The high surface to volume ratio which is a property of nanoscale devices means the interfacial effects from these devices on the mass transport of analyte can be significant. Quartz nanopipette effect on the mass transport behavior of inorganic monovalent salts such as potassium chloride is shown to differ from those of conical nanopore. Quartz nanopipettes demonstrate a more significant interfacial impact on the mass transport behavior of inorganic salts. This is evidenced by significant impacts on ionic transport even at high electrolyte concentration where nanopore interfacial effects do not significantly impact the ion transport. Nanopipettes have been use to precipitate salts such as lithium chloride in bulk concentrations three orders of magnitude below the saturation concentration. These novel interfacial interactions have opened new avenues for crystallization of more complex organic biomolecules using inorganic systems as model systems on which to base the approach for these more complex systems.

Multiple hurdles, such as drug solubility, stability, and physical barriers in the body, hinder bioavailability of many promising therapeutics. Polymeric nanocarriers can encapsulate the therapeutics to protect non-target areas from side effects but also protect the drug from premature degradation for increased circulation and bioavailability. To capitalize on these advantages, the polymer nanoparticle must be properly engineered for increased control in size distribution, therapeutic encapsulation, colloidal stability, and release kinetics. However, each application requires a specific set of characteristics and properties. Being able to tailor these by manipulation of different design parameters is essential to optimize nanoparticles for the application of interest. This study of nanoparticle fabrication and characterization takes us a step closer to building effective delivery systems tailored for specific treatments. Poly(ethylene oxide)-b-poly(D,L-lactic acid) (PEO-b-PDLLA) based nanoparticles were produced to range from 100-200 nm in size. They were fluorescently labeled with a hydrophobic dye 6-13 bis(triisopropylsilylethynyl) pentacene (TIPS pentacene) at an optimal loading of 0.5 wt% with respect to the core. Surfaces were successfully coated with streptavidin to be readily functionalized with various biotinylated compounds such as PD-L1 antibodies or A488 fluorophore. Using the same PEO-b-PDLLA, iron oxide and a conjugated polymer poly(2- methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) (MEH-PPV) were co-encapsulated to form fluorescently labeled magnetic particles. Using poly(lactic-co-glycolic acid), CRISPR-Cas9 plasmids were encapsulated at 1.6 wt% and most of the payload released within the first 24 hours. The incorporated plasmids were intact enough to have mammalian macrophages successfully express the bacterial protein Cas9. Using similar PLGA based particles, the surface was functionalized with streptavidin and bound to the surface of bacteria as an active carrier for increased penetration of solid tumors averaging ~23 particles per bacterium. PEO-b-PLGA based particles were used in conjunction with a hydrophobic salt former to encapsulate a peptide designed to reduce platelet binding to cancer cells and mitigate extravasation. The peptide encapsulated was increased from < 2 wt% without salt former to 8.5 wt% with the used of hexadecyl phosphonic acid. Although the applications across these projects can be broad, the fundamentals and important design parameters considered contribute to the overarching field of effective carriers for drug delivery. / Ph. D. / There are many reasons why many promising pharmaceutical formulations never make it through regulation and onto market, including low solubility of the drug, low absorbance by the body, and harmful side effects, to name a few. Using polymer drug carriers, these difficulties can be overcome by holding the drug in a more soluble carrier, releasing it on a certain timeline or to a specific location to increase absorbance and decrease side effects. When designing a carrier, the requirements for the product are dependent on the application and the disease of interest. This work looks at the material types and conditions during particle formation to see how it affects the final product to better define and understand how these parameters change the performance. This work shows that the carrier size can be manipulated depending on how much of one material is used versus the other, they can be labeled to fluoresce so they can be tracked during cell and animal studies, and they can be coated with targeting compounds on the surface to increase the specificity of the carrier to localize to a target location of interest. Different particles containing DNA for gene editing, peptides for cancer therapies, and magnetic iron oxides to increase transport across difficult cell barriers have all be fabricated and characterized. The lessons learned through these projects will help guide future work to more effective and efficient delivery of pharmaceuticals to the body.

