Focused ion beam-scanning electron microscopy provides novel insights of drug delivery phenomena.

Scanning electron microscopy (SEM) has long been a standard tool for morphological analyses, providing sub micrometer resolution of pharmaceutical formulations. However, analysis of internal morphologies of such formulations can often be biased due to the introduction of artifacts that originate from sample preparation. A recent advancement in SEM, is the focused ion beam scanning electron microscopy (FIB-SEM). This technique uses a focused ion beam (FIB) to remove material with nanometer precision, to provide virtually sample-independent access to sub-surface structures. The FIB can be combined with SEM imaging capabilities within the same instrumentation. As a powerful analytical tool, electron microscopy and FIB-milling are performed sequentially to produce high-resolution 3D models of structural peculiarities of diverse drug delivery systems or their behavior in a biological environment, i.e. intracellular or -tissue distribution. This review paper briefly describes the technical background of the method, outlines a wide array of potential uses within the drug delivery field, and focuses on intracellular transport where high-resolution images are an essential tool for mechanistical insights.

(Solvato-) polymorphism of formulations of rifampicin for pulmonary drug delivery prepared using a crystallization/spray drying process.

Rifampicin is an antibiotic used in tuberculosis therapy showing extensive (solvato-) polymorphism. Per oral administration of high doses is recommended, but application as dry powder for inhalation at the site of infection being the lungs, is desirable. Recrystallization from ethanol and consecutive spray drying is reported to yield a rifampicin dihydrate with suitable aerosol performance and stability. Nevertheless, the origin of water in the crystal remained unclear and demanded further investigation so to clarify its solid state throughout manufacture and storage. The present study reports the relationship of (solvato-) polymorphs occurring during manufacture and storage of samples recrystallized and spray dried from ethanol, methanol and water and it was concluded that processes involving a recrystallization from EtOH and MeOH produce particles of a common isostructural group of channel solvates. Samples recrystallized and spray dried from water were identified as members of another isostructural group, which was already characterized in literature. As a second aim, aerosol performance and storage stability of the formulations were investigated and all samples showed stable aerosol performance. Chemical stability of samples spray dried from ethanol was found suitable over a period of six months, whereas samples spray dried from methanol or water showed significant degradation.

Inhalable formulations of rifampicin by spray drying of supersaturated aqueous solutions.

Tuberculosis is still one of the leading causes of death from a single infectious agent (i.e. Mycobacterium tuberculosis). First line therapy includes per oral administration of high doses of rifampicin over several months and is often times accompanied by the occurrence of unwanted side effects that might limit the patient's adherence to the therapy. Thus, local antibiotic treatment at the site of infection i.e. the lungs is desirable. Amongst other approaches, spray drying of solutions of rifampicin has been shown as suitable method to produce respirable dry powders. In this work, we present inhalable formulations manufactured via spray drying of aqueous solutions of rifampicin. Powders manufactured were characterized for their aerodynamic and solid state properties, as well as their physical and chemical stability. The main focus of this study was to investigate the mechanism of particle formation using an acoustic levitator. Fine particle fractions of the test formulations ranged from 80 to 89% whereas a reference formulation (a spray dried isopropyl alcoholic solution of rifampicin) showed a lower fine particle fraction of 37%. Acoustic levitator and surface tension experiments showed that interfacial properties of rifampicin lead to early crust formation upon drying of the droplets, which eventually decoupled from the liquid core and formed highly collapsed, low apparent density powders with excellent aerosol properties.

Cascade Impactor Performance of Commercial pMDI Formulations Using Modified Induction Ports.

