, and a denser surface in PEC41 samples. An expansion in size of PEC41 depots suggested that the polymer barrier of PEC41 impeded the diffusion of solvent out of the samples effectively. In conclusion, the initial burst of protein from ISFD of PEC41 was significantly reduced, which would be a promising candidate as polymeric carrier.

Enzyme-responsive surface erosion of poly(ethylene carbonate) for controlled drug release.

Cholesterol esterase (CE) induced surface erosion of poly(ethylene carbonate) (PEC) and drug release from PEC under mild physiological environment was investigated. The degradation process was monitored by changes of mass and molecular weight (MW) and surface morphology of polymer films. During the whole period of degradation, MW of PEC was unchanged. Water uptake of the polymer was only 2.8% and 0.2% for PEC with the MW of 200 kDa (PEC200) and PEC with the MW of 41 kDa (PEC41), respectively. Degradation of less hydrophilic PEC41 with higher density was slower than that of PEC200. By this mechanism, CE-responsive drug in vitro release from PEC in situ forming depots (ISFD) was conducted successfully. As expected, less bovine serum albumin (BSA) was released from PEC41 compared with that of PEC200 in the same time period. In conclusion, this work enabled the in vitro drug release evaluation of existing PEC devices and implied a new candidate for the development of enzyme-responsive systems.

Ex vivo human trabecular bone model for biocompatibility evaluation of calcium phosphate composites modified with spray dried biodegradable microspheres.

Our aim was to study the suitability of the ex-vivo human trabecular bone bioreactor ZetOS to test the biocompatibility of calcium phosphate bone cement composites modified with spray dried, drug loaded microspheres. We hypothesized, that this bone bioreactor could be a promising alternative to in vivo assessment of biocompatibility in living human bone over a defined time period. Composites consisting of tetracycline loaded poly(lactic-co-glycolic acid) microspheres and calcium phosphate bone cement, were inserted into in vitro cultured human femora head trabecular bone and incubated over 30 days at 37°C in the incubation system. Different biocompatibility parameters, such as lactate dehydrogenase activity, alkaline phosphatase release and the expression of relevant cytokines, IL-1ß, IL-6, and TNF-α, were measured in the incubation medium. No significant differences in alkaline phosphatase, osteocalcin, and lactate dehydrogenase activity were measured compared to control samples. Tetracycline was released from the microspheres, delivered and incorporated into newly formed bone. In this study we demonstrated that ex vivo biocompatibility testing using human trabecular bone in a bioreactor is a potential alternative to animal experiments since bone metabolism is still maintained in a physiological environment ex vivo.

In situ forming nimodipine depot system based on microparticles for the treatment of posthemorrhagic cerebral vasospasm.

The present study was conducted to examine the feasibility of nimodipine-loaded PLGA microparticles suspended in Tisseel fibrin sealant as an in situ forming depot system. This device locally placed can be used for the treatment of vasospasm after a subarachnoid hemorrhage. Microparticles were prepared via spray-drying by using the vibration mesh spray technology of Nano Spray Dryer B-90. Spherically shaped microparticles with different loadings and high encapsulation efficiencies of 93.3-97.8% were obtained. Depending on nimodipine loading (10-40%), the particle diameter ranged from 1.9 ± 1.2 µm to 2.4 ± 1.3 µm. Thermal analyses using DSC revealed that nimodipine is dissolved in the PLGA matrix. Also, fluorescent dye loaded microparticles were encapsulated in Tisseel to examine the homogeneity of particles. 3D-pictures of the in situ forming devices displayed uniform particle homogeneity in the sealant matrix. Drug release was examined by fluorescence spectrophotometry which demonstrated a drug release proportional to the square root of time. A prolonged drug release of 19.5h was demonstrated under in vitro conditions. Overall, the nimodipine in situ forming device could be a promising candidate for the local treatment of vasospasm after a subarachnoid hemorrhage.

The use of isothermal titration calorimetry and molecular dynamics to show variability in DNA transfection performance.

The mechanism causing variability in DNA transfection efficacy for low-molecular-weight pDMAEMA (poly(2-(dimethylamino)ethyl methacrylate) and pDMAEMA-b-pHEMA (poly(2-(dimethyl amino)ethylmethacrylate)-block-poly(2-hydroxyl methacrylate)) has so far remained unclear, apart from the evidence of beneficial effects of the pHEMA grafting. This study has explicitly characterized the electrostatically driven self-assembly process of linear polymethacrylate polymers with DNA-generating nanocarriers for efficient gene transfection. Isothermal titration calorimetry (ITC) showed clear differences in binding-heat profiles of homo-polycationic and pHEMA grafted polymers with DNA. Polyethylene imine, a branched polycationic polymer of 25kDa with high transfection potential that has previously been successfully used in transfection experiments, demonstrated a heat flow profile that was partly identical to pDMAEMA-b-pHEMA. Computational molecular dynamics (MD) simulated the folding process of polymer in water from a linear to a coiled state: homo-pDMAEMA and pHEMA grafts reduced their overall positive charge accessibility upon folding, down to 45% and 63%, respectively. The homo-pDMAEMA formed the globular conformation more preferably than pHEMA grafts, thus impeding electrostatic interaction with DNA. These findings substantiate the known disadvantage of low-molecular-weight linear polymers compared to higher-molecular-weight polymers in transfection performance; here we have disclosed the ability of a non-cationic chain elongation to be beneficial for the self-assembly process. The combination of MD and ITC has proved to be a suitable approach for carrier-payload interaction studies and may be used to predict the efficacy of a polymer as a nanocarrier from the flexibility of its structure.