Cette thèse porte sur l’influence de la formulation de méthode de nanoprecipitation sur la taille et la structure interne de particules de dérivés squalénés. Ces composés introduits en 2006 par P. Couvreur sont basés sur l’association de principe actif au groupe squalène pour obtenir des propriétés d’auto-assemblage. L’augmentation de l’efficacité thérapeutique des nanoparticules par rapport au principe actif libre et la versatilité de fonctionnalisation ont ouvert une nouvelle stratégie pour les applications biomédicales. Cependant, la compréhension des mécanismes de formation des particules et de leur structure interne reste incomplète. Ces caractéristiques (taille et structure) pouvant influencer l’activité thérapeutique, il est crucial en vue d’une application médicale de déterminer les paramètres clef de la formation des nanoparticules. Dans cette thèse, les particules de squalène deoxycitidine ont été caractérisées par diffusion de neutrons et de rayons X aux petits angles et par cryo-microscopie électronique. Dans une première partie, nous avons identifié les paramètres optimums de formulation pour la nanoprecipitation. Dans une deuxième partie, l’étude de la nature du solvant organique sur la nanoprecipitation a mis en évidence pour certains solvants un mécanisme de nucléation croissance. Les effets de solvants sont cependant faibles par rapport à ce qui est classiquement observé pour les polymères. La troisième partie basée sur la cryo-TEM des particules oriente vers un mécanisme décrit pour les autres cubosomes d’attachements inter lamellaires pour les structures internes cubiques. Enfin, à l’issue de ce travail, nous avons étendu le protocole de nanoprecipitation au D α- Tocophérol Succinate en tentant de stabiliser la phase cristalline hexagonale présente dans le diagramme de phase binaire eau/ α-TOS. / This thesis investigates the influence of the formulation on the size and the internal structure of particles obtained by nanoprecipitation of squalene derivatives. These compounds were introduced in 2006 by P. Couvreur's team as part of a process called squalenoylation. Based on the association of drug with the squalene group it confers to these molecules self-assembling properties and it increased therapeutic efficacy. The particles are prepared by the solvent displacement method ("Ouzo" effect) and were characterized by neutron and X-ray scattering at small angles and by cryo-electron microscopy.We have highlighted the necessary preparation parameters for the reproducible preparation of cubosomes in a dimater of hundred nanometres. A study on the effect of the nature of the organic solvent on Squalene deoxycitidine has highlighted for some of them (isopropanol and DMSO) a nucleation mechanism that could be followed by growth (ethanol and acetone). TEM cryo-microscopy analysis suggests that cubic internal structures form by the mechanism of interlamellar attachments. Finally, we extended the nanoprecipitation method to Dα-Tocopherol Succinate, a derivative of Vitamin E

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, PhD. Student: Daniela Širajová Title of thesis: Preparation of polymeric fluorescent nanoparticles Nanoparticles based on biodegradable polyesters are a widely used platform for targeted drug delivery and subsequent controlled release. The aim of this diploma thesis was to prepare and optimize the preparation of polymeric nanoparticles with a fluorescent dye as a model substance. The nanoprecipitation method was used to prepare the nanoparticles. Nanoparticles prepared from two types of PLGA polymers (COOH terminated and ester terminated) were evaluated and compared. The surfactant and stabilizer were used in various concentration ratios to optimize the preparation. The surfactant was sodium cholate at concentrations of 0.1%, 0.5%, 1%, 2% and 5%. The nanoparticles were stabilized with Pluronic F-127 poloxamer at concentrations of 0.1%, 0.5% and 1%. Nanoparticles were compared in terms of encapsulation efficiency, particle size and zeta potential. In a dissolution experiment, the amount of fluorescein released was evaluated and compared as a function of time (48 hours) with acid-terminated PLGA and sodium cholate at concentrations of 0.1% and 2%....