The induction port (IP) for aerosol analysis with the Next Generation Pharmaceutical Impactor as monographed in the United States and European pharmacopoeia (USPIP) lacks physiological relevance, which, amongst other reasons, has been identified as critical for the predictability of in vitro aerosol data to lung deposition observed in vivo. In this publication, we report the impact of replacing the USPIP with two modified induction ports, which were designed based around geometries derived from a computer tomographic scan of a human trachea and the distal section of the USPIP. Test formulations were selected on the basis of availability of in vivo lung deposition data so that results obtained in vitro could be evaluated for their predictability. All formulations assessed showed increased deposition in the modified induction ports, and different mechanisms of particle deposition have been identified. In vitro predictions of the lung deposition were found to correlate well with the in vivo observations reported using the modified induction ports. Furthermore, the quality of the correlation was found superior to the one achieved with the USPIP with an average deviation of the predicted from observed values (n = 10) of 6 ± 4, 12 ± 6, and 16 ± 6% for the modified induction ports (mIP and mIPext) and the USPIP, respectively, when using a fine particle fraction (FPF) cutoff value of 5 µm. Using a FPF cutoff value of 3 µm yielded a more accurate in vitro-in vivo correlation with an average deviation of the predicted from observed values of 5 ± 4, 7 ± 5, and 8 ± 4% for the mIP, mIPext, and USPIP, respectively. For both FPF size cutoff values, the mIP yielded the most accurate in vitro-in vivo correlation.

Inhalable dry powders of rifampicin highlighting potential and drawbacks in formulation development for experimental tuberculosis aerosol therapy.

Introduction: Recently, tuberculosis was reported as the leading cause of death from a single infectious agent. Standard therapy includes administration of four first-line antibiotics, i.e. rifampicin, isoniazid, ethambutol, and pyrazinamide over a period of at least 26 weeks, which in case of rifampicin oftentimes is accompanied by unwanted side effects and variable bioavailability that compromise a positive therapeutic outcome. As the main site of infection is the lungs, it is desirable to develop a therapeutic formulation to be administered via the pulmonary route.Areas covered: This work presents a literature review on studies investigating inhalable dry powder formulations including rifampicin in the context of an experimental tuberculosis therapy, with a special focus on aerosol performance.Expert opinion: It was found that formulation approaches involving different strategies and functional excipients are under investigation but as of now, no formulation has managed to leap into commercial clinical testing. Reasons for this might not primarily be associated with a lack of suitable candidates, but amongst others a lack of suitable in vitro models to assess the efficacy, therapeutic benefit, and cost-effectiveness of the candidate formulations.

Investigating cascade impactor performance using a modified 3D printed induction port.

Based on a computer tomographic scan of a human trachea, a modified induction port (mIP), for use with the Next Generation Cascade Impactor, was manufactured using 3D printing technology. Standard United States Pharmacopoeia IP (USPIP) was compared to the mIP and a 3D printed version of the USPIP (USP3DIP) by analyzing different types of commercial salbutamol formulations for inhalation. Increased retention of particles in the mIP was found analyzing a pMDI formulation, leading to a decrease in the FPF from 28.8±2.0% to 14.2±1.2%, which correlates better to in vivo deposition data from literature. Increased deposition was found to be based on geometrical factors only. The impact of surface related effects was investigated by (a) comparing results obtained with the USPIP and USP3DIP (all formulations) and (b) generating another model IP (USP3DSEIP) with a surface area equivalent to the mIP but maintaining the geometry of the USPIP (pMDI only). USPIP, USP3DIP, and USP3DSEIP were found to perform equivalently. The impact of different geometries on airflow velocities in the USPIP and mIP was assessed using a computational fluid dynamics (CFD) model. Conclusively, this study shows that replacing the USP IP by the mIP can provide additional information in formulation assessment and in in vitro/in vivo correlation, when applied on pMDI formulations.

Nebulized coenzyme Q10 nanosuspensions: A versatile approach for pulmonary antioxidant therapy.

