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...

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Bibliographic Details
Main Author: Alawak, Mohamad
Contributors: Bakowsky, Udo (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2019
Online Access:PDF Full Text
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Summary: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.
Physical Description:136 Pages