Magnetoliposomes as new approach for bone cancer therapies

University of Minho School of Engineering João Carlos Macedo Barros Magnetoliposomes as New Approach for Bone Cancer Therapies january 2024 University of Minho School of Engineering João Carlos Macedo Barros Magnetoliposomes as New Approach for Bone Cancer Therapies Masters Dissertation Masters in Physics Engineering Dissertation supervised by Dr. Vanessa Fernandes Cardoso Professor Senentxu Lanceros Méndez january 2024 Copyright and Terms of Use for Third Party Work This dissertation reports on academic work that can be used by third parties as long as the internationally accepted standards and good practices are respected concerning copyright and related rights. This work can thereafter be used under the terms established in the license below. Readers needing authorization conditions not provided for in the indicated licensing should contact the author through the Repositório UM of the University of Minho. License granted to users of this work: CC BY https://creativecommons.org/licenses/by/4.0/ i Acknowledgements I would like to express my gratitude to my supervisors, Dr. Vanessa Cardoso, Professor Senentxu and also Dr. Beatriz Cardoso for their unwavering support throughout the last year of this project. I would also like to acknowledge Dr. Clarisse Ribeiro for her assistance at IBS, and Professor Elisabete Coutinho for providing access to the laboratory. Additionally, thanks to BCMaterials for conducting sample analysis. A heartfelt thank you to my family, girlfriend, and friends for their endless motivation, care, and patience during the execution of the project. ii Statement of Integrity I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. University of Minho, Braga, january 2024 iii Assinado por: João Carlos Macedo Barros Num. de Identificação: 14904569 Data: 2024.03.10 17:55:36+00'00' Abstract Cancer, notably osteosarcoma, poses detection and treatment challenges, with its relentless progression and resistance to conventional therapies. Innovative approaches are imperative to overcome limitations like toxicity and adverse effects on healthy cells. Nanotechnology, particularly magnetoliposomes, offers promise in this pursuit. Osteosarcoma’s intricate nature demands precision and enhanced effectiveness in treatment strategies. Conventional therapies, such as radiotherapy, often fall short of delivering optimal results due to their limitations, such as the resistance of bone cancer to radiation requiring higher doses, prompting the exploration of alternative methods. Notably, liposomes have proven to be a successful vehicle for drug delivery, significantly improving the efficacy of chemotherapeutic agents like doxorubicin. The integration of superparamagnetic nanoparticles further enhances the potential of these systems by enabling localized drug delivery. This innovative approach not only addresses the challenges associated with conventional treatments but also marks a significant stride towards improving the precision and overall effectiveness of combating osteosarcoma. This project involves the processing of magnetoliposomes containing encapsulated doxorubicin, magnesium, and calcium ferrites (with a diameter of 8.75 ±0.35nm, saturation magnetization of 4.39emu/g, and Eg= 1.35eV ), along with a comprehensive study of their respective physical characteristics. The results reveal magnetoliposomes with a diameter of 542.54 ±71.78nm and a polydispersity index of 27.48 ±3.84%. Notably, the systems exhibit nearly complete encapsulation of doxorubicin and a magnetic nanoparticle encapsulation efficiency of 49.00±5.17%. The investigation into drug release kinetics under static and dynamic conditions unveils significant variations in release rates. Particularly, when subjected to an alternating magnetic field with different frequencies and on-cycles (time the magnetic stimuli are applied), the drug release rates are nearly 30% higher under magnetic stimulation over 5hcompared to static conditions. This emphasizes how external stimuli affect controlled substance release, showcasing their potential in targeted drug delivery. Keywords: Magnetoliposomes, Osteosarcoma, Cancer, Doxorubicin, Magnetic Nanoparticles, Magnetic Bioreactor. iv Resumo O cancro, incluindo o osteossarcoma, apresenta desafios na deteção precoce e tratamento eficaz. Abordagens inovadoras são necessárias devido à resistência às terapias convencionais. A nanomedicina, como os magnetolipossomas, surge como uma alternativa promissora, superando limitações dos tratamentos tradicionais. O tratamento do osteossarcoma requer precisão e eficácia aprimoradas, pois terapias convencionais, como a radioterapia, são pouco eficazes. Isso impulsiona a exploração de métodos alternativos, como os magnetolipossomas na nanotecnologia. Lipossomas têm se mostrado eficazes na entrega de fármacos, melhorando a eficácia de agentes quimioterapeuticos como a doxorrubicina. A integração de nanopartículas superparamagnéticas eleva ainda mais o potencial desses sistemas ao permitir a entrega localizada de fármacos. Esta abordagem inovadora não só enfrenta os desafios associados aos tratamentos convencionais, mas também representa um avanço significativo para melhorar a precisão e a eficácia global no combate ao osteossarcoma. Este projeto envolve a síntese de magnetolipossomas contendo doxorrubicina encapsulada e nanopartículas de ferrites de cálcio e magnésio (com um diâmetro de 8.75 ±0.35nm, magnetização de saturação de 4.39emu/g, e Eg= 1.35eV ), juntamente com um estudo das suas características físicas. Os resultados revelam magnetoliposomas com um diâmetro de 542.54 ±71.78nm e um índice de polidispersividade de 27.48 ±3.84%. Notavelmente, os sistemas exibem uma encapsulação quase completa da doxorrubicina e uma eficiência de encapsulação de nanopartículas magnéticas de 49.00 ±5.17%. A investigação das cinéticas de libertação de fármacos sob condições estáticas e dinâmicas revela variações significativas nas taxas de libertação. Em particular, quando sujeitos a um campo magnético alternado com diferentes frequências e ciclos-ON (tempo em que os estímulos magnéticos são aplicados), as taxas de libertação de fármacos no caso com estimulação magnética é ≈30% superior relativamente ao estático ao fim de 5h. Isso realça o impacto de estímulos externos na libertação controlada de substâncias encapsuladas, destacando as potenciais aplicações desses sistemas na entrega direcionada de medicamentos. Palavras Chave: Magnetolipossomas, Osteossarcoma, Cancro, Doxorrubicina, Nanopartículas Magnéticas, Biorreator Magnético. v Contents 1 Introduction 1 1.1 Contextualization ................................... 1 1.2 Motivation and Objective ............................... 3 1.3 Dissertation Structure ................................. 4 2 Theoretical Framework 5 2.1 Nanomedicine .................................... 5 2.2 Osteosarcoma .................................... 6 2.2.1 Cancer Overview ............................... 6 2.2.2 Bone Cancer ................................. 7 2.2.3 Osteosarcoma ................................ 7 2.2.4 Current Osteosarcoma Therapies ....................... 9 2.2.5 Challenges in Cancer Therapy ........................ 10 2.2.6 New Approaches for Osteosarcoma Therapy .................. 11 2.3 Liposomes ...................................... 13 2.3.1 Phospholipids Composition .......................... 13 2.3.2 Liposome Organization ............................ 15 2.3.3 Liposomes as Nanocarriers .......................... 16 2.4 Magnetic Nanoparticles ................................ 18 2.4.1 Types of Magnetism ............................. 18 2.4.2 Superparamagnetism ............................. 23 2.4.3 Superparamagnetic Iron Oxide Nanoparticles ................. 25 2.4.4 Magnetic Hyperthermia ............................ 26 2.5 Magnetoliposomes .................................. 26 2.5.1 Mechanisms of Action of Magnetoliposomes ................. 28 vi List of Tables 2.1 Studies of immunotherapy adaptations for OS ..................... 12 2.2 Liposome-encapsulated drugs and their uses. ..................... 18 3.1 Reagents used with the respective molar mass, concentration used and mass. ..... 34 3.2 Components used in the procedure. .......................... 35 3.3 Quantities and volumes of each lipid from chloroform solution to achieve the desired percentage. ...................................... 35 3.4 Reagent for each solution with respective molar mass, concentration and mass used in the 200mL solution. ................................. 39 3.5 Resulting pH after combining the respective volumetric ratio of each solution. . . . . . . 39 4.1 Combined table with size and PDI. .......................... 55 4.2 Combined table with size and PDI. .......................... 56 4.3 Obtained results from the EE(%)of DOX. ....................... 66 4.4 Obtained results from the absorption spectroscopy measurements for the 4 different samples. ....................................... 67 xiii List of Abbreviations AML Aqueous Magnetoliposome AMF Alternating Magnetic Field CHEMS Cholesteryl Hemisuccinate DLS Dynamic Light Scattering DNA Deoxyribonucleic Acid DOX Doxorubicin DPPC Dipalmitoylphosphatidylcholine DSPE-PEG2000 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[amino(polyethylene glycol)-2000] DSMA Dimercaptosuccinic Acid DMSO Dimethyl Sulfoxide DSC Differential Scanning Calorimetry DSPC Distearoylphosphatidylcholine EE Encapsulation Efficiency EFS Event Free Survival EPR Enhanced Permeability and Retention FDA Food and Drug Administration FTIR Fourier Transform Infrared Spectroscopy HBP Hydroxybenzophenone IR Infrared MLP Magnetoliposome MPI Magnetic Particle Imaging MRI Magnetic Resonance Imaging NP Nanoparticle OS Osteosarcoma PDI Polydispersity Index RNA Ribonucleic Acid SML Solid Magnetoliposome SPION Superparamagnetic Iron Oxide Nanoparticle TEM Transmission Electron Microscopy UV-Vis Ultraviolet to Visible VSM Vibrating Sample Magnetometer XRD X-ray Diffraction xiv xv Chapter 1 Introduction 1.1 Contextualization In 2019, cancer emerged as the primary cause of death in 57 countries and the second most prevalent cause of mortality among individuals under 70 years old in 55 countries (Figure 1.1). Globally, it was estimated that there were approximately 19.3 million cases and 10 million deaths attributable to cancer in 2020 [1]. Figure 1.1: Global ranking of cancer as a cause of death at ages <70 years in 2019 [1]. Among all the cases of cancer in both children and adults, bone tumours make up a small percentage, approximately 3-5% in children and less than 1% in adults. Osteosarcoma (OS) is the most common type of malignant bone tumour that is diagnosed. [2]. The treatment methodology has remained largely unchanged for several decades. It yields a 5-year 1 event-free