Coenzyme Q10 (CoQ10) is an antioxidant substance indicated as a dietary supplement which has been proposed as adjuvant in the treatment of cardiovascular disorders and cancer for its protective and immunostimulating activities. The aim of this work was the production by high-pressure homogenization, characterization and stability investigation of three different CoQ10 nanosuspensions designed to be administered to the lungs by nebulization. Three surfactants, i.e. lecithin, PEG32 stearate and vitamin-E TPGS, were selected to stabilize CoQ10 formulations. Preparations were identified as nanosuspensions (particle size in the range 35-60nm): the smallest particles were obtained with vitamin-E TPGS and denoted a core-shell structure. The CoQ10 delivered from a commercial air-jet nebulizer was in all the cases around 30% of the loaded dose. The nanosuspension containing PEG32 stearate presented the highest respirable fraction (70.6%) and smallest MMAD (3.02µm). Stability tests showed that the most stable formulation, after 90days, was the one containing vitamin-E TPGS, followed by the CoQ10-lecithin formulation. Interestingly, those formulations were demonstrated to be suitable also for nebulizers using other mechanisms of aerosol production such as ultrasound and vibrating mesh nebulizers. Studies focused on in vitro cellular toxicity of the formulations and their single components using A549 human lung cells showed no obvious cytotoxicity for the formulations containing lecithin and PEG 32 stearate. Vitamin-E TPGS alone was shown to be able to damage the plasma membrane, nevertheless, cell damage was decreased when vitamin-E TPGS was present in the formulation with CoQ10.

Challenges and Future Prospects for the Delivery of Biologics: Oral Mucosal, Pulmonary, and Transdermal Routes.

Biologic products are large molecules such as proteins, peptides, nucleic acids, etc., which have already produced many new drugs for clinical use in the last decades. Due to the inherent challenges faced by biologics after oral administration (e.g., acidic stomach pH, digestive enzymes, and limited permeation through the gastrointestinal tract), several alternative routes of administration have been investigated to enable sufficient drug absorption into systemic circulation. This review describes the buccal, sublingual, pulmonary, and transdermal routes of administration for biologics with relevant details of the respective barriers. While all these routes avoid transit through the gastrointestinal tract, each has its own strengths and weaknesses that may be optimal for specific classes of compounds. Buccal and sublingual delivery enable rapid drug uptake through a relatively permeable barrier but are limited by small epithelial surface area, stratified epithelia, and the practical complexities of maintaining a drug delivery system in the mouth. Pulmonary delivery accesses the highly permeable and large surface area of the alveolar epithelium but must overcome the complexities of safe and effective delivery to the alveoli deep in the lung. Transdermal delivery offers convenient access to the body for extended-release delivery via the skin surface but requires the use of novel devices and formulations to overcome the skin's formidable stratum corneum barrier. New technologies and strategies advanced to overcome these challenges are reviewed, and critical views in future developments of each route are given.

Development of aqueous dispersions of Coenzyme Q10 for pulmonary delivery and the dynamics of active vibrating-mesh aerosolization.

Coenzyme Q10 (CoQ10) is a poorly-water soluble compound that is being investigated for the treatment of carcinomas. The aim of this study was to investigate the feasibility of preparing phospholipid-stabilized dispersions of the anticancer agent for continuous pulmonary delivery using a vibrating-mesh nebulizer. We determined the physicochemical properties (drug particle size distribution in dispersion, zeta potential, surface tension, and rheology) and compared the aerosolization profiles (nebulization performance, aerodynamic drug deposition and total emitted dose) of dispersions of CoQ10 prepared with different phospholipids. The hydrodynamic sizes of the drug particles in dispersion were primarily in the submicron range, but formulations with drug particle sizes greater than the aperture size of the nebulizer presented superior aerosolization profiles. At high shear rates, certain formulations presented increased shear-thickening behavior, which was connected to a decrease in mass and drug output over time, and with decreased aerodynamic and geometric sizes. Other formulations presented shear-thinning behavior and showed similarly high drug depositions. In this investigation, we found that dispersed formulations of CoQ10 presented different in vitro performance for pulmonary delivery based on their rheological behavior. In conclusion, this characterization methodology provides an innovative approach to screen formulations of poorly-water soluble compounds for continuous (no clogging) active vibrating-mesh nebulization.

The function and performance of aqueous aerosol devices for inhalation therapy.

