49 research outputs found

    Asymmetric particles for pulmonary drug delivery

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    Targeted drug delivery and controlled release are current challenges in pulmonary drug delivery. The deposition pattern and clearance from deposition site are two key parameters for drug delivery carrier design. Asymmetric particles allow an increase in peripheral drug delivery compared to spherical particles and furthermore, affect particle clearance mechanisms from the lung. Therefore, the main aim of this thesis was to develop new synthesis strategies to produce well-dispersible, biocompatible, biodegradable microfibers with a variety of aspect ratios and porosities. The macrophage response to the resulting microfibers was investigated. The aerosolization properties of the resulting microfibers were examined. From the obtained results it can be concluded that: 1. A new template-assisted synthesis strategy to produce monodisperse microfibers with defined dimensions has been developed. 2. The technique has been extended to various materials and process parameters for cell testing, drug loading and aerosolization tests. 3. Microfibers were successfully taken up by macrophages, only when they were approached from the pointy end. 4. Aerosolization studies showed good dispersion properties of microfibers with relatively high fine particle fractions. In summary, this new technique may allow to produce microfibers for pulmonary drug delivery, which will lead to a better understanding of their in vivo behaviour such as mucoadhesion, macrophage interaction and deposition behaviour.Die aktuellen Herausforderungen der inhalativen Therapie sind die gezielte Wirkstoffdeposition und die kontrollierte Wirkstofffreisetzung in der Lunge. Asymmetrische Partikel haben dabei durch ihre erhöhte tiefe Lungendeposition und ihren Einfluss auf die Clearance-Mechanismen erhöhtes Interesse gefunden. Ziel dieser Arbeit war daher die Entwicklung einer neuen Herstellungsmethode, um gut vereinzelte, biokompatible, bioabbaubare Mikrofasern mit variablen Aspektverhältnissen und Porositäten zu generieren. Weiteres Ziel war die Testung der Makrophagen-Mikrofaser-Interaktion und des Aerosolisierungsverhaltens. Die gewonnenen Ergebnisse führen zu folgenden Aussagen: 1. Es wurde eine neue Methode zur Herstellung monodisperser Mikrofasern mit definierten Maßen entwickelt. 2. Mikrofasern aus diversen Materialien wurden in späteren Versuchen für Zelltests, Wirkstoffbeladung und Aerosolisierungsstudien verwendet. 3. Die Aufnahme von Mikrofasern durch Makrophagen zeigte eine Korrelation zum Faserdurchmesser, wobei diese nur vom spitzen Ende her aufgenommen wurden. 4. Aerosolisierungsstudien zeigten eine gute Dispergierung der Mikrofasern mit hohen Fine-Particle-Fractions. Die entwickelte Methode kann zu einer Optimierung der pulmonalen Wirkstoffapplikation und einem besseren Verständnis des Verhaltens asymmetrischer Partikel im Körper beitragen. Die Mukoadhesion, die Makrophagen-Interaktion und das Depositionsverhalten in der Lunge können mittels dieser Fasern weiter untersucht werden

    Developing Print Dry Powders for Pulmonary Protein Delivery

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    Pulmonary delivery is an attractive route of administration that can be used for the local delivery of therapeutics for respiratory conditions or to non-invasively deliver sufficiently low molecular weight therapeutics to systemic circulation. There is a particular interest in protein delivery, however, many respirable formulations are inefficient at delivering therapeutics to the desired region of the lungs, which precludes the development of costly biologics for inhalation. Particle engineering, a strategy that aims to rationally and precisely control particle size, shape, density, and composition, has been utilized to design high-performance dry powder aerosols that deposit efficiently and precisely in the desired area of the lungs. However, current fabrication methods offer limited control of particle geometry and impose unfavorable stresses on proteins during manufacturing. The overall goals of this dissertation were to fabricate and characterize protein-based microparticles with Particle Replication In Non-wetting Templates (PRINT) technology and engineer these particles into high-performance protein dry powder aerosols. We hypothesized that the precise control of particle geometry afforded by PRINT along with the low physical stress imparted by the process would allow for the stable incorporation of proteins into precisely engineered particles, resulting in high-performance protein dry powder aerosols. A generalizable formulation strategy to micromold a variety of proteins into precisely engineered PRINT particles was developed, and the incorporated proteins were found to retain their native structure and function. Following lyophilization into dry powders, these formulations were found to fluidize, aerosolize, and deposit with high efficiency and precision. We then expanded the formulation strategy to fabricate multiple PRINT particle shapes, which were used to explore the impact of particle shape on dry powder performance in an effort to inform and improve particle engineering strategies. Informed by the formulation development and particle shape studies, dry powder formulations of two therapeutic proteins were developed and the delivery of one formulation was demonstrated in vivo. Overall, we have demonstrated the utility of PRINT as a platform to manufacture high-performance protein dry powders and we have furthered understanding of the impact of particle shape on aerosol performance, both of which contribute to the advancement of particle engineering strategies for inhalable formulations.Doctor of Philosoph