survival (EFS) rate of approximately 70% for localized tumours, while patients with metastatic or recurrent disease experience less than 20% [3]. The current treatment approach for OS involves performing a surgical resection to eliminate all cancerous tissue, in combination with systemic chemotherapy and occasionally radiotherapy to address any micrometastatic disease (cases where cancer cells spread to distant areas of the body unrelated to the primary tumours) [1]. Despite its effectiveness in eliminating significant cancer cell masses, surgical intervention is an invasive procedure that carries a heightened risk of infections and the potential for incomplete tumour removal, which may result in cancer reappearing [4]. Furthermore, chemotherapy poses an additional challenge for OS therapy. The drugs typically exhibit systemic effects, affecting the entire body rather than exclusively targeting cancer cells, leading to significant side effects [2]. Magnetoliposomes (MLPs) have emerged as a highly promising and versatile tool in the field of oncology, offering a large potential for the treatment of various types of cancer. MLPs possess unique characteristics that enable them to transport anti-cancer drugs, facilitate hyperthermia treatment [5], and aid in disease detection through advanced imaging techniques [6]. They demonstrate exceptional biocompatibility, ensuring their compatibility with biological systems. The use of biodegradable and non-toxic lipid components in MLP formulations significantly reduces the risk of adverse reactions and immune responses. This biocompatibility profile enhances the safety of these systems, making them suitable for clinical applications [7]. MLPs offer a valuable contribution to disease detection through their incorporation of superparamagnetic nanoparticles (NPs). These NPs enable the visualization of MLPs using imaging techniques such as magnetic resonance imaging (MRI) or magnetic particle imaging (MPI) [8]. This allows for accurate and non-invasive assessment of tumour size, location, and response to therapy. MLPs serve as effective imaging agents, providing critical information for monitoring disease progression and evaluating treatment effectiveness. Integrating MLPs with imaging techniques represents a significant advancement in diagnostic capabilities, facilitating personalized and precise imaging [9]. One of the most significant advantages of MLPs lies in their ability to act as carriers for anti-cancer drugs. They can also offer a controlled and targeted drug delivery system by encapsulating therapeutic agents within their lipid bilayer structures. This approach allows for the precise administration of high drug concentrations directly to the tumour site while minimizing systemic toxicity and reducing adverse side effects. MLPs effectively enhance drug efficacy and bio-availability, thus improving therapeutic outcomes [10]. MLPs can be classified based on the molecules added to their structure. Active targeting MLPs 2 can be modified with targeting compounds that have a higher affinity for specific types of cells, such as cancerous cells. On the other hand, they can be locally administered, and possess a passive targeting [11]. MLPs and similar systems tend to accumulate more in tumorous tissues compared to normal tissues. This phenomenon is known as the Enhanced Permeability and Retention (EPR) effect. It is caused by abnormal molecular and fluid transport dynamics in tumour tissues [12]. The magnetic targeting of MLPs can enhance the therapeutic agents of these systems in tumourous regions using an external magnetic field. This helps guide the MLPs to the targeting site, thereby synergizing the EPR effect [13]. In addition to their drug delivery capabilities, MLPs have shown great promise in hyperthermia treatment, a therapeutic approach that utilizes heat to selectively target and destroy cancer cells. By taking advantage of their magnetic properties, MLPs can be guided and localized within the tumour region using external magnetic fields. Once accumulated at the desired site, MLPs can be subjected to an alternating magnetic field, leading to the generation of heat and the induction of hyperthermia. This localized hyperthermia treatment approach holds immense potential as a non-invasive and targeted strategy for eliminating cancer cells while minimizing damage to surrounding healthy tissues [14]. 1.2 Motivation and Objective The motivation behind this project lies in the necessity to develop new methods for more efficient cancer therapy, particularly for targeting OS. Drug delivery systems have become increasingly important, particularly in the field of nanomedicine. Their primary purpose is to improve the pharmacokinetic and pharmacodynamic properties of drugs by ensuring targeted drug delivery, controlled drug release, and improved drug stability. Overall, these systems have been instrumental in enhancing therapeutic efficacy [15]. The chosen approach for study involves the use of MLPs due to their numerous advantages, as outlined in the contextualization and further discussed in upcoming chapters. The objective of the project is to develop these systems and characterize them physically and chemically, while also analysing their ability to release drugs passively and in a controlled manner through magnetic stimulation using a magnetic bioreactor. The study on MLPs is a promising approach for improving cancer treatment. By studying their physicochemical properties and drug-release capabilities, the research of these systems hopes to develop more effective methods for combating OS. 3 1.3 Dissertation Structure The dissertation will consist of the following chapters, which can be summarized as follows: Chapter 1 - Introduction This chapter will provide context, motivation and objective for the project, as well as present the structure of the dissertation. Chapter 2 - Theoretical Framework This chapter will present the theoretical concepts of the project, including nanomedicine, OS, liposomes, NPs, doxorubicin (DOX), MLPs and the magnetic bioreactor. Chapter 3 - Experimental Procedures, Methods and Characterization Techniques In this part it will be presented the various laboratory techniques and materials used for processing superparamagnetic NPs and MLPs. Also, this chapter explores theoretical concepts related to physically characterizing systems and equipment used in the project. Chapter 4 - Results and Discussion This chapter will present and discuss the results and their analysis. Chapter 5 - Conclusion and Future Prespectives In the final chapter, the project’s conclusion and future work will be presented. 4 Chapter 2 Theoretical Framework 2.1 Nanomedicine Nanotechnology is a rapidly advancing interdisciplinary field that consists in the manipulation and creation of systems and devices at the nanoscale [16]. It holds the potential to revolutionize various domains such as medicine [17], communication [18], electronics [19], robotics [20] and many more. It possesses evident advantages such as cost-effectiveness, due to less material used, and accelerated functionality in mechanical, chemical, and biological components [21]. Furthermore, nanotechnology offers a group of advantages when materials are approached to the nanoscale. However, as materials shrink to this scale, their properties change. These changes encompass alterations in structural, magnetic, thermal, optical, electronic, mechanical, and even biological aspects [22]. These modifications are intricately tied to the interactions of a limited number of atoms at surfaces and interfaces [22]. At this scale, the dominance of electromagnetic energy quantization plays a more pronounced role, molecular motion follows a more unpredictable pattern, and the concept of wave-particle duality becomes increasingly apparent. This realm showcases a pivotal shift in the ratio of surface area to volume, significantly influencing reactivity and the point at which materials melt. The phenomenon of quantum confinement emerges, imposing restrictions on the movement of electrons. Simultaneously, fluctuations in ionization energy occur as a function of the size of these types of materials. The ionization energy can either increase or decrease depending on the specific material and its size at the nanoscale [23,24]. Nanotechnology has revolutionized various fields, including nanomedicine, by introducing innovative techniques and tools. This branch of medicine uses nanotechnology in order to prevent diseases [25] and it is applied to other fields such as imaging [26], diagnosis [27], monitoring [28], treatment [29], repair, and regeneration of biological systems [30]. Research so far has focused on the development of biosensors to aid in diagnostics and vehicles to administer vaccines, medications [15], and gene therapy, 5 including the development of nanosystems to assist in cancer treatment [31,32]. It is crucial to the evolution of disease treatment, especially in the case of oncologic diseases such as OS, that is understood and utilized the concept of nanomedicine. Traditional methods have shown limited success in treating this condition, highlighting the need for new and improved ways to combat this disease. 2.2 Osteosarcoma 2.2.1 Cancer Overview Cancer is a term used to describe a group of diseases that can affect any part of the body. It is also known as malignant tumours or neoplasms [33]. Typically, human cells undergo growth and multiplication, achieved through the process of cell division, to generate new cells in accordance with the body’s needs. In cases where cells age or become damaged, they enter a state called apoptosis (cell death) followed by the introduction of fresh cells to take their place. However, there are instances when this well-regulated process malfunctions, leading to the abnormal proliferation of flawed or impaired cells in situations where such growth should not occur [34]. A fundamental indicator of cancer is the accelerated proliferation of irregular cells that surpass their typical confines and have the capacity to infiltrate neighbouring body regions. This infiltration can result in metastasis, a phenomenon where cancer spreads to distant organs. Generally, metastases are the leading cause of death from these diseases. This process is described by multiple stages: from 0 to 4 describing the evolution of the disease in the body [35]: •Stage 0 signifies a localized state of cancer referred to as ” in situ ”, indicating that cancer remains confined to its original location. At this point, cancer has not extended to neighbouring tissues and is frequently treatable. Surgical intervention typically results in the complete removal of the tumour. •Stage I characterizes cancer that has not significantly penetrated surrounding tissues and has not metastasized to lymph nodes or distant areas within the body. This phase is commonly identified as early-stage cancer. •Stage II and Stage III denote cancers that have penetrated surrounding tissues to a greater extent. In some cases, these stages also involve the spread to nearby lymph nodes but not to other body regions. •Stage IV designates a critical point where cancer has disseminated to other organs or body parts. This phase is also referred to as advanced or metastatic cancer, indicating an advanced spread 6 beyond the original site. The development of cancer involves a multi-stage process, starting from a pre-cancerous lesion and progressing to a malignant tumour. The change in a person’s genetic configuration is influenced by external factors that fall into three categories: physical carcinogens (like UV and ionizing radiation), chemical carcinogens (such as asbestos, tobacco smoke, alcohol, aflatoxin, and arsenic), and biological carcinogens (including infections caused by viruses, bacteria, or parasites) [36]. 