Immuno Magnetic Thermosensitive Liposomes For Cancer Therapy

The present work describes the encapsulation of the drug doxorubicin (DOX) in immuno paramagnetic thermosensitive liposomes. DOX is the most common chemotherapeutic agent for the treatment of a variety of carcinomas. However, the pure drug has high cytotoxicity and therefore requires a targeted and biocompatible delivery system. The introduction includes concepts, modalities, and functionalities of the project. First, a detailed description of the cell type (triple-negative breast cancer) is given. Furthermore, the importance of liposomal doxorubicin is explained and the current state of research is shown. The importance of modification to achieve thermosensitive properties and the procedure for co-encapsulation with Gd chelate to achieve paramagnetic properties is also discussed. In addition, the first part describes the surface modification with ADAM8 antibodies, which leads to improved targeting. The second part of the thesis covers the different materials and methods used in this paper. The production of the liposomes LipTS, LipTS-GD, LipTS-GD-CY, LipTS-GD-CY-MAB and the loading of DOX using an ammonium sulfate gradient method were described in detail. The results part deals with the physicochemical characterization using dynamic light scattering and laser Doppler velocimetry, which confirmed a uniform monodisperse distribution of the liposomes. These properties facilitate the approach of liposomes to target cancer cells. The influence of lipid composition of liposomes, co-encapsulation with Gd chelate and surface modification of liposomes was evaluated and described accordingly. The size and structure of the individual liposomal formulations were determined by atomic force microscopy and transmission electron microscopy. Morphological examination of the liposomes confirmed agreement with the sizes obtained by dynamic light scattering. Temperature-dependent AFM images showed an intact liposome structure at 37 °C, whereas heating by UHF-MRI led to a lipid film indicating the destruction of the lipid bilayer. Furthermore, TEM images showed the morphological properties of the liposomes and gave a more precise indication of how Gd-chelate accumulates within the liposomes. Liposomes with Gd-chelate showed well-separated vesicles, suggesting that Gd- chelate is deposited in the lipid bilayer of the liposomes. Gd was encapsulated in the hydrophilic core whereas chelate was extended into the lipid bilayer. By differential scanning calorimetry and drug release, the heat-sensitive functionality of the liposomes could be determined. Liposomes showed a beginning of phase transition temperature at about 38 °C, which can be achieved by UHF-MRI exposure. The maximum phase transition temperature in the case of LipTS-GD and LipTS-GD-CY-MAB was 42 °C and 40 °C, respectively. A proof of concept study for the thermosensitive properties of liposomes and a time-dependent DOX release profile in hyperthermia was performed. Gd-chelate is encapsulated in both LipTS-GD and LipTS-GD-CY-MAB and led to paramagnetic properties of the liposomes. This facilitates imaging mediated DOX delivery and diagnosis of the solid tumor and metastatic cells. The change in relaxation rate R1 of liposomes was quantified before and after heating above Tm (T> Tm). The relaxivity of the liposomes was obtained from the adapted slope of the relaxation rate against the Gd