Nanoparticles have the potential to revolutionize medicine, but many obstacles complicate the translation of nanoparticles from the bench to the clinic. A deeper understanding of nanoparticle synthesis parameters that influence nanoparticle size, drug loading, and surface chemistry is needed to accelerate the design of efficacious therapeutic nanoparticle systems. In this work, organic and inorganic nanoparticles were prepared with hydrodynamic diameters below 200 nm for applications in cancer treatment and immunology. Hydrophobic ion pairing was applied to enhance the loading capacity of drugs and peptides in polyester and polysaccharide nanoparticles systems. Polyester nanoparticles were successfully functionalized with streptavidin-Cy3, interferon gamma (IFN-γ), and CX3CL1. Poly(methacrylic acid), chitosan, and polyinosinic-polycytidylic acid (poly(I:C)) were successfully adsorbed to the surfaces of nanoparticles to enhance particle stability and targeting. Iron-based coupling media capable of eliminating ~ 90% of the water signal from an acoustic coupling bath during gradient echo magnetic resonance imaging (MRI) thermometry was successfully designed using magnetic iron oxide nanoparticles to improve the clinical efficacy of MRI-guided focused ultrasound surgery (MRI-FUS). While the critical nanoparticle design criteria may change depending on the biomedical application, fundamental concepts of nanoparticle design and synthesis can be applied across applications. The projects presented here help to bridge the knowledge gap regarding the use of flash nanoprecipitation (FNP) for nanoparticle synthesis. FNP is a scalable nanoparticle fabrication method that produces small, well-defined nanoparticle populations through rapid, turbulent mixing of multiple solvent streams. This work elucidates nanoparticle design concepts that can be applied across a wide variety of biomedical applications. / Doctor of Philosophy / Cancer remains a critical public health issue worldwide because many promising therapies never make it from the lab into the hospital. Many chemotherapeutic drugs are hindered by poor solubility and serious, undesirable side effects. In the past few decades, new production techniques have been developed to create carriers for these drugs to help overcome these obstacles. These carriers can be made from a variety of materials including metals and biodegradable polymers. In fact, it is even possible to create "smart" carriers that react to their environment to travel within the body or release the drugs they contain. Understanding how to design these carriers for different biomedical applications is critical. This work shows how carriers made from metal or polymer can be designed to exhibit desirable characteristics for use in biomedical applications ranging from vaccines to cancer treatment. Various ways to modify the surfaces of these carriers to tailor them for different applications are presented. This work provides valuable information that can help drive the next generation of biomedical innovation.

No / This study employs artificial neural networks (ANNs) to create a model to identify relationships between variables affecting drug nanoprecipitation using microfluidic reactors. The input variables examined were saturation levels of prednisolone, solvent and antisolvent flowrates, microreactor inlet angles and internal diameters, while particle size was the single output. ANNs software was used to analyse a set of data obtained by random selection of the variables. The developed model was then assessed using a separate set of validation data and provided good agreement with the observed results. The antisolvent flow rate was found to have the dominant role on determining final particle size.