OBJECTIVES: In this review paper, we explore the interaction between the functioning mechanism of different nebulizers and the physicochemical properties of the formulations for several types of devices, namely jet, ultrasonic and vibrating-mesh nebulizers; colliding and extruded jets; electrohydrodynamic mechanism; surface acoustic wave microfluidic atomization; and capillary aerosol generation. KEY FINDINGS: Nebulization is the transformation of bulk liquids into droplets. For inhalation therapy, nebulizers are widely used to aerosolize aqueous systems, such as solutions and suspensions. The interaction between the functioning mechanism of different nebulizers and the physicochemical properties of the formulations plays a significant role in the performance of aerosol generation appropriate for pulmonary delivery. Certain types of nebulizers have consistently presented temperature increase during the nebulization event. Therefore, careful consideration should be given when evaluating thermo-labile drugs, such as protein therapeutics. We also present the general approaches for characterization of nebulizer formulations. SUMMARY: In conclusion, the interplay between the dosage form (i.e. aqueous systems) and the specific type of device for aerosol generation determines the effectiveness of drug delivery in nebulization therapies, thus requiring extensive understanding and characterization.

Devices for dry powder drug delivery to the lung.

Dry powder inhalers (DPIs) are an important and increasingly investigated method of modern therapy for a growing number of respiratory diseases. DPIs are a promising option for certain patient populations, and may help to overcome several limitations that are associated with other types of inhalation delivery systems (e.g., accuracy and reproducibility of the dose delivered, compliance and adherence issues, or environmental aspects). Today, more than 20 different dry powder inhalers are on the market to deliver active pharmaceutical ingredients (APIs) for local and/or systemic therapy. Depending on the mechanism of deagglomeration, aerosolization, dose metering accuracy, and the interpatient variability, dry powder inhalers demonstrate varying performance levels. During development, manufacturers focus on improving aspects characteristic of their specific DPI devices, depending on the intended type of application and any particular requirements associated with it. With the wide variety of applications related to specific APIs, there exists a range of different devices with distinct features. In addition to the routinely used multi-use DPIs, several single-use disposable devices are under development or already approved. The recent introduction of disposable devices will expand the range of possible applications for use by including agents such as vaccines, analgesics, or even rescue medications. This review article discusses the performance and advantages of recently approved dry powder inhalers as well as disposable single-use inhalers that are currently under development.

Pulmonary delivery of anti-inflammatory agents.

INTRODUCTION: Respiratory infections and diseases are accompanied by or exhibit inflammation. Recent advances in nanoparticle engineering technology, together with the increased knowledge of inflammatory pathophysiology, have ignited interest in the pulmonary delivery of anti-inflammatory agents (AIAs) to achieve local treatment of pulmonary inflammatory disorders. AREAS COVERED: This review summarizes and discusses the investigated formulation approaches for the pulmonary delivery of AIAs, including: inhalation of actives as suspensions or dry powder formulations, with polymeric micro- and nano-delivery carriers, or within liposomes and lipid nanoparticles. Some recent approaches for targeting AIAs to the pulmonary endothelium have also been reviewed. The discussion focuses on finding out whether the investigated approaches were really able to achieve lung targeting and reduce the side effects associated with the systemic administration of AIAs. EXPERT OPINION: The use of the inhalation route for the pulmonary delivery of AIAs is facing several challenges. Some of the investigated formulation approaches appear to be promising in overcoming these challenges. However, in order to create products that reach patients, more therapeutically oriented studies are still needed to ensure formulation stability, in-vivo sustained release behavior, pulmonary retention, and bypassing lung clearance mechanisms.

Films loaded with insulin-coated nanoparticles (ICNP) as potential platforms for peptide buccal delivery.