2.2.2 Bone Cancer Primary bone cancers are very uncommon tumours, making up less than 0.2% of all cancers. However, it is hard to determine their true incidence due to their rarity. These cancers have a diverse range of clinical presentations, but with appropriate treatment, they are usually curable. The most frequently occurring types of bone cancer are OS (35%), chondrosarcoma (30%), and Ewing sarcoma (16%) [37]. Tumours of this type often cause a range of symptoms, including pain, hypercalcaemia (abnormally high levels of calcium in the blood), bone fractures, and more. These symptoms have a significant impact on the quality of life of patients [38]. Diagnosing these specific tumours is frequently challenging due to their initial symptoms resembling those of other muscle and skeletal conditions. X-rays are commonly relied upon for diagnosis, given their widespread utilization in such cases [39]. 2.2.3 Osteosarcoma The most widespread form of bone cancer among children and adolescents is OS, a type of primary malignant bone tumour. This disease causes abnormal bone cell growth, resulting in the creation of malignant bone tissues in the affected area, as seen in Figure 2.1 (provided as an example). OS primarily targets the bones surrounding the knees, tibias, femurs, legs, or arms, but it can also appear in rare instances in the spinal cord, pelvis and other regions. One of the most common signs of OS is the presence of a mass, which is almost always firm and tender to the touch [41]. Patients with OS may also experience functional impairments, such as limping or difficulty moving the affected limb. These limitations in mobility can arise from the tumour’s location and size, impacting the surrounding tissues and bone structure [42]. Consequently, loss of function and a decreased range of motion are often observed in affected individuals. 7 lipid’s physical state from the ordered gel phase, where the hydrocarbon chains are fully extended and closely packed, to the disordered liquid crystalline phase, where the hydrocarbon chains are randomly oriented and fluid [81]. Dipalmitoylphosphatidylcholine (DPPC) (Figure 2.3) is the most commonly used phospholipid due to its drug encapsulation capacity, thermosensitivity (Tm= 41◦C), and suitability for biomedical applications and controlled release [82]. Figure 2.3: DPPC molecular structure. Also, Distearoylphosphatidylcholine (DSPC) (Figure 2.4) has a higher transition temperature (Tm=55°C). When combined with DPPC in a binary formulation, it results in more rigid vesicles, which in turn enhances the amount of drug release [83]. It has also been found that adjusting the ratio between DPPC and DSPC can regulate the number of nanoscale gaps induced in liposome membranes, leading to improved release kinetics. These findings suggest that the careful selection and manipulation of lipid components can significantly impact drug delivery efficacy [84]. Figure 2.4: DSPC molecular structure. Additionally, when 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DPSE-PEG2000) (Figure 2.5) is added to the lipid formulation it prevents the aggregation and enhances stability and half-life comparatively to conventional liposomes [85]. Figure 2.5: DSPE-PEG2000 molecular structure. 14 Furthermore, an acidic cholesterol ester known as cholesteryl hemisuccinate (CHEMS) can also be used, whose molecular structure is presented in Figure 2.6. This will allow for the acquisition of a lamellar structure of liposomes upon hydration in aqueous solutions with a neutral or alkaline pH [86]. Figure 2.6: CHEMS molecular structure. Thus, within the scope of this dissertation and considering the aforementioned, the previously mentioned combination was utilized due to the benefits presented by the combination of these lipids. 2.3.2 Liposome Organization Phospholipids, when in contact with an aqueous environment, arrange themselves spontaneously. This organization is a consequence of the hydrophilic interaction among polar groups and the Van der Waals forces that come into play between hydrocarbon chains. This intricate process can be explained from a thermodynamic perspective: it is primarily driven by the natural tendency of hydrophobic lipid chains to avoid contact with water, causing them to repel each other [87]. The result of this interaction is the formation of thin, flexible lipid bilayers, as illustrated in Figure 2.7. These bilayers establish a unique structure that acts as a semi-permeable barrier, selectively allowing certain substances to pass through while restricting the movement of ions and other compounds such as polar molecules. These bilayers are immersed in an aqueous medium on both their outer and inner sides, effectively creating a water-filled environment on both surfaces. This arrangement has significant implications in various biological and chemical contexts [87]. 15 Figure 2.7: Illustration of phospholipid self-arrangement in an aqueous solution. Furthermore, during their formation, liposomes can encapsulate hydrophilic materials such as certain drugs in the aqueous solution, or hydrophobic compounds within the layers of their lipid bilayer, as illustrated in Figure 2.8 [88]. Figure 2.8: Carrier liposome with hydrophilic drug and NPs loaded in the core region of liposome and hydrophobic drug loaded. The biomedical potential of various drugs and compounds can be significantly improved by enclosing these systems, which can help to protect them from degradation and enhance their therapeutic efficacy. 2.3.3 Liposomes as Nanocarriers Liposomes are versatile and promising nanocarriers for drug delivery and other biomedical applications. As research in this area continues, liposomes play an increasingly important role in nanomedicine due to numerous advantages presented next [89]: 16 • Biocompatibility: Liposomes are composed of the same material as cell membranes, making them highly biocompatible. • Drug Protection: It is possible to protect drugs from degradation caused by exterior factors by encapsulating them inside the bilayer or its core, extending the half-life of the drug. • Controlled Release: By exposing liposomes to external factors, such as heat, their contents can be released in a controlled manner. Additionally, they can offer a sustained release of medication passively or via stimulation. • Site-targeting: These systems can transport their contents to the affected area through either passive or active targeting methods. This can help to reduce side effects throughout the body and increase the maximum dose that can be tolerated, enhancing the therapeutic benefits. Although liposomes have been successful in therapeutic applications, they still present some disadvantages, such as [90,91]: • Low Lifespan: Despite extensive research, liposomes still face issues such as short shelf life and poor circulation in the blood. This can cause drugs to leak out too early or liposomes to fuse together. • High Production Cost: Phospholipids’ high cost makes mass production of these systems difficult. • Unwanted Reactions: At times, phospholipids can experience oxidation and reactions similar to hydrolysis with certain compounds. • Low solubility: Some liposomes may have low solubility, which can affect their ability to deliver drugs effectively. In medicine, liposomes are used as drug carriers to enhance drug effectiveness and minimize side effects. Liposomes have several uses, including chemotherapy, as presented in Table 2.2: 17 Table 2.2: Liposome-encapsulated drugs and their uses. Drug Application Article Doxil Ovarian, Kaposi’s sarcoma and breast cancer [92] Myocet Metastatic breast cancer [93] Marqibo Acute lymphoblastic Leukaemia [94] Onivyde Metastatic adenocarcinoma of the pancreas [95] Lipo-dox Ovarian and breast cancer [96] Although liposomes are extensively used in chemotherapy, they are also widely used in delivering drugs for infections. For example, Mosquirix is used in the treatment of malaria via vaccination [97], and AmBisome is used for fungal infections [98]. The research on new liposome formulations is ongoing, as their potential for cancer therapy continues to be proven. Promitil and Lipocurc are two such formulations being studied for the treatment of solid tumours [99,100], which are abnormal masses of tissue without cysts or liquid areas. Other liposomebased in development include PTX-LDE for ovarian carcinoma [101]. 2.4 Magnetic Nanoparticles Superparamagnetic NPs are extensively researched systems with large potential in the field of biomedical applications. Their capabilities encompass the detection of bacteria, viruses, and proteins, as well as their utility in advancing MRI technology among other areas [102]. 2.4.1 Types of Magnetism In other to understand superparamagnetism it is important to aboard the different types of magnetism, and their differences in aspects of electron spin, their orbital movement and how an external magnetic field (H) affects it. Magnetization (M), in Ampere per meter (A/m), can be defined by the following Equation 2.1: M=Hχm(2.1) Where χmis the magnetic susceptibility of a certain material that indicates a measure of how susceptible the material is to becoming magnetized [103]. 