    Statische und dynamische Magnetfelder fĂĽr die Nanopartikel-basierte zielgerichtete Wirkstofffreisetzung

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    Although medicine has made great progress in the last centuries and decades, it is still facing basic challenges that make doctors fail to efficiently and successfully treat the continuously emerging diseases and ailments due to ageing, industrialization, pollution and resulting biological mutations. In this context, the systemic chemotherapeutic treatment of cancer seems to be one of the most fitting examples for the wide gap between the usually followed medical approach and the theoretically optimal solution. Extrapolating from in vitro experiments and mouse models to humans, treating children as “miniaturized” adults when analyzing therapeutic effects, estimating drug doses based on relatively coarse processes like up scaling on weight, volume or area, and flooding the human body with drugs to solely achieve a minimal effect at the ailment site are just few examples for improvement needs in medical methods. One of the most promising approaches intended to bring more specificity and precision into the therapeutic toolbox is the directed delivery of drugs, already prophesized and described one hundred years ago by the German immunologist and Nobel Laureate in Medicine (1908) Paul Ehrlich (1854-1915) as the “magic bullet” principle. It is a visionary medical method in which active agents -such as drugs or antibodies- are guided within the human body and brought to bind directly and exclusively to their biological target. This approach was triggered and has been remarkably promoted by the introduction and continuous development of nano-sized medical systems since the 1950s, and is expected to experience a real breakthrough by the clinical validation of the so called “Magnetic Drug Targeting”. According to this technique, magnetically active nanoparticles are coated with a therapeutically active biomaterial and guided through external magnetic fields in the natural transport pathways of the body, then retained and concentrated at target sites where the biologically active load is set free. The delivered dose is augmented, side effects are lowered and the overall therapeutic efficiency is enhanced. Especially for cancer treatment, the magnetically guided drug delivery represents a huge potential. In fact, conventional chemotherapy methods are used systemically and succeed in best cases in delivering only a fractional amount of the drug to the target sites, while the rest is absorbed by the healthy tissue of the treated body. This is so inefficient that dose levels of about 50 to 100-fold those of conventional doses need to be administered to achieve cures of cancer cells (T. A. Connors 1995). As a result, blood filtering and trafficking organs, such as the liver, the kidneys, the spleen and most importantly the heart, are the direct victims of the highly toxic substances used in chemotherapy. Even the apparently more gentle approach of applying the maximum tolerated dose at defined intervals -in order to avoid toxicity- can unintentionally lead to a chemoresistance of the tumor (C. Damyanov 2009). These shortcomings of the chemical therapy further aggravate the fact that cancer is still the worldwide deadliest disease, with an upward trend. For instance, around 25 % of all registered death cases in the European Union are reported by the World Health Organization to be caused by tumors. Despite the development of advanced anti-cancer medicine, it still remains a difficult challenge to keep costs at an affordable level. For that reason, new and more efficient cancer treatment methods with higher success rates and lower side effects and costs are urgently needed and would help physicians cope with an ever ageing world population. In this work, we report improvements achieved in the understanding and control of the magnetically targeted drug delivery, mainly realized by the consideration of time issues and the investigation of dynamic magnetic fields. New approaches to assess the magnetic behavior of nanoparticles in suspensions as well as an advanced examination of the lung drug targeting and the mechanisms of cellular drug uptake after successful localized delivery represent the major achievements compiled in this manuscript. The registered improvements are an important contribution to the further development of the idea of directed therapies promoted by the emerging nanomedicine. This