title: Multifunctional thermosensitive liposomes for antitumor therapy
title: Multifunktionale thermosensible Liposomen für die Antikrebstherapie
titleType: AlternativeTitle
Creators: name: Shi, Tan
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: Brustkrebs;
Thermosensitive liposomes
Arzneimittelverabreichungssystem
Doxorubicin
Thermosensible Liposomen
3D cell culture
Drug delivery system
Mild hyperthermia
Breast cancer
Mehrzellige Tumorsphäroide
3D-Zellkultur
Pharmacology &amp; therapeutics, prescription drugs
Pharmakologie, Therapeutik
Solid
Leichte Hyperthermie
Multicellular tumor spheroids

description: n this dissertation, thermosensitive liposomal drug delivery systems for anticancer therapy were developed and investigated in 3D cell culture models. Multicellular tumour spheroids (MCTS) were generated to mimic solid tumour environments in vivo as closely as possible. Spheroid culturing offers many advantages over conventional 2D cell cultures, including a 3D cell architecture comparable to a tumour, more natural cell morphology, nutrient and drug diffusion gradients, as well as cell-cell junctions and cell-ECM interactions. The TNBC cell line model MDA-MB-231 was used to generate the spheroids in all experiments. MDA-MB-231 spheroids were generated in agarose-coated multi-well plates and supplemented with collagen I. The spheroid is divided into three zones from the inner to the outer region: the proliferation zone, the resting zone and the necrosis zone. The first project dealt with the generation of thermosensitive liposomes (TSL) and their loading with the chemotherapeutic drug doxorubicin (DOX), a commonly used chemotherapeutic agent for various types of cancer. The use of pure DOX leads to severe side effects such as cardio- and hepatotoxicity, which can be reduced by advanced, biocompatible drug delivery systems that transport DOX directly to the tumour site. For this purpose, thermosensitive liposomes containing DOX were prepared (DOX-TSL). The empty liposomes were prepared by the conventional thin-layer hydration method. DOX was then incorporated by "remote loading" method, which uses an ammonium sulphate gradient between the inside and outside of the liposome for loading. The ultracentrifuge method was used to separate unencapsulated DOX and purify the DOX-TSL liposomes.
The hydrodynamic diameter and zeta potential of the liposomes were determined by dynamic light scattering (DLS) and laser Doppler velocimetry (LDV). The phase transition temperature (Tm) of the thermosensitive liposomes was measured by differential scanning calorimetry (DSC). 
The entrapment efficiency (EE %) of DOX in the liposomal formulations was determined by UV-VIS spectrophotometry after purification of unencapsulated DOX. The long-term storage stability in an aqueous medium and short-term stability in a serum-supplemented cell culture medium of DOX-TSL were monitored and further analysed by DLS and LDV.
Morphological characteristics of each DOX-TSL formulation were visualised by atomic force microscopy (AFM) and transmission electron microscopy (TEM) before and after temperature elevation and drug release, respectively. The results showed a well-separated spherical structure of DOX-TSL. The AFM results were comparable to the size distribution obtained by DLS.
The DLS and LDV analytical results showed a uniform mono-disperse size distribution of DOX-TSL liposomes. This was maintained for six weeks in an aqueous storage solution and for 24 hours in a serum-supplemented cell culture medium. The phase transition temperature Tm of DOX-TSL liposomes was determined by DSC at 39.8 °C. Mild hyperthermia at 40 °C was chosen to trigger the release. Before increasing the temperature above Tm, DOX-TSL showed intact structures, whereas after heating, the integrity of DOX-TSL was destroyed. Simultaneously with the deformation of the liposome, the encapsulated DOX was released.

DOX-TSL was further applied to MDA-MB-231 spheroids to evaluate their efficacy in a 3D cell culture model. Quantification was performed using 3D cell viability assays and live/dead staining assays, which demonstrated the increased cytotoxicity of DOX-TSL on spheroids at elevated temperatures (40, 41 and 42 °C). The penetration ability of DOX on spheroids was temperature dependent, with higher temperatures (40 °C) providing deeper penetration than at 37 °C.
The second project addressed the triggering of DOX-TSL drug release using near-infrared (NIR) irradiation. To make DOX-TSL liposomes sensitive to NIR, indocyanine green (ICG) and DOX were entrapped in thermosensitive liposomes. NIR irradiation effectively disrupted liposomes, triggering the release of DOX at the tumour site.
In summary, the developed thermosensitive liposomes with DOX inclusion showed high efficacy against 3D cell culture models as tumour models. The integration of ICG introduced phototherapeutic properties into the new drug delivery system (ICG/DOX-mTSL), offering the potential to improve treatment precision at the target site. Further studies on 3D cell culture models under more physiological conditions are promising and could open a new chapter for drug evaluation in 3D cell culture.