concentration. Remarkably, the relaxation rate and relaxivity increased after heating the liposomes above Tm (T> Tm), suggesting that the liposomes opened, released Gd chelate, and the exchange of water molecules became faster and more practicable. Toxicity studies describe the different mechanisms for induced DOX toxicity. The increased cytotoxic effect at elevated temperatures showed that the induced toxicity is thermally dependent, i.e. DOX was released from the liposomes. The high viability of the cells at 37 °C indicates that the liposomes were intact at normal physiological temperatures. Under UHF-MRI treatment, cell toxicity due to elevated temperature was observed. The cellular uptake of liposomes under UHF-MRI was followed by a confocal laser scanning microscope. An increase in fluorescence intensity was observed after UHF-MRI exposure. The study of the uptake pathway showed that the majority of liposomes were mainly uptake by clathrin-mediated endocytosis. In addition, the liposomes were modified with anti-ADAM8 antibodies (MAB 1031) to allow targeted delivery. The cellular binding capabilities of surface-modified and non-modified liposomes were tested on cells that had ADAM8 overexpression and on ADAM8 knockdown cells. Surface-modified liposomes showed a significant increase in binding ability, indicating significant targeting against cells that overexpress ADAM8 on their surface. In addition, cells with knockdown ADAM8 could not bind a significant amount of modified liposomes. The biocompatibility of liposomes was assessed using a hemolysis test, which showed neglected hemolytic potential and an activated thromboplastin time (aPTT), where liposomes showed minimal interference with blood clotting. Hemocompatibility studies may help to understand the correlation between in vitro and in vivo. The chorioallantois model was used in ovo to evaluate systematic biocompatibility in an alternative animal model. In the toxicity test, liposomes were injected intravenously into the chicken embryo. The liposomes showed a neglectable harmful effect on embryo survival. While free DOX has a detrimental effect on the survival of chicken embryos, this confirms the safety profile of liposomes compared to free DOX. LipTS-GD-CY-MAB were injected into the vascular system of the chicken embryo on egg development day 11 and scanned under UHF-MRI to evaluate the magnetic properties of the liposomes in a biological system with T2-weighted images (3D). The liposomal formulation had distinct magnetic properties under UHF MRI and the chick survived the scan. In summary, immunomagnetic heat-sensitive liposomes are a novel drug for the treatment of TNBC. It is used both for the diagnosis and therapy of solid and metastasizing tumors without side effects on the neighboring tissue. Furthermore, a tumor in the CAM model will be established. Thereafter, the selective targeting of the liposomes will be visualized and quantitated using fluorescence and UHF-MRI. Liposomes are yet to be tested on mice as a xenograft triple-negative breast cancer model, in which further investigation on the effect of DOX-LipTS-GD-CY-MAB is evaluated. On one hand, the liposomes will be evaluated regarding their targetability and their selective binding. On the other hand, the triggered release of DOX from the liposomes after UHF-MRI exposure will be quantitated, as well as evaluate the DOX-Liposomes therapeutic effect on the tumor