modern medicine is expected to provide techniques that can act on a cellular and even sub-cellular level, treating ailments with considerably more accuracy. Gradually, modern diagnostic and therapeutic techniques should elevate us slowly to the point where we can start thinking more in terms of real “regenerative” medicine. That means, we should be able to precisely and directly address pathologic tissues, save cells and organs, repair and heal them, rather than extinguish them.Mehr als hundert Jahre nach dem Tod von Paul Ehrlich, dem bedeutendsten deutschen Immunologen, verfolgt die "Nachwelt" noch mit großen Schritten eine seiner wichtigsten Visionen, die er während seiner Arbeiten zur Behandlung der Syphilis entwickelte: eine „Zauberkugel“ (magic bullet), die einen gegebenen krankmachenden Erreger gezielt abtöten kann. Ganz nach diesem noch -mehr denn je- aktuellen Prinzip, entwickeln Forscher heutzutage weltweit neue Methoden, um nicht nur Krankheitserreger, sondern auch befallene Gewebe, spezifisch zu behandeln. In den letzten Jahren entwickelte sich dadurch die Medizin von der konventionellen Anwendung, über die personalisierte Behandlung, wo die genetische Information eines jeden Patienten präventiv untersucht werden kann und die Ergebnisse zur Auswahl und Anpassung der Therapie-Art herangezogen werden, bis hin zur "Nanomedizin", einer neuen Ära der Arzneimittel-Konzipierung, -Synthese, -Dosierung und -Verabreichung, die Therapien auf zellulärer und sub-zellulärer Ebene ermöglichen sollte. Mediziner sind heutzutage weit entfernt von der Darstellung von Christian Friedrich Hebbel (18.03.1813 - 13.12.1863), dass "ein Arzt eine Aufgabe hat, als ob ein Mensch in einem dunklen Zimmer in einem Buche lesen sollte". Sie sind in der Lage, durch die Integration der Nanotechnologie im biomedizinischen Bereich, Gewebe und Zellen, die durchschnittliche Dimensionen von 10 µm haben, mit Nanosystemen im Submikrometer-Bereich zu adressieren und gezielt zu behandeln. In diesem Rahmen präsentiert sich das Magnetic Drug Targeting (MDT) als besonders wirksamer Therapie-Ansatz. Dabei werden Wirkstoff-beladene magnetische Nanopartikel über externe Magnetfelder im Körper geführt und an einem gegebenen Krankheitsort lokal angereichert. Die verabreichte Wirkdosis wird dadurch erhöht, Nebeneffekte minimiert. Besonders in der Krebsbekämpfung verspricht dieser Ansatz hohe Erfolgsquoten und eine Reduzierung der ohnehin enormen Chemo- und Radiotherapie-Kosten, die meistens einen bremsenden Effekt auf die Entwicklung und Verbreitung zahlreicher Behandlungsmethoden haben. An dieser Stelle sei daran erinnert, dass Krebs nach wie vor die weltweit wichtigste Todesursache ist, an der schätzungsweise 11.5 Millionen Weltbewohner im Jahre 2030 sterben werden, was einem Anstieg von 45% zum Jahre 2007 darstellt. Die zielgerichtete Arzneimittel-Applikation, zu Englisch "Directed Drug Delivery", soll hierfür Lösungen anbieten, die Tumore spezifisch angreifen und ausschalten können. Durch eine magnetische Lenkung und Anreicherung wird dieses Verfahren weiter optimiert. Die somit entstehende MDT-Methode eignet sich für Anwendungen in der Blutbahn, sowie in den Atemwegen von Patienten, mit entsprechenden Anpassungen. Entscheidend ist hierbei vor Allem das eingesetzte Magnetfeld, in Bezug auf Amplitude, Homogenität und Dynamik. In zahlreichen wissenschaftlichen Arbeiten, wurden bisher Erfolg versprechende Ergebnisse präsentiert, die überwiegend durch die Manipulation und Aufkonzentrierung von Nanopartikel-Wirkstoff-Komplexen mit statischen Magnetfeldern realisiert wurden. Eine hierzu komplementäre Betrachtung mit dynamischen Magnetfeldern wird in dieser Arbeit untersucht. Im Rahmen dieses Forschungsprojekts wurden Ansätze mit statischen und dynamischen Magnetfeldern zur Verbesserung des Magnetic Drug Targeting theoretisch überprüft, simulativ validiert und systemtechnisch umgesetzt. Nach einer ausführlichen Untersuchung der Nanopartikel-Eigenschaften, die den MDT-Effekt überhaupt ermöglichen und besonders beeinflussen, wurde der Anreicherungsprozess unter Magnetkraftwirkung modelliert und ein für Anwendungen in der Blutbahn optimiertes Magnetsystem simuliert, konstruiert und bei in-vivo-Versuchen eingesetzt. Dadurch konnte eine aktive und vor Allem reproduzierbare Retention von beladenen Nanopartikel-Komplexen in den Arterien und Venen der Rückenhaut einer Maus verzeichnet werden

    Development and Characterization of PRINT® Particles as Drug Delivery Vehicles in the Lung