18 There are materials with five different types of magnetic properties: ferromagnetism, antiferromagnetic, ferrimagnetism, diamagnetism and paramagnetism. Materials that exhibit ferromagnetism have positive magnetic susceptibilities and high values, in the order of 102and 106, so they are strongly attracted to external magnetic fields and have the ability to retain magnetic properties after the removal of an external field like it is observed in Figure 2.9. The remnant magnetic flux density, Br, is the magnetic flux density that remains in a ferromagnetic material after an external magnetic field has been removed. The coercive field strength, Hc, is the value of the external magnetic field that must be applied in the opposite direction to reduce the magnetic flux density in the material to zero. Figure 2.9: Schematic representation of the B−Hcurve of a ferromagnetic material. The magnetic flux density that remains in a ferromagnetic material after removing an external magnetic field is represented by Br, while Hcrepresents the coercive field strength, adapted from [104]. This happens because these materials, such as iron or nickel, have some unpaired electrons in their constitution and, as a result, their atoms have a permanent atomic magnetic dipole. Since the magnetic dipole moments of the atoms exert strong forces on the moments of neighbouring atoms, there is a small region of space where the moments are aligned with each other, even without an external field, as shown in Figure 2.10, the phenomenon occurs. 19 Figure 2.10: Microcrystalline grains within a piece of Nd2F e14B(the alloy used in neodymium magnets) with magnetic domains made visible with a Kerr microscope. The domains are the light and dark stripes visible within each grain [105]. Another important aspect of ferromagnetic materials is a specific temperature known as the Curie point, which varies for each substance. At this temperature, ferromagnetic materials undergo a transformation, losing their distinctive magnetic properties and no longer exhibiting magnetism. Nevertheless, upon cooling, they once again acquire their ferromagnetic attributes [106]. For the case of antiferromagnetic materials, such as manganese oxide (MnO). Neighbouring ions act as tiny magnets and spontaneously arrange in opposing, or anti-parallel, orientations at lower temperatures within the material as demonstrated in Figure 2.11. As a result, the material does not exhibit significant external magnetism. Under specific conditions, the material demonstrates antiferromagnetic characteristics, where the magnetism from aligned magnetic atoms or ions in one direction is balanced by those in the opposite direction [107]. Figure 2.11: Schematic of alignment of the material’s polarization for antiferromagnetic and ferromagnetic materials. 20 This anti-parallel alignment of atomic magnets can be disrupted by heating and disappears completely beyond a distinct temperature known as the Néel temperature (TN). This temperature, which depends on the antiferromagnetic material, was named after the pioneering work of French physicist Louis Néel in 1936, who played a key role in understanding antiferromagnetism. While some antiferromagnetic materials have Néel temperatures that go well beyond room temperature, more commonly, these temperatures are lower [108]. When antiferromagnetic solids are exposed to an external magnetic field, their behaviour varies depending on the temperature. At extremely low temperatures, the material remains unresponsive to the external field due to the unwavering anti-parallel alignment of the atomic magnets. As the temperatures increase, certain atoms break free from this organized arrangement and align themselves with the external field. The atoms line up and create some magnetism in the solid, strongest at the Néel temperature. But when the temperature increases past that point, the excessive movement of atoms prevents them from aligning with the magnetic field, causing the small amount of magnetism present in the solid to gradually decreases. Ferrimagnetism is a specific type of permanent magnetism observed in solids. In this type of material, individual atomic magnetic fields arrange themselves spontaneously, with some aligning parallel or in the same direction (similar to ferromagnetism), while others adopt an anti-parallel configuration, pairing off in opposite directions (similar to antiferromagnetism). This behaviour is particularly evident in single crystals of ferrimagnetic substances, where the dominant influence comes from the parallel alignment described in Figure 2.12. With this, the presence of atoms in the anti-parallel arrangement limits the overall magnetic strength of these materials compared to purely ferromagnetic materials. This spontaneous alignment responsible for ferrimagnetism is disrupted entirely when the material surpasses a specific Curie temperature, just like ferromagnetic materials. Once the material’s temperature drops below the Curie point, the phenomenon of ferrimagnetism becomes active again [109]. Figure 2.12: Schematic of the alignment of the material’s polarization for antiferromagnetic, ferromagnetic materials and ferrimagnetic material. 21 In the case of diamagnetic materials, they exhibit negative susceptibility, with magnetic susceptibility values ranging from −10−6to −10−3[110]. They are slightly repelled in the presence of external magnetic fields. The susceptibility of diamagnetic materials arises from the realignment of electron orbitals under the influence of a magnetic field. This results in a weak magnetization that opposes the applied external magnetic field as observed in Figure 2.13. As all their electrons are paired, they lack a permanent magnetic moment. Furthermore, they do not retain their magnetic properties when the external magnetic field is removed [111,103]. Figure 2.13: Schematic of the alignment of the material’s polarization for antiferromagnetic, ferromagnetic materials and diamagnetic materials. Finlay paramagnetic materials exhibit a positive magnetic susceptibility with reduced values (10−5< χm<10−3), causing them to be slightly attracted to external magnetic fields [112]. Their magnetic properties arise due to the presence of some unpaired electrons and the realignment of electrons caused by the external magnetic field. In the absence of these fields, the magnetic moments tend to align with the field, but this tendency is counteracted by the random orientations of the moments due to thermal motion described in Figure 2.14. The fraction of moments that become aligned with the field depends on the field’s intensity and temperature. After the field is removed, they do not retain their magnetic properties [113,103]. Figure 2.14: Schematic of the alignment of the material’s polarization before, during and after applying an external magnetic field represented as the two red arrows. 22 With a better understanding of the complexities of paramagnetism, it is now possible to comprehend materials with superparamagnetism. These phenomena occur at the nanometric level and result from the small size of particles, which give rise to this effect. . 2.4.2 Superparamagnetism Ferromagnetic materials consist of discrete regions known as domains, each comprising a large number of atomic dipoles, typically around 1012 to 1018 [114], with inherent magnetic moments. In the absence of an external magnetic field, the orientations of these moments within a domain are randomly oriented. However, when an external magnetic field is applied, the domains begin to align their magnetic moments in parallel with the field direction. This alignment process continues until all the domains are aligned, resulting in saturation magnetization. At this point, the material’s overall magnetic properties cannot be significantly enhanced by increasing the external magnetic field strength (saturation magnetization) [115]. Between these aligned domains, there exist interfaces referred to as Bloch walls. These Bloch walls represent regions of magnetic transition between neighbour domains. They are essential for accommodating the gradual change in magnetization orientation from one domain to the next [116]. This transition can be visualized in Figure 2.15. Figure 2.15: Illustration representing a Bloch Wall featuring the magnetization transition between two distinct domains. The emergence of domains is a result of a delicate equilibrium between magnetostatic energy, which originates from an applied external magnetic field (and increases proportionally with the material’s volume), and the energy associated with Bloch walls (which escalates with the surface area between the domains) [117]. This interplay is responsible for the formation process and since the creation of domains is defined by the particle size, there is a specific size at which the magnetostatic energy becomes equal to the energy 23 Figure 2.20: Schematic representation of the molecular structure from DOX [144]. daunosamine molecule, composed of carbon, hydrogen, oxygen, and nitrogen atoms. The daunosamine and the anthracycline ring are connected through a glycosidic bond, which is a specific type of chemical linkage connecting a sugar molecule to another molecule. Additionally, the molecule includes a carboxylic acid group located at the end of the anthracycline ring [145]. This functional group consists of a carbon atom, two oxygen atoms, and a hydrogen atom. It plays an important role in rendering DOX soluble in water, which helps it to dissolve and disperse effectively in aqueous solutions [146]. 2.6.1 Mechanism of Action of Doxorubicin DOX mechanism of action is complex and not fully understood, but it is known to interact with DNA in several ways. This drug can insert itself between the base pairs of the DNA double helix. This can cause breaks in the DNA and inhibit the replication of both DNA and ribonucleic acid (RNA). This will inhibit normal cellular processes leading to cellular death (apoptosis) [147]. Furthermore, DOX also inhibits the function of topoisomerase II, an enzyme that helps to relax the double helix formation in DNA during transcription, as illustrated in Figure 2.21. By inhibiting this enzyme, DOX can prevent the DNA from being properly transcribed, which can lead to cell death [147]. DOX can also generate free radicals and cause oxidative stress, which can damage multiple cell components. [143]. The administration is commonly given intravenously at 21-day intervals, typically, in liposomal formulations. This approach serves the purpose of mitigating the numerous side effects associated with its use. They include a large number of conditions such as fatigue, vomiting, nausea, oral sores, bone marrow suppression, hair loss, and the potential emergence of new tumours. Additionally, in the case of some patients, cardiac toxicity is a side effect that limits its extended usage [143]. In unfortunate instances of extravasation, where the intravenously administered drug leaks into sur30 Figure 2.21: Schematic representation inhibition of Topoisomerase II by its combination with DOX. rounding tissues, leads to severe tissue ulceration and, in the most severe cases, necrosis, underscoring the importance of careful administration procedures [148]. DOX is an effective chemotherapy drug, but its systemic action can cause severe side effects, as previously mentioned. Such side effects can restrict the use of the drug and lead to discontinuation of treatment. Therefore, enclosing DOX in liposomes, such is the case of DOXIL, is a better approach. This encapsulation technique has already been proven to safeguard patients from side effects and ensure the completion of treatments [92]. 