The goal of this investigation was to develop films containing insulin-coated nanoparticles and evaluate their performance in vitro as potential peptide delivery systems. To incorporate insulin into the films, a new antisolvent co-precipitation fabrication process was adapted to obtain insulin-coated nanoparticles (ICNPs). The ICNPs were embedded in polymeric films containing a cationic polymethacrylate derivative (ERL) or a combination of ERL with hydroxypropyl methylcellulose (HPMC). ICNP-loaded films were characterized for morphology, mucoadhesion, and insulin release. Furthermore, in vitro insulin permeation was evaluated using a cultured tridimensional human buccal mucosa model. The antisolvent co-precipitation method was successfully adapted to obtain ICNPs with 40% (w/w) insulin load, achieving 323±8nm particles with a high zeta potential of 32.4±0.8mV, indicating good stability. High yields were obtained after manufacture and the insulin content did not decrease after one month storage. ICNP-embedded films using ERL as the polymer matrix presented excellent mucoadhesive and insulin release properties. A high permeation enhancement effect was observed for ICNP-loaded ERL films in comparison with ICNP-loaded ERL-HPMC films and a control insulin solution. ICNP-loaded ERL formulations were found to be more effective in terms of film performance and insulin permeation through the human buccal mucosa model, and thus are a promising delivery system for buccal administration of a peptide such as insulin.

Novel strategies for the buccal delivery of macromolecules.

For years now, the delivery of small molecules through the buccal mucosal route has been described in the literature, but it has only been over the past decade that investigations into macromolecule delivery via the buccal route have sharply increased. The administration of macromolecules such as proteins and peptides, antibodies, or nucleic acids by buccal administration would be greatly enhanced due to the avoidance of the gastrointestinal conditions, rapid uptake into systemic circulation, as well as the potential for controlled drug delivery. Since macromolecules are faced with a number of specific challenges related to permeation through the epithelium, several strategies have been employed historically to improve their buccal absorption and subsequent bioavailability. Several conventional strategies to improve macromolecule penetration include the use of chemical permeation enhancers, enzyme inhibitors and the use of mucoadhesive materials acting as carriers. More recent approaches include the incorporation of the macromolecule as part of nanostructured delivery systems to further enhance targeting and delivery. This review focuses on the different permeation enhancing strategies as well as formulation design that are tailored to meet the challenges of active macromolecule delivery using the buccal mucosal route of administration.

Development and characterization of phospholipid-stabilized submicron aqueous dispersions of coenzyme Q10 presenting continuous vibrating-mesh nebulization performance.

Coenzyme Q10 (CoQ10) is a poorly-water soluble compound that is being investigated for the treatment of carcinomas. The aim of this research was to develop a suitable formulation for pulmonary delivery of this anticancer agent. An appropriate selection of excipients (phospholipids) and a suitable device (Aeroneb Pro® vibrating-mesh nebulizer) were selected initially after reviewing the literature. After characterization of the bulk drug, a feasible manufacturing process was selected to obtain small particle size dispersions of CoQ10. Following selection of an appropriate process, the parameters affecting drug particle size were studied. Using LD and gravimetrical analysis, nebulization was evaluated to assess the performance of the inhalation system triad: drug-excipients-device. CoQ10 powder studied was crystalline with a melting point approximately at 51 °C and with a particle size of 30 µm. Microfluidization was found to be a suitable method to prepare submicron drug particles in aqueous dispersions. Increasing microfluidization processing to more than 50 passes did not provide further particle downsizing for both soya phosphatidylcholine (lecithin) and dipalmitoyl phosphatidylcholine (DPPC) dispersions of CoQ10, presenting Z-average values of approximately 130 and 70 nm, respectively. Nebulization performance of lecithin-stabilized CoQ10 dispersions varied according to number of passes in the microfluidizer. Formulations processed with 10 passes presented steadier nebulization over time and different rheological behavior compared to those processed with 30 or 50 passes. In conclusion, aqueous dispersions of CoQ10 were adequately produced using a microfluidizer with characteristics that were suitable for pulmonary delivery with an Aeroneb Pro® nebulizer. Furthermore, the rheology of these dispersions appeared to play a significant role in the aerosol generation from the active vibrating-mesh nebulizer used.