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    The aim of this dissertation is to develop and investigate the utility of the Particle Replication in Non-wetting Templates (PRINT®) technology as a toolbox to generate precisely defined particles in shape, matrix, and surface functionality to further the understanding of particulate drug delivery to the lung. The pulmonary route of administration is of particular interest in analyzing the effects of particles due to the vast exposure of our lungs to a wide array of airborne particulates. From pollen to bacteria to diesel exhaust, the fate and physiological impacts of these particulates cause a multitude of disease states in the human body. Understanding, and even harnessing, the specific characteristics which make these airborne invaders so potent at evading or wreaking havoc on the body's defense systems will hopefully lead to the development of more safe and efficient drug delivery vehicles. Particles of varying geometries were fabricated and shape effects on macrophage internalization in vitro were investigated to explore physical particle characteristics that may impart the ability to tailor alveolar macrophage uptake for use in pulmonary therapeutics. PEG particles ranging from 80x320 nm to 6 µm in diameter were instilled into the lungs of mice and cellular uptake, residence time, and inflammatory responses in the lung were analyzed. Being able to precisely tune individual particle parameters allowed for the determination of specific characteristics that could target or de-target specific cell populations in the lung. These PRINT particles were also shown to reside in the lung out to twenty-eight days without inducing an inflammatory response which demonstrates the potential of these particles as immunologically inert drug carriers to the lung.Doctor of Philosoph

    End-of-life analysis of nanotechnology products

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    Previous research has shown that thermodynamic properties including melting point and specific heat capacity of nanomaterials may be higher than that of their corresponding bulk materials. The melting point elevation and specific heat capacity enhancement of nanomaterials may result in increased energy consumption and waste gases emission at the end-of-life (EOL) stage where the products containing nanomaterials are recycled by high temperature metal recovery (HTMR) process. In this dissertation, the effect of physical characteristics of nanomaterials, referred to as physicochemical parameters, on their melting temperature and specific heat capacity was investigated. In addition, physical, chemical, and thermodynamic properties of nanomaterials embedded inside commercially available lithium-ion (Li-ion) battery were examined by various characterization techniques including scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). Thermodynamic analysis techniques with life cycle assessment (LCA) were used to investigate the environmental impacts of nanomaterials during the EOL material recovery stage due to their unusual thermodynamic properties. As opposed to the energy analysis result, the exergy analysis showed that the chemical reactions that occur during the reduction and smelting processes are the primary sources of exergy loss. If the smelting temperature is increased to fully melt down nanomaterials with unusually high melting point, under assumptions of constant heat flux, the smelter may operate for a longer period of time resulting in substantial amount of exergy loss and carbon dioxide emission. It was also shown that the reduction process consumes larger amount of energy to raise the temperature of nanomaterials with specific heat capacity enhancement, as opposed to bulk materials. Design for environment (DFE) guideline was developed to improve process performance and risk management. Potential vulnerabilities to recycling of nanomaterials as well as recommended product design and process modifications are summarized. Finally, a novel exergy footprint was formulated as a sustainable and environmental impact metric that provides a meaningful understanding of the environmental impact of a product or a process. The consumption and flow of exergy in the US economy is defined in terms of five functional categories: materials, transportation, food, water, and direct energy carriers. To illustrate the exergy footprint calculation, the environmental impact associated with the HTMR process measured in terms of exergy loss and exergy consumption were compared to the exergy consumption at a national level

    Nanotoxicology : pulmonary toxicity studies on self-assembling rosette nanotubes

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    A growing demand for information on the human health and environmental effects of materials produced using nanotechnology has led to a new area of investigation known as nanotoxicology. Research in this field has widespread implications in facilitating the medical applications of nanomaterials but also in addressing occupational and environmental toxicity concerns. Improving our understanding of these issues also has broad appeal in the stewardship of nanotechnology and its acceptance by the public. This work represents some of the early research in burgeoning field of nanotoxicology. Using a variety of in vivo and in vitro models, as well as cellular and molecular techniques I first studied a possible role for the novel cytokine endothelial monocyte activating polypeptide-II (EMAP-II) in acute lung inflammation in rats (Chapter 2). This work demonstrated a significant increase in total EMAP-II concentration in lipopolysaccharide inflamed lungs as early as 1h post-treatment (

    Novel Drug Carriers for Pulmonary Administration Utilising a Template-Assisted Approach

