Development of Imaging Paradigms for Drug Distribution and Fate in the Eye

Aging-associated vision loss is increasingly prevalent in our population and intravitreal injections are commonly used to administer ocular drugs to the posterior segment of the eye. This work aims to visualize and predict the delivery of ocular drugs by combining micro- computed tomography (micro-CT) imaging and computational fluid dynamics (CFD) modeling. Intravitreal injections were administered into ex vivo porcine eyes and imaged for an extended period of time to track the progression of the injected drug mimic. Non-invasive imaging allowed for precise determination of contrast agent concentration, flow patterns and fate. A computational model was developed that provided quantitative agreement with the concentration values found in the experimental study and allowed for easy manipulation of parameters. The ability to accurately model drug transport following an intravitreal injection provides vital information to better understand the specific concentration and time frame for the drug to reach the target sit

The study of renal function and toxicity using zebrafish (Danio rerio) larvae as a vertebrate model

Zebrafish (Danio rerio) is a powerful model in biomedical and pharmaceutical sciences. The zebrafish model was introduced to toxicological sciences in 1960, followed by its use in biomedical sciences to investigate vertebrate gene functions. As a consequence of many research projects in this field, the study of human genetic diseases became instantly feasible. Consequently, zebrafish have been intensively used in developmental biology and associated disciplines. Due to the simple administration of medicines and the high number of offspring, zebrafish larvae became widely more popular in pharmacological studies in the following years. In the past decade, zebrafish larvae were further established as a vertebrate model in the field of pharmacokinetics and nanomedicines. In this PhD thesis, zebrafish larvae were investigated as an earlystage in vivo vertebrate model to study renal function, toxicity, and were applied in drug-targeting projects using nanomedicines. The first part focused on the characterization of the renal function of three-to four-dayold zebrafish larvae. Non-renal elimination processes were additionally described. Moreover, injection techniques, imaging parameters, and post-image processing scripts were established to serve as a toolbox for follow-up projects. The second part analyzed the impact of gentamicin (a nephrotoxin) on the morphology of the pronephros of zebrafish larvae. Imaging methodologies such as fluorescent-based laser scanning microscopy and X-ray-based microtomography were applied. A profound comparison study of specimens acquired with different laboratory X-ray-based microtomography devices and a radiation facility was done to promote the use of X-ray-based microtomography for broader biomedical applications. In the third part, the toxicity of nephrotoxins on mitochondria in renal epithelial cells of proximal tubules was assessed using the zebrafish larva model. Findings were compared with other teleost models such as isolated renal tubules of killifish (Fundulus heteroclitus). In view of the usefulness and high predictability of the zebrafish model, it was applied to study the pharmacokinetics of novel nanoparticles in the fourth part. Various in vivo pharmacokinetic parameters such as drug release, transfection of mRNA/pDNA plasmids, macrophage clearance, and the characterization of novel drug carriers that were manipulated with ultrasound were assessed in multiple collaborative projects. Altogether, the presented zebrafish model showed to be a reliable in vivo vertebrate model to assess renal function, toxicity, and pharmacokinetics of nanoparticles. The application of the presented model will hopefully encourage others to reduce animal experiments in preliminary studies by fostering the use of zebrafish larvae

A review of emerging technologies enabling improved solid oral dosage form manufacturing and processing

Tablets are the most widely utilized solid oral dosage forms because of the advantages of self-administration, stability, ease of handling, transportation, and good patient compliance. Over time, extensive advances have been made in tableting technology. This review aims to provide an insight about the advances in tablet excipients, manufacturing, analytical techniques and deployment of Quality by Design (QbD). Various excipients offering novel functionalities such as solubility enhancement, super-disintegration, taste masking and drug release modifications have been developed. Furthermore, co-processed multifunctional ready-to-use excipients, particularly for tablet dosage forms, have benefitted manufacturing with shorter processing times. Advances in granulation methods, including moist, thermal adhesion, steam, melt, freeze, foam, reverse wet and pneumatic dry granulation, have been proposed to improve product and process performance. Furthermore, methods for particle engineering including hot melt extrusion, extrusion-spheronization, injection molding, spray drying / congealing, co-precipitation and nanotechnology-based approaches have been employed to produce robust tablet formulations. A wide range of tableting technologies including rapidly disintegrating, matrix, tablet-in-tablet, tablet-in-capsule, multilayer tablets and multiparticulate systems have been developed to achieve customized formulation performance. In addition to conventional invasive characterization methods, novel techniques based on laser, tomography, fluorescence, spectroscopy and acoustic approaches have been developed to assess the physical-mechanical attributes of tablet formulations in a non- or minimally invasive manner. Conventional UV-Visible spectroscopy method has been improved (e.g., fiber-optic probes and UV imaging-based approaches) to efficiently record the dissolution profile of tablet formulations. Numerous modifications in tableting presses have also been made to aid machine product changeover, cleaning, and enhance efficiency and productivity. Various process analytical technologies have been employed to track the formulation properties and critical process parameters. These advances will contribute to a strategy for robust tablet dosage forms with excellent performance attributes

Accomplishments and challenges in stem cell imaging in vivo.