Desarrollo de nanocápsulas lipídicas como estrategia para facilitar el paso a través de la barrera hematoencefálica de fármacos que actúan a nivel del sistema nervioso central

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Farmacia Galénica y Tecnología Alimentaria, leída el 17/09/2018. Tesis formato europeo (compendio de artículos)Diseases affecting the central nervous system (CNS) should be regarded as a major health challenge due to their steadily rising incidences and to the current lack of effective treatments given the hindrance to brain drug delivery imposed by the blood-brain barrier (BBB). Some of the described delivery strategies to circumvent the BBB such as the direct intracerebral administration and the artificial disruption of the tight junctions involve high risk of neurological damage. Hence, every effort is currently being devoted to achieving efficient transport across the brain endothelium with targeted drug carriers following minimally-invasive intravenous injection. In particular, nanomedicine is chiefly germane to the field of chemotherapy wherein dose availability at the target site cannot be enhanced by dose increase for fear of severe side effects. Since efficient brain targeting should not solely rely on passive targeting, brain active targeting of nanomedicines into the CNS is being explored...Las patologías que afectan al sistema nervioso central representan un desafío terapéutico por su incidencia creciente y la limitación del acceso a sistema nervioso central de la mayoría de fármacos administrados por vía sistémica por parte de la barrera hematoencefálica. Algunas de las estrategias para sortear esta barrera, incluyendo la administración intracerebral o la disrupción artificial de sus uniones estrechas, suponen un elevado riesgo de daño neurológico. Por ello, actualmente se persigue diseñar transportadores de fármacos capaces de atravesar de manera eficiente el endotelio cerebral tras su administración intravenosa. En concreto, la vectorización de antineoplásicos en nanotransportadores para el tratamiento de tumores cerebrales supondría un sustancial avance en terapéutica por la reducción de efectos secundarios derivados de su distribución sistémica. Dado que la distribución de transportadores a sistema nervioso central no puede depender en exclusiva de la vectorización pasiva, a fin de favorecer su paso a través de la barrera hematoencefálica, se está investigando la incorporación de distintos ligandos a estos sistemas. El objetivo global de esta tesis doctoral es diseñar, desarrollar y evaluar a nivel preclínico un nanotransportador lipídico capaz de atravesar la barrera hematoencefálica para vectorizar fármacos a nivel del sistema nervioso central tras una administración intravenosa. Este objetivo general se desglosa en tres objetivos específicos Estudiar los parámetros experimentales determinantes en la obtención de nanocápsulas lipídicas mediante el método térmico de inversión de fases para habilitar la producción de nanocápsulas lipídicas bajo demanda. Desarrollar una novedosa estrategia de funcionalización de nanocápsulas lipídicas con cannabidiol para favorecer su distribución a sistema nervioso central y evaluar su potencial in vitro e in vivo. Encapsular cannabidiol en el núcleo oleoso de las nanocápsulas y evaluar in vitro su eficacia como sistemas de liberación prolongada con actividad frente a la línea celular U373MG de glioblastoma humano. Asimismo, se persigue evaluar la estrategia de funcionalización con cannabidiol para potenciar la captación por células de glioma. En cuanto a los resultados, la obtención de nanocápsulas lipídicas con tamaños de partícula predeterminados para aumentar las posibilidades de éxito de tratamientos de patologías del sistema nervioso central puede conseguirse mediante el método térmico de inversión de fases, pues el diámetro volumen se ajusta a un modelo matemático en una variable (el cociente másico entre la fase interna oleosa y el tensioactivo). Este modelo es válido para nanocápsulas blancas y cargadas con fármaco. Además, una disminución de tamaño de las nanocápsulas lipídicas en el intervalo 20-60 nm incrementa en 2,5 y 1,6-2,5 veces su paso a través de la barrera hematoencefálica in vitro e in vivo, respectivamente. Por otra parte, la funcionalización de las nanocápsulas lipídicas con el fitocannabinoide cannabidiol aumenta el paso a través de barrera hematoencefálica 4,3 y 2,5 veces in vitro e in vivo, respectivamente. Una disminución de tamaño de las nanocápsulas lipídicas incrementa 3 veces la captación por células de glioma. Asimismo, el tamaño de las nanocápsulas lipídicas condiciona la liberación de fármacos: nanocápsulas de 20 nm cargadas con cannabidiol reducen invariablemente 3 veces los valores de concentración inhibitoria 50 en comparación con sus homólogas de 50 nm. Además, la funcionalización de nanocápsulas lipídicas con cannabidiol aumenta la captación por células de glioma 3,4 veces. Como conclusión, las nanocápsulas lipídicas, cargadas y funcionalizadas con cannabidiol, son prometedores candidatos para el tratamiento de gliomas con capacidad de vectorización a través de barrera hematoencefálica y de células de glioma. Su potencial terapéutico debe ser evaluado en modelos animales de glioma.Depto. de Farmacia Galénica y Tecnología AlimentariaFac. de FarmaciaTRUEunpu

Folate-conjugated nanoparticles as a potent therapeutic approach in targeted cancer therapy

The selective and efficient drug delivery to tumor cells can remarkably improve different cancer therapeutic approaches. There are several nanoparticles (NPs) which can act as a potent drug carrier for cancer therapy. However, the specific drug delivery to cancer cells is an important issue which should be considered before designing new NPs for in vivo application. It has been shown that cancer cells over-express folate receptor (FR) in order to improve their growth. As normal cells express a significantly lower levels of FR compared to tumor cells, it seems that folate molecules can be used as potent targeting moieties in different nanocarrier-based therapeutic approaches. Moreover, there is evidence which implies folate-conjugated NPs can selectively deliver anti-tumor drugs into cancer cells both in vitro and in vivo. In this review, we will discuss about the efficiency of different folate-conjugated NPs in cancer therapy. © 2015, International Society of Oncology and BioMarkers (ISOBM)