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    The template technique was selected for the generation of monodisperse fibres, intended for pulmonary administration. The deposition site in the inhalation tract is strongly governed by the geometry (size and shape) of the particle, whereas the precision of targeting is linked to their homogeneity. Since conventional carrier systems are not formed within precisely defined templates, such as the track-etched membranes with cylindrical pores used herein, the geometry is less defined. Despite their largely irregular shape, conventional carriers are described as spherical. Two major benefits of fibrous shape have been identified for pulmonary administration, promising advantages over conventional drug carriers. Firstly, the residence time of the therapeutic in the target region, the deep lung, is extended because of the shape and orientation dependent delay of cellular uptake. Secondly, the load of peripheral delivery is increased through fibrous shape; more material is transported per filament in comparison a spherical particle with identical diameter due to alignment in the airstream. Experiments confirm that the engulfment exclusively occurred from the tips of the cylindrical particles, delaying the uptake until this orientation was reached by the phagocyte. The aerodynamic properties of the cylindrical particles depend on the diameter of the filaments and not on the length, which was constant for all tested filaments. Cylinders with lower diameter proceed to deeper stages in the impactor, implying alignment with the airstream. The physiological conditions in the peripheral lung with scarce lining fluid, acting as the solvent, and low enzymatic activity of the fragile tissue largely restrict the selection of compounds for the design of pulmonary carriers. Only a few substances have been approved for this route of administration. Filamentous particles were formed from the FDA-approved excipient lactose, APIs and blends of various ratios. These cylinders dissolved instantaneously upon contact with aqueous media. In contrast, longer residence time is desired for prolonged release systems. This can be achieved by the incorporation of hydrogels into the matrix of the cylindrical particles. The biocompatible hydrogel alginate, degrading as a function of the phosphate concentration, was utilized in order to form the backbone of the carrier system. This mode of degradation reduces the likelihood of detrimental long-term accumulation in the peripheral lung because phosphate is ubiquitous in the body. The template technique allows for the embedding of NPs into the cylinders, too. These hierarchical microfibres were formed from silica NPs and were interconnected with biocompatible hydrogels (alginate and agarose). As a proof of concept, macrophage uptake experiments were performed in order to verify the paradigm of shape and orientation dependent uptake; this could be confirmed for fibres formed with the template technique. Uptake was quantified using the novel correlative light and electron microscopy (CLEM). Through the combination of high resolution of EM and specificity of fluorescence, misleading quantification based upon the single techniques SEM and FLM could be corrected. Additionally, the adaptation of the preparation protocol allows for a straightforward generation of hydrogel surfaces carrying fibres in high abundance and fidelity in various diameters and compositions. Literature reports about implications of surface structure on fundamental cell behaviour and functions. Consequently, the adhesion of murine alveolar fibroblasts was scrutinized on hairy alginate sheets with various dimensions and quantities of the filaments. The more abundant and more delicate the filaments were, the more adhesion was observed; in addition to a preferential alignment along the filaments. Without these fibres fibroblasts did not adhere to the alginate hydrogel surface. Furthermore, the hairy sheets could be loaded with small molecules, as well as macromolecules; a fact that might proof beneficial for potential applications as a surrogate for the ECM, loading growth factors for instance. The release of these model compounds was quantified. It was depending on the molecular size and the phosphate concentration

    Aquasomes as a drug delivery system for proteins and peptides

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    Aquasomes are nanocarrier systems consist of three distinctive layers; an inner core, a polyhydroxy carbohydrate layer and an outer layer of an API (Kossovsky et al., 1991). Aquasomes have a unique structure and ability to carry active molecules through a non-covalent bounding and provide superior stability, especially for proteins and peptides (Masatoshi and Yongning, 1998; Kim and Kim, 2002; Khopade et al., 2002). Different core and coating materials were used to prepare aquasomes under different conditions to investigate the relationship between preparation conditions and loading efficiency. In terms of loading efficiency, hydroxyapatite aquasomes, with either lactose or trehalose as a coating material, had the highest BSA loading (40%-60%) when compared to DSPA aquasomes. While DCPA aquasomes, with either lactose or trehalose as a coating material, had the lowest BSA loading (8%-16%). To investigate the interaction of the three layers of aquasomes, Surface analysis, docking and MD simulations were performed. Surface analysis performed by Discovery Studio showed that HA and trehalose interact by hydrogen bonding with the later acting as a hydrogen acceptor, while BSA displayed almost complete SAS and that there are numerous targets for trehalose attachments (no specific active site). MD simulations of BSA performed by AMBER 12 showed a stable MD simulation of BSA for 5 ns. Total energy analysis of BSA on the two conditions performed (300K and 280K) support the experimental data of lower BSA loadings of aquasomes prepared at 400C compared to those manufactured at 250C (p5.5 and steadily release for 6 hr. Cell culture studies were conducted to demonstrate the controlled release effect of aquasomes using Caco-2 cell lines. The release of metronidazole (model drug) from aquasomes post 2 hr started to slow gradually until it reached its highest difference at 6 hr (p<0.05) when compared to the control

    Electron Microscopy of Nanostructures in Cells

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