Over recent years the delivery of nanosized drug particles has shown potential in improving bioavailability. Drug nanosizing is achieved by 'top-down' and by 'bottom-up' approaches. Owing to limitations associated with the top-down techniques, such as high energy input, electrostatic effects, broad particle size distributions and contamination issues, great interest has been directed to alternative bottom up technologies. In this study, the hypothesis that microreactors can be used as a simple and cost-effective technique to generate organic nanosized products is tested using three steroids (hydrocortisone, prednisolone and budesonide). Arrested antisolvent nanoprecipitation using ethanol (solvent) and water (antisolvent) was conducted within the microreactors. To enable experimental design for the microreactor studies, solubility profiles in different ethanol-water combinations at 25 °C were explored. All three drugs' solubility increased with increasing ethanol concentration showing maxima at 80-90 % v/v ethanol-water mixtures. Because of the complex multivariate microfluidic process, artificial neural network modelling was then employed to identify the dominant relationships between the variables affecting nanoprecipitation (as inputs) and the drug particle size (as output). The antisolvent flow rate was found to have the major role in directing drug particle size. Based on these successful findings, the potential of preparing pharmaceutical nanosuspensions using microfluidic reactors was researched. A hydrocortisone (HC) nanosuspension (NS) was prepared by introducing the generated drug particles into an aqueous solution of stabilizers stirred at high speed with a propeller mixer. A tangential flow filtration system was then used to concentrate the prepared NS. Results showed that a stable narrow sized HC NS of amorphous spherical particles 500 ± 64 nm diameter and zeta potential -18 ± 2.84 mV could be produced. The ocular bioavailability of a microfluidic precipitated HC NS (300 nm) was assessed and compared to a similar sized, milled HC NS and HC solution as a control. The precipitated and the milled NS achieved comparable AUC0-9h of 28.06 ± 4.08 and 30.95 ± 2.2, respectively, significantly (P < 0.01) higher than HC solution (15.86 ± 2.7). These results illustrate the opportunity to design sustained release ophthalmic formulations. Going nano via microfluidic precipitation was also exploited to tailor budesonide (BD) NS for pulmonary administration. The in vitro aerosolization by nebulization of a BD NS was studied in comparison with a commercial BD microsuspension. Overall, the fine particle fraction generated from BD NS (56.88 ± 3.37) was significantly (P < 0.05) higher than the marketed BD (38.04 ± 7.81). The mean mass aerodynamic diameter of BD NS aerosol (3.9 ± 0.48 μm) was significantly smaller (P < 0.05) than the microsuspension (6.2 ± 1.09 μm) indicating improved performance for BD NS. In conclusion, findings of this study support the hypothesis of using microfluidic nanoprecipitation as a promising and economical technique of drug nanosizing.

L’objectif de ce travail de thèse est d’étudier la relation entre la méthode de préparation des nanoparticules, les propriétés colloïdales et l’encapsulation d’un principe actif à usage pédiatrique. Dans ce but, le diclofenac a été utilisé comme molécule modèle et les nanoparticules ont été préparées via la nanoprécipitation et l’émulsification-diffusion. Des études fondamentales et systématiques ont permis de mettre en évidence l'existence de différences notables entre les propriétés électrocinétiques et l'efficacité d’encapsulation en fonction du procédé utilisé pour la préparation des particules. Ces propriétés colloïdales et physico-chimiques sont primordiales pour la bonne stabilité des dispersions de nanosphères et des nanocapsules et pour le comportement de ces vecteurs lors d’utilisation in vivo. Cette étude a permis de proposer et de discuter le mécanisme de formation des nanoparticules en se basant sur le comportement des variables critiques du procédé et de la formulation, les propriétés de surface et l'efficacité d’encapsulation de l’actif modèle / The objective of this PhD thesis is to point out the relationship between the preparation method of the nanoparticles, the colloidal properties and the encapsulation efficiency of a given active molecule for paediatric purpose. In this direction, diclofenac was used as model molecule and the nanoparticles were prepared via the nanoprecipitation and the emulsification-diffusion processes. The conducted fundamental and systematic studies rend evident notable differences between the two processes, particularly in the electrokinetic properties of the particles and the effectiveness of the drug encapsulation. These colloidal and physicochemical properties are paramount for the good stability of the nanoparticles and their in vivo use. This research work made it possible to propose and to discuss the mechanism of nanoparticle formation from the behavior of key variables of the process and the recipe used, the surface properties of the particles and the effectiveness of encapsulation of the model drug