Design of Nanoparticle-Based Carriers for Targeted Drug Delivery

Nanoparticles have shown promise as both drug delivery vehicles and direct antitumor systems, but they must be properly designed in order to maximize efficacy. Computational modeling is often used both to design new nanoparticles and to better understand existing ones. Modeled processes include the release of drugs at the tumor site and the physical interaction between the nanoparticle and cancer cells. In this paper, we provide an overview of three different targeted drug delivery methods (passive targeting, active targeting, and physical targeting) and compare methods of action, advantages, limitations, and the current stages of research. For the most commonly used nanoparticle carriers, fabrication methods are also reviewed. This is followed by a review of computational simulations and models on nanoparticle-based drug delivery

Nanopharmaceuticals: A Boon to the Brain-Targeted Drug Delivery

Brain is well known for its multifarious nature and complicated diseases. Brain consists of natural barriers that pose difficulty for the therapeutic agents to reach the brain tissues. Blood-brain barrier is the major barrier while blood-brain tumor barrier, blood-cerebrospinal (CSF) barrier and efflux pump impart additional hindrance. Therapeutic goal is to achieve a considerable drug concentration in the brain tissues in order to obtain desired therapeutic outcomes. To overcome the barriers, nanotechnology was employed in the field of drug delivery and brain targeting. Nanopharmaceuticals are rapidly emerging sub-branch that deals with the drug-loaded nanocarriers or nanomaterials that have unique physicochemical properties and minute size range for penetrating the CNS. Additionally, nanopharmaceuticals can be tailored with functional modalities to achieve active targeting to the brain tissues. The magic behind their therapeutic success is the reduced amount of dose and lesser toxicity, whereby localizing the therapeutic agent to the specific site. Different types of nanopharmaceuticals like polymeric, lipidic and amphiphilic nanocarriers were administered into the living organisms by exploiting different routes for improved targeted therapy. Therefore, it is essential to throw light on the properties, mechanism and delivery route of the major nanopharmaceuticals that are employed for the brain-specific drug delivery

Morphologies and functionalities of polymeric nanocarriers as chemical tools for drug delivery: A review

In these years a variety of polymeric nanocarriers such as dendrimers, polymeric micelles, nanoparticles, nanogels, nanocapsules and vesicles are widely investigated as potential drug delivery systems. In addition to the different morphologies and sizes, these carriers may have on their surfaces specific functionalizations to improve the drug loading and controlled release and specific ligands for cell receptors, in order to achieve a precise targeting. This review focuses on recent functionalized polymeric nanomaterials used as drug delivery systems, with an emphasis on morphology and surface modifications of polymeric nanocarriers to improve controlled drug delivery. Moreover, this work offers a number of suggestions on how to achieve the systematization of data on the most relevant physico-chemical parameters, which govern and control the interaction between carrier and drug, with the aim to give the reader an overview of the most significant advances in this field. Keywords: Nanostructured polymers, Drug delivery systems, Dendrimers, polymeric micelles, Polymeric nanoparticles, Nanogels, Polymeric nanocapsules, Vesicle

Multifunctional Nanocarriers for diagnostics, drug delivery and targeted treatment across blood-brain barrier: perspectives on tracking and neuroimaging