Stem cell therapies have demonstrated promising preclinical results, but very few applications have reached the clinic owing to safety and efficacy concerns. Translation would benefit greatly if stem cell survival, distribution and function could be assessed in vivo post-transplantation, particularly in patients. Advances in molecular imaging have led to extraordinary progress, with several strategies being deployed to understand the fate of stem cells in vivo using magnetic resonance, scintigraphy, PET, ultrasound and optical imaging. Here, we review the recent advances, challenges and future perspectives and opportunities in stem cell tracking and functional assessment, as well as the advantages and challenges of each imaging approach

Superparamagnetic iron oxide nanoparticles for imaging and drug delivery

In the past years, nanoparticle usage and research increased enormously in different fields, especially in the clinical sciences for drug delivery and imaging. Selection of the most suitable type of nanoparticle is not always easy because of a broad material variety and different physicochemical characteristics. Depending on the purpose, the safe usage of nanoparticles in vivo needs to be ensured first to predict and eliminate unwanted effects like agglomeration, loss of function, immune system responses (i.e. inflammation), or toxicity after intravenous application. To guarantee the safe usage of nanoparticles, there are various hazard evaluation strategies for different scenarios for example for nanomedicines. Drug delivery with nanomedicines has advantages like increased absorbability, increased in vivo half-life, and decreased drug dosage needs to reach the same therapeutic effect. Amongst others, superparamagnetic iron oxide nanoparticles (SPIONs) and liposomes are already used as medicinal nanoparticles and are approved from the US Food and Drug Administration (FDA). Examples are Feraheme®, which is a ferumoxytol injection for iron deficiency treatments and Doxil®, a liposomal doxorubicin hydrochloride chemotherapy drug. SPIONs are also used as contrast agents because of their high-dense core and the possibility to synthesize very small diameters below 10 nm, which can even penetrate into smallest fenestrations of i.e. the kidneys. This work is divided into two main parts: Part one was the analysis of different nanoparticle safety evaluation strategies to propose a new hazard evaluation guideline for intravenously applied nanoparticles. This was part of the NanoREG II European Union’s Horizon 2020 research and innovation program under grant agreement 646221. Part two started with the synthesis and physicochemical characterization of hybrid nanoparticles made out of SPION cores and liposome coatings. The coatings were differently modified with additions like polyethyleneglycol for increased in vivo half-life or folic acid for renal targeting. Injected into zebrafish (Danio rerio) embryos, their biodistribution and toxicity was analyzed. Various methods like confocal laser scanning microscopy and synchrotron X-ray radiation micro-computed phase-contrast tomography were used and compared with each other. Finally, those hybrid nanoparticles were manipulated in vivo with external magnets to increase phagocytic uptake and also with electromagnetic fields and acoustic waves for controlling the nanoparticles in vivo in terms of agglomeration and rotation