2.7 Magnetic Bioreactor Magnetic stimulation is a possible way to stimulate drug release from MLPs. When a low-frequency AMF is applied to these systems, it can induce motion of the magnetic NPs embedded in the liposome membrane. This movement can cause changes in the structure and permeability of the membrane, resulting in the release of the encapsulated drug. The motion of the magnetic NPs generates mechanical stress, which can cause the formation of local pores or defects in the liposome membrane. The alignment of the magnetic NPs in the direction of the applied magnetic field increases the permeability of the membrane. According to [149], the rate of drug release from MLPs can be controlled by varying the frequency and intensity of the applied AMF. The authors suggested that the slow release of the drug observed at low frequencies is due to the formation of local pores or defects in the liposome membrane. These changes 31 increase the permeability of the membrane, allowing the drug to diffuse out more easily. Magnetic stimulation of MLPs has the potential to be a valuable tool for controlled drug delivery, as it is non-invasive and contactless. It can also be used to deliver drugs to specific tissues or organs in the body. Additionally, the rate of drug release can be controlled by varying the parameters of the AMF, such as the frequency and intensity. Bioreactor systems have become increasingly important in tissue engineering applications, as they can accurately replicate the native cell/tissue microenvironment. By exposing the tissue culture to various chemical or physical stimuli, bioreactors can create a synergistic environment that stimulates cell response. The effectiveness of this approach depends on the bioreactor technology used and the response of scaffold structures to the stimuli. Bioreactors have an important application in bone tissue engineering. Bone material is piezoelectric, which means that it can create an electric signal when it undergoes mechanical stress and vice-versa. This effect is essential for bone growth and development. Bioreactors that can electromechanically stimulate bone cells can mimic this important element of the bone microenvironment, and encourage tissue growth and differentiation. In [150] it is described a magnetic bioreactor that can be used for bone tissue engineering. In their study, they used a sinusoidal magnetic field with an amplitude of 0.22T and a frequency of 0.3Hz. This device applies magnetic, mechanical, and electrical stimuli to the cells in culture. It works by utilizing a magnetic field to a magnetically responsive scaffold that contains magnetostrictive nano/microparticles embedded in a specific matrix. The matrix can be electrically responsive or not, depending on the desired application frequency until certain limits, in order to get the required alternated magnetic field at the culture plate. To create an AMF a magnet is moved at a specific frequency for a set amount of time. To ensure mechanical protection, limit switches and precision sensors were implemented. These components permitted electronic control of the table’s position through a linear sensor and magnetic encoder for speed. These components are illustrated in Figure 2.22. 32 Figure 2.22: Magnetoelectric bioreactor system operating principle through the use of electrical and mechanical controls to produce an AMF [150]. Bioreactors such as this one offer a number of advantages for tissue engineering applications. First, they allow us to recreate the complex physical and chemical environment of the native tissue, which is essential for promoting cell growth and differentiation. Second, they allow the application of controlled stimuli to the cells, which can be used to direct their behaviour in a desired way. Third, they allow us to scale up tissue production, which is important for clinical applications. 33 Chapter 3 Experimental Procedures, Methods and Characterization Techniques 3.1 Materials and Methods 3.1.1 Magnesium and Calcium Ferrite Magnetic Nanoparticles Synthesis In order to prepare magnesium and calcium ferrite (Ca0.25Mg0.75Fe2O4) magnetic NPs with shape anisotropy, a number of steps, were taken. First it was measured the following reagents from Sigma-Aldrich in Table 3.1. Table 3.1: Reagents used with the respective molar mass, concentration used and mass. Reagent Molar Mass (g/mol) Molar Ratio (mM) Mass (mg) Magnesium acetate tetrahydrate 214.45 0.75 161 Calcium acetate hydrate 158.16 0.25 39 Iron(III) citrate tribasic monohydrate 544.9 2 526 Oleic acid 282.46 3.1 875 Next, 15mL of octadecene was poured into a double-neck flask, which was then heated to 120◦C while being stirred continuously with a magnetic stirrer. The pre-measured reagents were then added to the preheated octadecene, and the temperature was maintained at 120◦Cfor an hour. Following this, the reflux condenser was attached, and the temperature was gradually increased to 200◦Cat a rate of 5◦C/min. It was kept at 200◦Cfor 10min before the temperature was raised to 290◦Cat a rate of 1C/min and refluxed for 60min. The NPs obtained were then washed with dimethyl sulfoxide (DMSO) and ethanol in order to remove oleic acid residues. Oleic acid was then replaced with dimercaptosuccinic acid (DMSA) to make the NPs hydrophilic. This was 34 done by preparing a solution of 60mg DMSA in 5mL of DMSO and combining it with 50mL of toluene. The mixture was then subjected to sonication for 5min and mechanical stirring for 24h. Toluene was introduced, and the mixture was centrifuged. The supernatant containing oleic acid-coated particles was discarded, and multiple washing cycles with ethanol and acetone were performed to remove any remaining oleic acid molecules. Finally, the particles were dried and immersed in ultrapure water. By following these steps carefully, the desired magnetic Ca0.25Mg0.75Fe2O4NPs with shape anisotropy were obtained. 3.1.2 Magnetoliposome Synthesis The Sigma-Aldrich lipids were separately dissolved in chloroform to create a 20mM solution. The respective lipids used are present in Table 3.2. Table 3.2: Components used in the procedure. Lipid Molar Mass (g/mol) Lipid Mass (mg) Volume of Chloroform (µL) DPPC 734.53 0.147 10 DSPC 746.94 0.158 10 DSPE-PEG2000 3532.41 0.2 5 CHEMS 1112.79 0.0974 10 A lipid composition was formulated with each lipid present in Table 3.3 in the quantities specified. Table 3.3: Quantities and volumes of each lipid from chloroform solution to achieve the desired percentage. Lipid Name Lipid Ratio (%) Volume (mL) DPPC 60 90 DSPC 20 30 DSPE-PEG2000 5 7.5 CHEMS 15 22.5 The chloroform solvent was completely removed from the lipid solution using a nitrogen stream. Next, it was introduced 150µL of ethanol to the lipid mixture and in a separate procedure, it was combined 2mg of magnetic NPs with 3mL of ultrapure water. Then, the suspension of water and magnetic NPs was heated to 50◦Cand added DOX in water to achieve a concentration of 50µM. 35 Gradually, the lipid solution was added gradually into the NP solution, using constant vortexing, at ≈1500rpm, to ensure a uniform and controlled incorporation of the lipids into the magnetic NPs. By slowly adding the lipid solution to the water solution, the formation of MLPs is observed around the DOX and magnetic NPs as shown in Figure 3.1. Finally, the resulting formulation was sonicated for 30min, ensuring the proper MLP size. Figure 3.1: Process of formation of lipids into liposomes around the magnetic NPs and DOX and the remaining non-encapsulated components. It is possible to repeat this procedure using varying quantities of lipids, magnetic NPs, and DOX. 3.2 Characterization Methods 3.2.1 Dynamic Light Scattering Dynamic Light Scattering (DLS) is a powerful technique used to accurately determine the size of particles in solutions and emulsions at the nanometric scale. The principle of this technique lies in the Brownian motion, also called ”random walk”, exhibited by particles, where smaller particles exhibit faster, and more erratic movements compared to larger ones [151]. Exploiting the phenomenon of Brownian motion, DLS utilizes a laser beam that interacts with the particles in the sample. As the laser light encounters the particles, it undergoes scattering, resulting in a pattern of scattered light that contains valuable information about the particles’ size distribution [151,152]. By analysing the intensity fluctuations of the scattered light, the DLS instrument can derive the particles’ diffusion coefficient, which is directly related to their size as depicted in Figure 3.2. The technique provides a detailed size distribution profile of the particles present, offering valuable insights into their characteristics [152]. 36 One of these parameters is the polydispersity Index (PDI). It is a metric used to measure the heterogeneity of particle sizes within a sample. It helps to determine the diversity in particle dimensions among a population of molecules, particles, or other entities [153]. On the other hand, it is also possible to obtain the hydrodynamic radius that represents the average radius of the vesicles. Figure 3.2: A basic representation of the frequency dependence on the particle size in a DLS system. The connection between the velocity of particles and their size can be determined using the StokesEinstein equation (Equation 3.1). The velocity of particles is indicated by the diffusion coefficient, D. This equation takes into account the viscosity of the dispersant (η) and the temperature (T), since these factors directly impact particle motion. The Stokes-Einstein equation must be valid that particle movement is solely governed by Brownian motion: D=kBT 6πηRH (3.1) Where RHis the hydrodynamic radius. Figure 3.3 illustrates the configuration of a DLS device. In this setup, a laser emitting light at a specific frequency is directed towards the sample contained within a cuvette. If the sample contains particles, the incoming laser light is scattered in various directions. The scattered light is captured and detected at a specific angle over some time. This collected signal is then utilized to calculate the diffusion coefficient and particle size using the Stokes-Einstein equation [154]. 