Nanotechnology has brought a variety of new possibilities into biological discovery and clinical practice. In particular, nano-scaled carriers have revolutionalized drug delivery, allowing for therapeutic agents to be selectively targeted on an organ, tissue and cell specific level, also minimizing exposure of healthy tissue to drugs. In this review we discuss and analyze three issues, which are considered to be at the core of nano-scaled drug delivery systems, namely functionalization of nanocarriers, delivery to target organs and in vivo imaging. The latest developments on highly specific conjugation strategies that are used to attach biomolecules to the surface of nanoparticles (NP) are first reviewed. Besides drug carrying capabilities, the functionalization of nanocarriers also facilitate their transport to primary target organs. We highlight the leading advantage of nanocarriers, i.e. their ability to cross the blood-brain barrier (BBB), a tightly packed layer of endothelial cells surrounding the brain that prevents high-molecular weight molecules from entering the brain. The BBB has several transport molecules such as growth factors, insulin and transferrin that can potentially increase the efficiency and kinetics of brain-targeting nanocarriers. Potential treatments for common neurological disorders, such as stroke, tumours and Alzheimer's, are therefore a much sought-after application of nanomedicine. Likewise any other drug delivery system, a number of parameters need to be registered once functionalized NPs are administered, for instance their efficiency in organ-selective targeting, bioaccumulation and excretion. Finally, direct in vivo imaging of nanomaterials is an exciting recent field that can provide real-time tracking of those nanocarriers. We review a range of systems suitable for in vivo imaging and monitoring of drug delivery, with an emphasis on most recently introduced molecular imaging modalities based on optical and hybrid contrast, such as fluorescent protein tomography and multispectral optoacoustic tomography. Overall, great potential is foreseen for nanocarriers in medical diagnostics, therapeutics and molecular targeting. A proposed roadmap for ongoing and future research directions is therefore discussed in detail with emphasis on the development of novel approaches for functionalization, targeting and imaging of nano-based drug delivery systems, a cutting-edge technology poised to change the ways medicine is administered

Graphene Oxide as a Nanocarrier for Biochemical Molecules: Current Understanding and Trends

The development of an advanced and efficient drug delivery system with significant improvement in its efficacy and enhanced therapeutic value is one of the critical challenges in modern medicinal biology. The integration of nanomaterial science with molecular and cellular biology has helped in the advancement and development of novel drug delivery nanocarrier systems with precision and decreased side effects. The design and synthesis of nanocarriers using graphene oxide (GO) have been rapidly growing over the past few years. Due to its remarkable physicochemical properties, GO has been extensively used in efforts to construct nanocarriers with high specificity, selectivity, and biocompatibility, and low cytotoxicity. The focus of this review is to summarize and address recent uses of GO-based nanocarriers and the improvements as efficient drug delivery systems. We briefly describe the concepts and challenges associated with nanocarrier systems followed by providing critical examples of GO-based delivery of drug molecules and genes. Finally, the review delivers brief conclusions on the current understanding and prospects of nanocarrier delivery systems.O

In-Vitro Application of Magnetic Hybrid Niosomes: Targeted siRNA-Delivery for Enhanced Breast Cancer Therapy

Even though the administration of chemotherapeutic agents such as erlotinib is clinically established for the treatment of breast cancer, its efficiency and the therapy outcome can be greatly improved using RNA interference (RNAi) mechanisms for a combinational therapy. However, the cellular uptake of bare small interfering RNA (siRNA) is insufficient and its fast degradation in the bloodstream leads to a lacking delivery and no suitable accumulation of siRNA inside the target tissues. To address these problems, non-ionic surfactant vesicles (niosomes) were used as a nanocarrier platform to encapsulate Lifeguard (LFG)-specific siRNA inside the hydrophilic core. A preceding entrapment of superparamagnetic iron-oxide nanoparticles (FexOy-NPs) inside the niosomal bilayer structure was achieved in order to enhance the cellular uptake via an external magnetic manipulation. After verifying a highly effective entrapment of the siRNA, the resulting hybrid niosomes were administered to BT-474 cells in a combinational therapy with either erlotinib or trastuzumab and monitored regarding the induced apoptosis. The obtained results demonstrated that the nanocarrier successfully caused a downregulation of the LFG gene in BT-474 cells, which led to an increased efficacy of the chemotherapeutics compared to plainly added siRNA. Especially the application of an external magnetic field enhanced the internalization of siRNA, therefore increasing the activation of apoptotic signaling pathways. Considering the improved therapy outcome as well as the high encapsulation efficiency, the formulated hybrid niosomes meet the requirements for a cost-effective commercialization and can be considered as a promising candidate for future siRNA delivery agents