Multimodal nanoparticles for quantitative imaging

The scope of this thesis is research related to applications of nanoparticles in quantitative preclinical imaging. Nanoparticles are a versatile platform that can interact with biological systems at many different length scales and can furthermore be rendered visible for basically any medical imaging technique by modification with appropriate contrast providing moieties. Thus, nanoparticles can be used as a new class of contrast agents for basically all imaging modalities, e.g. as long circulating blood pool agents in CT, or as MRI contrast agents. Vice versa, non-invasive imaging techniques can be used to for example follow the biodistribution of nanoparticles in vivo and apply nanoparticles as a tool to investigate biological processes related to disease processes. Dual modal imaging applying multifunctional and dual-labeled nanoparticles offer new approaches to quantitative imaging, giving new insights into technology development on one side and biological read-outs on the other. For instance, quantification of biological processes that lie at the basis in the development of disease may lead to earlier detection and better disease diagnosis and treatment. Results and concepts presented in this thesis have high impact on therapeutic application of nanoparticles, for example when they are used as drug delivery systems. Imaging can provide valuable information on drug delivery and biodistribution in a quantitative manner, which may help in development of new therapeutic strategies. Nanoparticles are promising structures for quantitative imaging. Its surface can be utilized to attach almost any desirable molecule. Nanoparticles are relatively large in size (typically 10-200 nm) and can for instance accommodate a high payload of contrast agent per particle on its surface or inside the particle, thereby increasing the signal/particle by five orders of magnitude. In addition, also multiple imaging probes for different imaging modalities can be incorporated providing a double read-out. For the understanding of biological processes, targeting ligands such as antibodies, proteins and peptides can be attached to its surface. Despite the wide variety of possibilities with nanoparticles, they have hardly been studies for quantitative imaging purposes. Therefore, the aim of the research described in this thesis was to explore and develop several nanoparticles for quantitative imaging by using existing or newly developed imaging techniques. Chapter 1 gives a general introduction in the field of nanoparticles for quantitative imaging. Several imaging techniques are described such as CT, Spectral CT, SPECT and MRI, and how nanoparticles can play an important role in research. Chapter 2 describes the development of a novel nanoparticulate CT contrast agent. Several amphiphilic molecules were investigated in this chapter in the combination with different iodinated oils for their influence on the size stability of the nanoparticles. In Chapter 3, the dose dependent biodistribution of the nanoparticles is investigated as well as strategies to vary the biodistribution. The effect of a co-injection with liposomes and soy bean oil emulsions was investigated using CT, SPECT and ¿-counting. The final optimized blood pool CT contrast agent from chapter 2 and 3 can be used for qualitative imaging in CT as well as in quantitative imaging in Spectral CT. Chapter 4 describes the very first use of this novel imaging technique Spectral CT in quantitative imaging. For this, the nanoparticles of chapter 2 were extended to a multimodal nanoparticulate contrast agent for CT, Spectral CT and SPECT. Spectral CT quantification was compared to quantification using SPECT and ICP-MS to demonstrate the correlations and accuracy of the techniques. In Chapter 5, the development is described of a dual-isotope SPECT imaging protocol as a tool for pre-clinical testing of new molecular imaging tracers. New molecular targeting probes are consistently investigated as a tool to enable target specific binding of nanoparticles to cellular surfaces of interest. Dual-isotope SPECT can be used in which the biodistribution of two different ligands labelled with two different radionuclides can be studied in the same animal, thereby excluding experimental and physiological inter-animal variations. The developed dual-isotope protocol was tested using a known angiogenesis specific ligand (cRGD peptide) in comparison to a potential non-specific control (cRAD peptide). Chapter 6 describes the use of a multimodal radiolabeled paramagnetic liposomal contrast agent that allows simultaneous imaging with SPECT and MRI. A double read-out is then possible and demonstrates the additional advantages of the combination of the two techniques. SPECT can for instance quantify the nanoparticle concentration and MRI can spatially localize the nanoparticle. The combination however gives an indirect read-out of the water exchange, which in return reveals insights in biological processes and environments. Chapter 7 describes a study that investigates the use of nanoparticles in the quantitative imaging technique fluorine MRI. The use of gadolinium-complexes as signal modulating ingredients into the nanoparticle formulation has emerged as a promising approach towards improvement of the fluorine signal. Paramagnetic lipids based on gadolinium complexes can be incorporated to increase the 19F MR signal per particle. Here, 3 different paramagnetic lipids were investigated on its influence at five different field strengths. This furthermore also provides important insights in the dependency of the magnetic field on fluorine signal intensity. The final Chapter 8 describes the future perspectives of the use of multimodal nanoparticles for quantitative imaging

Doctor of Philosophy

dissertationTreatment of cancer is a significant challenge due to the heterogeneity of both tumors and patients. This realization has led to the field of personalized medicine in which patients can be selected for a therapy based on the specific needs. One potential area for personalized medicine is utilizing medical imaging to predict and monitor the therapeutic efficacy and safety of a particular targeted therapy. Development of image- guided therapeutics based on macromolecular carriers such as N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers is advantageous because they are water-soluble and can contain a wide range of comonomers to confer multifunctionality. HPMA copolymers are water-soluble nano-sized constructs which can improve the delivery of therapeutics to tumors by passive targeting via the enhanced permeability and retention effect. They can also increase tumor uptake using targeting ligands that are conjugated to the backbone of the copolymer. This dissertation focuses on the development of targeted HPMA copolymers for delivery of both therapeutics and imaging agents to solid tumors. Barriers to delivery of these constructs were addressed in various tumor models. In pancreatic tumors, the desmoplastic response, or dense extracellular matrix prevents delivery of drugs and macromolecules alike. Treating hyaluronic acid, a component of desmoplasia, with hyaluronidase allowed for increased delivery of HPMA copolymers based on HER2 and αvβ3 integrin targeting strategies for HPMA copolymers. Based on the selection of HER2 as a viable tumor targeting strategy, iv an image-guided drug delivery (IGDD) system was synthesized, characterized and evaluated in vitro in pancreatic tumor cell lines. In vitro results suggest that the designed construct was potentially capable of targeting, binding, treating and imaging pancreatic tumors for an IGDD approach. Lastly, a study was conducted in a prostate tumor model for localized tumor delivery of a 90Y radiolabeled HPMA copolymers for radiotherapy. Imaging tumor localization and biodistribution was accomplished using an equivalent 111In radiolabeled HPMA copolymer. Targeting and efficacy were accomplished via gold nanorod (GNR)-mediated hyperthermia and demonstrated antitumor efficacy in the prostate tumor mouse model. The combined studies demonstrate the current progress for development of an HPMA copolymer conjugate for image-guided therapy of solid tumors