37 Figure 3.3: Basic setup of a DLS measurement system where the sample is contained in a cuvette. The scattered light of the incident laser can be detected at different angles. Aside from its dimensions, this DLS allows for the determination of the Zeta potential. This particular factor is linked to the surface charge of particles when they are suspended in a fluid. It offers valuable information about the stability of the dispersion and its units are in Volt (V). The extent of electrostatic repulsion and, therefore, the quality of particle dispersion improves as the absolute value of the Zeta potential increases and when this value approaches figures closer to zero, the probability of aggregate formation becomes more pronounced [155]. Magnetoliposome Average Size Procedure As previously mentioned, DLS is a powerful technique that can be used to determine the average size of particles, such as MLPs. In this study, it was used a DLS Litesizer 500 to estimate the size of MLPs. To ensure the accuracy and reliability of the obtained measurements, multiple tests were conducted on a single sample with a concentration of 50µM of DOX and 2mg of NPs. Stability Over Time Procedure As part of the research to evaluate the shelf life of these systems, the zeta potential of the sample was studied over a month-long period, including storage in a refrigerator and periodic measurements at room temperature. Understanding the temporal behaviour of zeta potential is key to assessing the stability and performance of these systems over time. During the observation period, zeta potential measurements were conducted at specified intervals in the DLS Litesizer 500 to comprehensively track the evolution of the sample’s surface charge after synthesizing the MLP sample with 50µM of DOX and 2mg of NPs. 38 Storing the sample in a refrigerator was a significant choice, as it simulates a controlled storage environment that is relevant for various applications, such as pharmaceuticals, cosmetics, or colloidal suspensions. Moreover, bringing the sample back to room temperature before each measurement mimics real-world scenarios where products are stored under different temperature conditions and are then subjected to environmental changes during use. Influence pH in Magnetoliposomes Procedure The aim of this study was to investigate the impact of pH on different factors in the systems, ranging from pH 7.3 to simulate physiological conditions [156]. In this procedure, two 200mL aqueous solutions were prepared, namely ”Solution A” and ”Solution B”, using the components listed in Table 3.4. Table 3.4: Reagent for each solution with respective molar mass, concentration and mass used in the 200mL solution. Reagent Name Molar Mass (g/mol) Concentration (M) Mass (g) Solution A Sodium Phosphate 177.9 0.1 3.56 Solution B Boric Acid 61.83 0.2 2.47 Citric Acid 210.14 0.05 2.10 Using the volume ratios of each solution presented in Table 3.5, multiple buffer solutions were produced with varying pH values. Table 3.5: Resulting pH after combining the respective volumetric ratio of each solution. pH 3 4 5 5.5 6 6.5 7 7.4 8 9 Solution A 12 22.5 33 37 41 45.75 50.5 53.4 57.5 65.5 Solution B 88 77.5 67 63 59 54.25 49.5 46.6 42.5 34.5 600µL of the respective pH buffer solution and 300µL of the MLP sample were applied into its respective Eppendorf tube. The DLS Litesizer 500 was used to analyze the zeta potential of each sample three times to ensure more accurate results. 3.2.2 Transmission Electron Microscopy Transmission Electron Microscopy (TEM) is an important technique used to study structures at a nanometer scale by using an electron beam. It provides insights into the morphology, size, and distribution 39 highest absorbance peak at 344nm. Using this wavelength, values were obtained and a calibration slope was processed with a Python script. Figure 3.10: Layout used in the microplate for processing the calibration slope. After it was synthesized MLPs under standard conditions, using 2mg of magnetic NPs, and repeating this process in quadruplicate. Subsequently, it was magnetically decanted the non-encapsulated NPs from the MLPs (depicted in Figure 3.11), and pipet the remaining encapsulated magnetic NPs and the non-encapsulated ones while evaporating any remaining water. Figure 3.11: Process of magnetic decantation that involves using a magnet to separate non-encapsulated NPs at a faster rate than encapsulated ones. Next, it was introduced 1.2mL of a 37% HCl solution to the dried SMLs and non-encapsulated NPs. The solution was sonicated for 10min and allowed the NPs to digest overnight. Then, dilute the acid to a 4Mconcentration by adding 1.8mL of ultrapure water to each sample. The NPs were analysed using the same equipment and conditions, and the non-encapsulated mass was determined with a Python script. 46 Drug Release Over Time Under Static and Dynamic Conditions As previously highlighted, a primary objective of this project is to investigate the effects of magnetic stimuli on drug release from the MLPs, utilizing the magnetic bioreactor detailed in Section 2.7. This study aims to compare these effects with those observed under static conditions. To achieve this, absorption spectroscopy was utilized as the analytical technique. For the procedure it was prepared several Eppendorf tubes with predetermined concentrations of DOX, including 100µM,50µM,25µM,12.5µM,6.25µM,3.125µM,1.5625µM, and 0M. Subsequently, each of these solutions was meticulously placed into five wells of a microplate, as depicted in Figure 3.12, and subjected to analysis in a microplate reader ®Infinite M Nano+. The mean values for each concentration were calculated, and the calibration slope was determined using the data obtained. Figure 3.12: Layout used in the microplate for processing the calibration slope. Subsequent to this, drug release under static conditions involved the preparation of a 6 mL solution of MLPs with the standard concentration of NPs, lipids, and DOX. Utilizing a larger microplate, 550µL of the solution was dispensed into each well. The microplate was then incubated at 37◦Cto mimic physiological conditions. After 30min, the contents of the first three wells were filtered using a 0.45µm syringe filter, determined to be the best means of filtration in the Appendix A, and transferred to an Eppendorf tube. This procedure was repeated 2 more times at 3hintervals and 5hintervals. For dynamic conditions, the same protocol was implemented on 3 separate days. On each day, the microplate was positioned above a homemade magnetic bioreactor (similar to Figure 3.13) with distinct settings. 47 Figure 3.13: (a) Illustration of the bioreactor assembled with a microplate; (b) detailed mechanism of the bioreactor; (c) Schematic display of the disassembled primary electric and mechanical components; and (d) Cross-sectional view of the mechanical component [174]. On the first day, a magnetic field was applied at a frequency of 0.3Hz, with a 20min ”on” period followed by a 40min ”off” period. On the second day, the same frequency was maintained, but the ”on” period extended to 40min, followed by a 20min ”off” period. On the last day, the frequency was increased to 0.6Hz, with a 40min ”on” period and a 20min ”off” period. Following the collection of filtered samples, each specimen was distributed into 4 wells of a microplate for subsequent analysis using the microplate reader. 3.2.5 Differential Scanning Calorimetry Differential Scanning Calorimetry (DSC) is a thermal analysis technique frequently used in the pharmaceutical field. It can also be used for studying the influence of temperature on systems (such as MLPs). It is essential to observe their behaviour in various conditions, including room temperature and physiological environments. It works via a dual chamber configuration: in one chamber, there is a sample to be analyzed while the other serves as a reference. This technique effectively distinguishes between endothermic (heat-absorbing) and exothermic (heat-releasing) transformations. This makes it easier to determine critical parameters such as transformation temperatures and enthalpy changes in both solids and liquids, as they relate to changes in temperature [175]. The system shown in Figure 3.14 is a simplified DSC system. The oven heats the sample and reference crucibles to a desired temperature. The sample crucible holds the material being analyzed, while the reference crucible holds a baseline reference material. The 48 thermometer, denoted as PT100, measures the temperature of both crucibles. The heating and cooling system controls the temperature of the oven, allowing it to be heated or cooled as needed. The purge gas system removes any unwanted gases or vapours from the oven that may interfere with the analysis. The temperature difference measurement system measures the difference in temperature between the sample and reference crucibles, which is used to calculate the heat flow into or out of the sample. Figure 3.14: Simplified DSC setup. The setup consists of an oven, a sample crucible, a reference crucible, a PT100, a heating and cooling system, a purge gas system, and a temperature difference measurement system, adapted from [176]. In order to observe the influence of temperature on MLPs, the DSC samples included one MLP sample with DOX and another without any drug. These samples underwent drying until approximately 500µL of water evaporated. The samples were analysed in a DSC822e from Mettler Toledo DSC822e using N2as a purging gas at a temperature range of 30 −60◦C. 3.2.6 Fourier Transform Infrared Spectroscopy Fourier Transform Infrared (FTIR) spectroscopy is a technique with a wide range of applications including analysis of small molecules and complexes, cells and tissues, and mapping of cellular components like carbohydrates, lipids, and proteins. This even helps in the detection of abnormal cells, such as cancerous ones. Moreover, FTIR spectroscopy has progressively expanded to encompass protein investigations, delving into their conformation, folding dynamics, and intricate molecular attributes. When a sample is traversed by infrared (IR) radiation, a portion of the radiation is absorbed, while the 49 remainder is transmitted. The ensuing signal captured by the detector manifests as a spectrum, akin to a molecular ”fingerprint” unique to the sample. By employing the Fourier Transform, the output from the detector is translated into a comprehensible spectrum [177]. A simple and possible FTIR can be depicted in Figure 3.15. Figure 3.15: Basic FTIR system. This test aims to detect the presence of Iron oxide (from the Ca0.25Mg0.75Fe2O4NPs), phospholipids, and DOX. In order to detect the presence of the multiple compounds from these systems (DOX, phospholipids and iron oxide form the Ca0.25Mg0.75Fe2O4), the FTIR sample involved a sample of 2mg of magnetic NPs, two samples of MLPs with DOX, and two samples without DOX. These samples were dried in an oven overnight at a temperature of 90◦C. FTIR-ATR spectrometer model Jasco FT/IR-4100. The spectrum was recorded in the 4000–600cm−1wavenumber range for 64 scans with a resolution of 4cm−1. 