Protein-coated nanoparticles embedded in films as delivery platforms.

OBJECTIVES: This work aimed to evaluate the performance of nanoparticle-loaded films based on matrices of polymethacrylates and hydroxypropylmethylcellulose (HPMC) intended for delivery of macromolecules. METHODS: Lysozyme (Lys)-loaded nanoparticles were manufactured by antisolvent co-precipitation. After size, loading efficiency and stability characterization, the selected batch of particles was further formulated into films. Films were characterized for mechanical properties, mucoadhesion, Lys release and activity after manufacture. KEY FINDINGS: We found that protein-coated nanoparticles could be obtained in USP phosphate buffer pH 6.8. Particles obtained at pH 6.8 had a z-average of 347.2 nm, a zeta-potential of 21.9 mV and 99.2% remaining activity after manufacture. This formulation was further studied for its application in films for buccal delivery. Films loaded with nanoparticles that contained Eudragit RLPO (ERL) exhibited excellent mechanical and mucoadhesive properties. Due to its higher water-swelling and solubility compared with ERL, the use of HPMC allowed us to tailor the release of Lys from films. The formulation composed of equal amounts of ERL and HPMC revealed a sustained release over 4 h, with Lys remaining fully active at the end of the study. CONCLUSIONS: Mucoadhesive films containing protein-coated nanoparticles are promising carriers for the buccal delivery of proteins and peptides in a stable form.

The influence of recrystallized caffeine on water-swellable polymethacrylate mucoadhesive buccal films.

The aim of this work was to investigate the influence of particles on the properties of polymethacrylate films intended for buccal delivery. A solvent casting method was used with Eudragit RS and RL (ERS and ERL, respectively) as film-forming rate-controlling polymers, with caffeine as a water-soluble model drug. The physicochemical properties of the model films for a series of formulations with increasing concentrations of caffeine were determined in terms of morphology, mechanical and mucoadhesive properties, drug content uniformity, and drug release and associated kinetics. Typically regarded as non-mucoadhesive polymers, ERS and mainly ERL, were found to be good mucoadhesives, with ERL01 exhibiting a work of mucoadhesion (WoA) of 118.9 µJ, which was about five to six times higher than that observed for commonly used mucoadhesives such as Carbopol(®) 974P (C974P, 23.9 µJ) and polycarbophil (PCP, 17.4 µJ). The mucoadhesive force for ERL01 was found to be significantly lower yet comparable to C974P and PCP films (211.1 vs. 329.7 and 301.1 mN, respectively). Inspection of cross-sections of the films indicated that increasing the concentration of caffeine was correlated with the appearance of recrystallized agglomerates. In conclusion, caffeine agglomerates had detrimental effects in terms of mucoadhesion, mechanical properties, uniformity, and drug release at large particle sizes. ERL series of films exhibited very rapid release of caffeine while ERS series showed controlled release. Analysis of release profiles revealed that kinetics changed from a diffusion controlled to a first-order release mechanism.

A design of experiments to optimize a new manufacturing process for high activity protein-containing submicron particles.

A novel method for the manufacture of protein/peptide-containing submicron particles was developed in an attempt to provide particles with increased activity while using high energy input technologies. The method consists of antisolvent co-precipitation from an aqueous solution containing both an amino acid core material (e.g. D,L-valine), and either bovine serum albumin (BSA) or lysozyme (Lys) as model proteins. The aqueous solution was added to the organic phase by means of a nebulizer to increase the total surface area of interaction for the precipitation process. Sonication proved to be an effective method to produce small particle sizes while maintaining high activity of Lys. The use of a polysorbate or sorbitan ester derivatives as stabilizers proved to be necessary to yield submicron particles. Particles with very high yields (approximately 100%) and very high activity after manufacture (approximately 100%) could be obtained. A particle size of 439.0 nm, with a yield of 48.8% and with final remaining activity of 98.7% was obtained. By studying various factors using a design of experiments strategy (DoE) we were able to establish the critical controlling factors for this new method of manufacture.