Development and in vitro characterization of three dimensional biodegradable scaffolds for peripheral nerve tissue engineering

Tissue engineering emerges nowadays to seek new solutions to damaged tissues and/or organs by replacing or repairing them with engineered constructs or scaffolds. In nerve tissue engineering, scaffolds for the repair of peripheral nerve injuries should act to support and promote axon growth following implantation. It is believed that substantial progress can be made by creating scaffolds from biomaterials, with growth-promoting molecules and spatially-controlled microstructure. To this end, this research aims to develop three dimensional (3D) scaffolds for peripheral nerve tissue regeneration by focusing on studies on the axon guidance, development and characterization of a novel 3D scaffold, and visualization of scaffolds by means of synchrotron-based diffraction enhanced imaging (DEI). Axon guidance is one of crucial considerations in developing of nerve scaffolds for nerve regeneration. In order to study the axon guidance mechanism, a two dimensional (2D) grid micropatterns were created by dispensing chitosan or laminin-blended chitosan substrate strands oriented in orthogonal directions; and then used in the in vitro dorsal root ganglion (DRG) neuron culture experiments. The results show the effect of the micropatterns on neurite directional growth can preferentially grow upon and follow the laminin-blended chitosan pathways. A novel 3D scaffold was developed for potential applications to peripheral nerve tissue engineering applications. The scaffolds were fabricated from poly L-lactide (PLLA) mixed with chitosan microspheres (CMs) by using a rapid freeze prototyping (RFP) technique, allowing for controllable scaffold microstructure and bioactivities protein release. The scaffold characterization shows that (1) the mechanical properties of the scaffolds depend on the ratio of CMs to PLLA as well as the cryogenic temperature and (2) the protein release can be controlled by adjusting the crosslink degree of the CMs and prolonged after the CMs were embedded into the PLLA scaffolds. Also, the degradation properties of the scaffolds were investigated with the results showing that the addition of CMs to PLLA can decrease the degradation rate as compared to pure PLLA scaffolds. This allows for another means to control the degradation rate. Visualization of polymer scaffolds in soft tissues is challenging, yet essential, to the success of tissue engineering applications. The x-ray diffraction enhanced imaging (DEI) method was explored for the visualization of the PLLA/CMs scaffolds embedded in soft tissues. Among various methods examined, including conventional radiography and in-line phase contrast imaging techniques, the DEI was the only technique able to visualize the scaffolds embedded in unstained muscle tissue as well as the microstructure of muscle tissue. Also, it has been shown that the DEI has the capacity to image the scaffolds in thicker tissue, and reduce the radiation doses to tissues as compared to conventional radiography. The methods and results developed/obtained in this study represent a substantial progress in the development and characterization of 3D scaffolds. This progress forms a basis for the future tests on the scaffolds as applied for peripheral nerve injuries

Understanding Nanoparticle Toxicity to Direct a Safe-by-Design Approach in Cancer Nanomedicine

Nanomedicine is a rapidly growing field that uses nanomaterials for the diagnosis, treatment and prevention of various diseases, including cancer. Various biocompatible nanoplatforms with diversified capabilities for tumor targeting, imaging, and therapy have materialized to yield individualized therapy. However, due to their unique properties brought about by their small size, safety concerns have emerged as their physicochemical properties can lead to altered pharmacokinetics, with the potential to cross biological barriers. In addition, the intrinsic toxicity of some of the inorganic materials (i.e., heavy metals) and their ability to accumulate and persist in the human body has been a challenge to their translation. Successful clinical translation of these nanoparticles is heavily dependent on their stability, circulation time, access and bioavailability to disease sites, and their safety profile. This review covers preclinical and clinical inorganic-nanoparticle based nanomaterial utilized for cancer imaging and therapeutics. A special emphasis is put on the rational design to develop non-toxic/safe inorganic nanoparticle constructs to increase their viability as translatable nanomedicine for cancer therapies