3.2.7 Vibrating Sample Magnetometry The Vibrating Sample Magnetometry (VSM) technique is used to measure the magnetic properties of materials, including MLPs. It allows the determination of magnetic characteristics of these systems, such as magnetization, coercivity, and magnetic susceptibility [178]. When performing VSM, the sample is placed on a support capable of vibrating in a specific direction. Subsequently, a magnetic field is applied to the sample, and its magnetic response is measured while the field is varied. This allows for the determination of the sample’s magnetization in relation to the applied magnetic field [178,179]. In Figure 3.16, a simple VSM system is depicted. 50 Figure 3.16: Basic VSM system. Composed of a vibration unit, an electromagnet, and pickup coils, that pick up current created by the varying magnetic created by the sample. VSM offers crucial insights into the magnetic properties of materials and other systems such as MLPs, including the presence and strength of magnetization. This information can be utilized to assess the effectiveness of the magnetic particles incorporated into the liposomes, as well as to investigate the influence of parameters like particle size and concentration on the magnetization of the MLPs. For the VSM samples, a 2mg sample of magnetic NPs was combined with three MLP samples containing different amounts of NPs (1mg,2mg, and 4mg). The samples were dried in an oven at 90◦C overnight. The samples were analysed using a MicroSense EZ7 vibrating sample magnetometer. 3.2.8 Fluorescence Spectroscopy A spectrofluorometer uses fluorescence or phosphorescence properties in certain materials for measuring. Fluorescent molecules contain specific electronic structures called fluorophores, which are responsible for their fluorescence. These fluorophores are usually found in their lowest energy state, known as the ground state, at room temperature. Within this ground state, there are several vibrational energy levels, with molecules naturally residing in the lowest one. When exposed to UV or visible light, the fluorophore absorbs photons. The absorption elevates the molecule’s electrons to higher molecular and vibrational states, often referred to as S1 and S2, the first and second excited singlet states, respectively. With this extra energy, the molecule can dissipate some of it as kinetic energy. As a result, these energized molecules collide with other molecules within the sample, resulting in the loss of their vibrational energy. These energy losses are called non-radiative transitions. Once a certain threshold of energy loss is reached, the molecule returns to the lowest vibrational level within the excited state. At this point, no further energy 51 can be lost kinetically, and the molecule must undergo relaxation through the process of fluorescence to return to the ground state. This process lifetime is relatively slow, taking about 10ns. Phosphorescence, similar to fluorescence, involves specific molecules called phosphors that emit light when exposed to energy sources like UV or visible light. These phosphors start in their lowest energy state, the ground state, and absorb photons to reach excited triplet states (T1). However, phosphorescence is distinct due to ”spin-forbidden” transitions, making the return to the ground state slower. This delay results in longer-lasting excitation and the emission of lower-energy light, often in the visible or IR range, making phosphorescence valuable in various scientific and practical applications. Compared to fluorescence, this process takes longer and can take seconds or even milliseconds to complete [180,181]. The types of emissions can be better understood by looking at the visual representation in Figure 3.17. Figure 3.17: Fluorescence emission and phosphorescent emission in the triplet state (T1). Also the wavelength pattern of fluorescence and phosphorescence emission A spectrofluorometer is a piece of equipment that includes a light source, usually a xenon arc lamp, although other options such as a laser port, NanoLED solid-state pulsed sources, and a triple-illuminator option are also available. The equipment also consists of an excitation monochromator that filters the source light and allows a single wavelength of light to reach the sample. Additionally, there is an emission monochromator that filters the light coming from the sample and transmits the filtered signal to a detector, which then transmits the signal to a controller. Lastly, the controller sends the signal to a computer to turn it into more usable data [182]. The system’s fundamental diagram is illustrated in Figure 3.18. 52 Figure 3.18: Basic spectrofluorometer system. Composed of a light source, an excitation monochromator, an emission monochromator, a detector and a controller. In a spectrofluorometer, a light source is filtered by an excitation monochromator, allowing only a single wavelength of light to reach the sample. If the sample is fluorescent or phosphorescent, it responds to the incoming radiation by emitting light after being excited with a certain wavelength. The resulting radiation from the sample is filtered by an emission monochromator. Finally, a spectrum is produced by recording the variation in intensity according to wavelength [182]. The system’s fundamental diagram is illustrated in Figure 3.18. Encapsulation Efficiency of Doxorubicin in Magnetoliposomes To determine the EE of DOX in MLPs fluorescence spectroscopy was used. In this experimental procedure, a series of aqueous DOX solutions with specific concentrations were meticulously prepared to establish a calibration curve. These concentrations ranged from 50µM down to significantly low concentrations, including 8.33µM,1.38µM,0.23µM,0.038µM,0.0064µM,0.0011µM,0.00017µM, 2.97×10−5µM,4.96×10−6µM, and 8.27×10−7µM. Each distinct DOX concentration was carefully divided into separate Eppendorf tubes. To determine the calibration slope, a highly sensitive fluorescence spectrometer was employed. Each Eppendorf tube, containing its respective DOX concentration, was placed within a quartz cell, and a fluorescence measurement was acquired. A fluorescence of DOX is structured, with peaks centred at 555 and 590nm and a slight increase at ≈630nm when excited at 480nm [183]. This fluorescence measurement was repeated three times for each concentration to ensure accuracy and reliability. 53 In a separate experiment, three distinct solutions of MLPs were prepared, each loaded with varying concentrations of DOX. These concentrations included 50µM,500µM, and 5mM. To isolate the DOX-loaded MLPs and remove any unincorporated drug, the solutions were processed through a 100nm Amicon filter. This filtration step was conducted using a centrifuge at 3000rpm for a duration of 8min, ensuring efficient separation of the components. This process can be visualized in Figure 3.19 Figure 3.19: Solution of MLPs was subjected to centrifugation both before and after passing through an Amicon. Subsequently, the non-isolated DOX solutions were subjected to fluorescence analysis in the same fluorescence spectrometer mentioned earlier. The resulting fluorescence data were collected and processed using a Python script, allowing for the quantification and comparison of DOX concentrations within the various MLP solutions. This comprehensive experimental approach ensures the accuracy and precision of the obtained results. 54 Chapter 4 Results and Discussion 4.1 Magnetoliposome Average Size This analysis provides a crucial role in understanding the physical characteristics and behaviour of MLPs, contributing to a broader comprehension of their properties and potential applications. The obtained results, of several syntheses carried out on different days, are presented in Table 4.1. Table 4.1: Combined table with size and PDI. Hydrodynamic Radius (nm) PDI (%) 467.9±95.93 31.2±2.26 523.3±105.26 29.2±2.05 469.2±129.28 31.0±2.59 643.1±114.3 24.1±2.94 469.6±92.34 31.3±2.68 577.7±90.82 27.9±2.97 630.6±106.3 22.7±3.13 560.5±149.0 22.4±4.37 Based on the analysis of the experimental results the hydrodynamic radius and PDI of the MLPs can be determined. The MLPs have a hydrodynamic radius of 542.74±110.40nm with a PDI of 27.48±2.87%. Upon comparison, it is possible to observe that the systems obtained are larger in size, and similar PDI compared to those presented in other research papers, as shown in Table 4.2. 55 4.7 Thermal Characteristics In this analysis, using a DSC model DSC822e, it was observed intriguing results that provide valuable insights into the behaviour of lipids at different temperatures, the results are presented in Figure 4.8. Figure 4.8: Results of the DSC analysis of the MLPs without DOX (orange) and MLPs with DOX (blue). In these results, the presence of two distinct peaks in the heat flow (measured in W/g) for both cases is observed, and it is particularly interesting to note that these peaks occur at around 47◦C. This temperature is significant because it closely aligns with the transition temperature of the lipids being studied. In the study [190], it was observed that DPPC vesicles exhibit a pronounced peak in heat flow at 41◦C, which aligns well with the known transition temperature of DPPC lipids, as described in Section 2.3.1. The systems under investigation contain other types of lipids which affect the transition behaviour of the vesicles. The presence of these additional lipids or molecules could cause the vesicles to transition at higher temperatures [190], indicating that they may be influencing the phase transition of the lipid bilayers within the vesicles. Moreover, it is worth noting that the presence of lipids with higher transition temperatures, such as DSPC, could be a key contributing factor to this phenomenon [191]. DSPC, being known for its higher transition temperature compared to DPPC, could be driving the overall transition of the lipid system to occur at higher temperatures. This finding underscores the intricate interplay between different lipid components within vesicles and their impact on the thermal behaviour of the system. Additionally, a higher overall heat flow in the MLPs containing DOX is observed. This suggests the influence of this molecule on the system. 