This work was performed as part of an EPSRC Leadership Fellowship [EP/I005420/1] for the study of irradiation damage in Zr alloys, and is supported heavily by industrial contributors and especially by Westinghouse, Studsvik and Rolls-Royce plc. for the investigation of mechanisms relating to irradiation-induced growth (IIG). This thesis is an analysis of the microchemical and microstructural evolution of Zircaloy-2 under both proton and neutron irradiation. Comparisons are made between the effects of the different irradiative species through the use of scanning transmission electron microscopy (STEM) and energy dispersive X-ray spectroscopy (EDS). The work takes advantage of advances in EDS capability with large solid angles of collection 0.7 srad coupled with an aberration-corrected FEI Titan ChemiSTEMTM with a high brightness X-FEG electron source.2 MeV proton irradiation experiments were performed to doses of 2.3, 4.7 and 7.0 displacements per atom (dpa) at a dose rate of ~6.7 x10-6 dpa s-1 and at 350 °C. Electropolished TEM foils from Zircaloy-2 cladding and channel components of a BWR were supplied by Westinghouse in the fluence range 8.7 to 14.7 x1025 n m-2 ~14.5 to 24.5 dpa. Comparisons have been made in relation to SPP chemical composition, grain boundary chemistry, dislocation density, correlations between dislocation evolution and microchemical segregations and the nature of irradiation-induced precipitates. Proton irradiation-induced dissolution was observed for both Zr(Fe,Cr)2 and Zr2(Fe,Ni) SPPs, the depletion of Fe was preferentially from the edge region in the former SPP and from throughout the whole SPP in the latter. While no proton-induced amorphisation was observed for the Zr(Fe,Cr)2, the compositional changes in all SPPs agreed well with the reports of other authors. All grain boundaries display Fe and Ni segregation prior to irradiation, which disperses into the matrix after both proton and neutron irradiation, while Sn segregates to the boundary. Sn and the light transition elements Fe, Cr and Ni have shown contrasting behaviour in the matrix also. After irradiation by both protons and neutrons, a-component dislocation loops (a-loops) align parallel to the basal plane and Fe, Cr and Ni segregate to the a-loop positions. Sn, conversely, segregates to between a-loop positions parallel to the basal trace. The threshold dose in c-component dislocation loop (c-loop) nucleation under proton irradiation (~4.5 dpa) is shown as similar to that due to neutron irradiation (~5 dpa). We observe that a-loop density decreases at the onset of c-loop nucleation and that the position of c-loops are in alignment with the a-loops but that they are anticorrelated in position along the basal trace. We therefore propose that chemical ordering promotes the alignment of a-loops, which then provides the conditions necessary for c-loop nucleation. Nanoprecipitation is evident in the matrix after both proton and neutron irradiation. After proton irradiation to ~2 dpa, parallel atom probe tomography and STEM-EDS investigations have shown the nano-rods to be of composition Zr4(Fe0.67Cr0.33), tending towards Zr3(Fe0.69Cr0.31) as the rod volume increases. The rods are higher in density than the a-loops by a factor of ~3 and so are likely to be a significant influence on mechanical properties and IIG phenomena.

The nasal mucosa provides a rapid, noninvasive route for drug administration to the systemic circulation and even potentially to the CNS. Nanoparticles made from the biodegradable polymer, PLGA, are of great interest for use in drug delivery systems due to PLGA’s relative safety and ease of surface modification. Nanoparticles may provide improved targeting and transport through the nasal mucosa. However, the optimal nanoparticle sizes and surface properties for intranasal delivery are unknown. In this study, we prepared PLGA nanoparticles within a size range of 50-70 nm containing the lipophilic fluorescent dye, Nile Red, using a surfactant-free nanoprecipitation method. The resulting nanoparticles were evaluated using dynamic light scattering and scanning electron microscopy. Nanoparticle uptake into the nasal mucosa was determined by exposing the tissues to nanoparticle dispersions for 30 or 60 minutes. The in vitro uptake of the nanoparticles by the nasal mucosal tissues revealed that the Nile Red-loaded PLGA nanoparticles were transported across the epithelial layer and accumulated in the sub-mucosal connective tissues. Nanoparticle uptake in the full thickness tissues was time dependent where 2% of the total loads of nanoparticles exposed to the tissues were measured in the mucosal tissue after 30 minutes and 4% were present in the tissues after 60 minutes. The rapid and measurable transfer of PLGA nanoparticles into the nasal mucosal tissues indicate that they may be an efficient delivery vehicle for drugs with either local or systemic activities.