62 4.8 Fourier Transform Infrared Spectroscopy This test aims to detect the presence of iron oxide (from the Ca0.25Mg0.75Fe2O4NPs), phospholipids, and DOX. A Python script was used to detect the most pronounced peaks of the obtained plot in Figure 4.9. Figure 4.9: Results of the FTIR analysis: CD1_data and CD2_data represent two batches of MLPs with DOX, SD1_data and SD2_data represent two batches of MLPs without DOX and NPs_data represent NPs. One of the significant findings was the presence of magnetic NPs, as evidenced by a strong band around 602cm−1. This band is a characteristic of vibrations between iron (Fe) and oxygen (O) atoms, indicating the existence of Fe-O bonds, which is a telltale sign of NPs in the sample [192]. Another observation is the absorption band at 3354cm−1, which is attributed to the moisture content. [192]. The FTIR spectrum also displayed distinct peaks in the range of 1095cm−1,1242cm−1, and 1222cm−1, indicating the presence of lipids. These peaks correspond to the stretching vibrations of the P=O group in the phosphate group of lipids, suggesting that lipids are a significant component of the sample. Moreover, The FTIR peaks at 2916cm−1and 2849cm−1are due to the C-H stretching of alkane groups. These peaks are usually medium to strong in intensity and indicate the presence of carbon atoms bonded to hydrogen atoms. This further supports the presence of lipids due to their composition of long hydrocarbon chains, which are essentially alkanes [193,194]. In [195], a graph is presented that compares the peaks of MLPs at analogous wavelengths. The article notes that the peaks at 575cm−1and 572cm−1are indicative of the Fe-O bonds in the NPs. 63 Additionally, a peak at 1092cm−1is observed, representing the P=O bonds in lipids. Another band in the 3200 −3600cm−1range, as well as a peak at 1239cm−1, represents other hydroxyl groups, including moisture. The peaks at 2848cm−1and 2921cm−1indicate the presence of C-H bonds, consistent with the presented results. 4.9 Magnetic Properties The test results showed that all samples exhibited superparamagnetic behaviour. This behaviour is due to the absence of hysteresis in the plot presented in Figure 4.10. Figure 4.10: Results of the VSM analysis: The samples are presented in blue (1mg of NPs), green (2mg of NPs), red (3mg of NPs) and black (only NPs) and it’s max value of magnetisation. The shaded region in the plots represents the associated error the mean moment. The plot displays a linear relationship between the addition of more magnetic NPs and its saturation magnetization. This linear relationship is clearly shown in Figure 4.11. The R-squared value of 0.97 indicates a strong linear approximation. This is due to a higher concentration of superparamagnetic NPs or, consequently, a lower concentration of lipids. 64 Figure 4.11: Linear regression applied to the higher mean moment value of each sample. The saturation magnetization values obtained in this study are higher than those reported in a previous study [196], where the saturation magnetization was estimated to be 0.23emu/g. Another study [197] found the saturation magnetization for the investigated systems to be 0.094emu/gand 0.089emu/g, respectively. However, the saturation magnetization observed in [198] is significantly higher, measuring 52.7emu/g. In the case of the NPs, similar results were presented in a previous study [199], where a value of 5.35emu/gwas reported compared to the obtained 4.39emu/g. 4.10 Encapsulation Efficiency of Doxorubicin To study the potential of encapsulation of these systems, 3 batches of MLPs were processed with different concentrations of DOX (5mM,50µM and 500µM). In this process, a spectrofluorometer Fluorolog 3 was used to obtain the calibration slope shown in Figure 4.15. The R-squared value obtained for this plot is 0.974, indicating a strong approximation as the value is nearly 1. In the same plot, it shows the results obtained from three different samples with varying concentrations. After filtration using a 100µm amicon filter and centrifugation at 3500rpm for 8min. 65 Figure 4.12: Each initial concentration measurement is denoted by a cross, with the calibration slope for DOX indicated by blue dots. The x-axis represents concentration in Molarity (M), while the y-axis depicts the average equipment reading. The results obtained from the process are shown in Table 4.3. In the first case, the value for the case of 50µM is negative due, possibly, to the resolution of the equipment, resulting in 100% encapsulation. For the remaining cases, it is concluded that these systems are capable of encapsulating large amounts of DOX, which makes them highly efficient in this regard. Table 4.3: Obtained results from the EE(%)of DOX. Concentration (µM) Non-encapsulated Amount (µM) EE(%) 50 0 100 500 1.33 99.73 5000 35.99µM 99.28 In the research paper [200], it was found that the result achieved a very high efficiency of 96%±2% encapsulation in MLPs that do not contain DPSE-PEG200 and 98.0%±0.6% in MLPs at a concentration of 10µM. However, in this project case, it was observed that the MLPs that contain DSPE-PEG2000 exhibited a higher encapsulation efficiency despite the higher concentration of DOX in the solution. The same for the case of [201], where an EE(%)ranging from 88.2% to 92.4% for a DOX concentration of 2mg/mL was obtained. 66 4.11 Encapsulation Efficiency of Nanoparticles Following the defined procedure, it was possible to calculate a calibration slope that resulted in an R-squared value of ≈0.98. This indicates a high degree of accuracy in estimating the results, as it is visualised in Figure 4.13, despite minor variations detected at some points. It is worth noting that these differences may indicate a reduced sensitivity for values over 2mg of magnetic NPs. Figure 4.13: Calibration slope for the encapsulation efficiency of NPs. The x-axis represents the concentration in Mand the y-axis represents the average value given by the equipment. The non-encapsulated NPs were measured on the same reader, and their values are presented in Table 4.4. The table displays the non-encapsulated content from the total 2mg added to each sample. Table 4.4: Obtained results from the absorption spectroscopy measurements for the 4 different samples. Sample Mean (mg) STD (mg) EE(%) STD(EE(%)) Sample 1 1.14955 0.0143682 42.73 0.72 Sample 2 1.03103 0.0102042 48.49 0.5 Sample 3 0.976827 0.0124202 51.61 0.62 Sample 4 0.917926 0.00472674 54.01 0.235 The process yielded (1.02 ±0.09)mg of non-encapsulated NPs from a total of (2 ±0.1)mg. Using 67 Equation 4.3 we conclude that EE(%) = (49.46 ±0.503)%. Encapsulation Efficiency (%) =Total Mass - Mass of Non-Encapsulated NPs Total Mass ×100 (4.3) In contrast to other research findings, our results reveal a marginally elevated EE(%)compared to the system presented in [202], which reported a value of 46.(9) ±14%, and [14], which achieved a 36% efficiency for a sample with 10.1mg/mol of Fe+. The synthesised systems also showed a lower EE(%) compared to the 58% efficiency reported by [203]. However, the latter study utilized a lower concentration of NPs (0.116mg/mL). 4.12 Drug Release Over Time Under Static and Dynamic Conditions To precisely measure the drug release rate, a calibration slope was created. This slope played a critical role in calculating the amount of drug released during the experiments. The calibration slope produced the following outcomes, illustrating the connection between drug concentration and measured values (Figure 4.14). Figure 4.14: The DOX calibration slope is represented by blue dots, and the resulting points. It shows a good linear approximation slope with a high R-squared value (0.9995). The slope was used as a reference for subsequent drug release experiments conducted under static and dynamic conditions. 68 Figure 4.15: The graph shows the concentration of drug release (µM) over time. The blue line represents the static condition, while the orange line shows the drug release concentration when a frequency of 0.3Hz was used with a 20min ON cycle. The green line represents the drug release concentration when a frequency of 0.3Hz was used with a 40min ON cycle. Finally, the red line represents the drug release concentration when a frequency of 0.6Hz was used with a 40min ON cycle. Under static conditions, the drug release rate stagnated at 10µM out of 50µM, which accounts for ≈20% over a 5hperiod. When a magnetic field was applied with an ON frequency of 0.3Hz, and with ON periods of 20min, it was noticed a slightly higher release at the 30min mark, possibly due to the magnetic stimuli over that period. It was also observed an almost linear drug release over 5h. It reached a final release of approximately 20µM, which is nearly 40%. In the case of the same ON frequency but with ON periods of 40min, an even higher initial release, just under 12µM, is observed, and there was a slightly higher release at the 2hmark. However, it achieved the same final concentration as the previous scenario. Finally, when the same ON cycle is used, but increasing the frequency of the applied magnetic field to 0.6Hz, at the initial 30min mark, it was observed a much higher initial release compared to the other scenarios. This was possibly due to the higher frequency of the magnetic stimuli. Despite that, it was observed a similar release at the 2hmark of the other dynamic conditions. However, the final release 69 exceeded 24µM (or ≈48%), which was much higher than all scenarios. It is reasonable to assume that these systems release a very small amount of DOX in the static condition. However, significant drug release can be observed by applying a magnetic stimulation. By increasing the ON cycle time, an increase in drug release over time was noticed. Increasing the frequency resulted in a higher initial release and an overall increase in the maximum drug release. Comparing the static and dynamic conditions it is observed an increase in drug release of ≈28%. 70 Chapter 5 Conclusion and Future Work 5.1 Conclusion The primary objective of this research project, centred on the development and characterization of MLPs for enhanced cancer therapy targeting OS, has been successfully achieved. Through rigorous experimentation and analysis, the study effectively established the physical and chemical properties of these innovative drug delivery systems. The developed systems are lipidic vesicles encapsulate both magnetic NPs and DOX, enhancing targeted delivery and therapeutic efficacy for this disease. The present study on superparamagnetic Ca0.25Mg0.75Fe2O4NPs provides a comprehensive characterization of their unique properties and potential applications. The TEM results showed a consistent size distribution of the magnetic NPs, averaging at around 8.75 ±0.35nm. This uniformity indicates a precise synthesis method and offers promising opportunities for controlled applications. The XRD analyses further confirmed the crystalline nature of these NPs, as peaks were present, indicating their structural integrity and potential stability under various environmental conditions. Moreover, magnetic characterization through VSM showcased a saturation magnetization of 4.39emu/g. Notably, the absence of hysteresis in the VSM curves emphasizes the superparamagnetic behaviour of the magnetic NPs, which is pivotal for applications like, in this case, drug delivery. Furthermore, the bandgap of these NPs, as determined through UV-visible absorption spectroscopy, was 1.35eV , indicating a strong absorption in the near-IR region. 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