Development of a localized drug delivery system with chitosan and ferromagnetic nanoparticles produced by wetspinning for cancer treatment

Universidade do Minho Escola de Engenharia Pedro José Ferreira da Silva Development of a localized drug delivery system with chitosan and ferromagnetic nanoparticles produced by wetspinning for cancer treatment october 2022 UMinho | 2022 Pedro Silva Development of a localized drug delivery system with chitosan and ferromagnetic nanoparticles produced by wetspinning for cancer treatment Pedro José Ferreira da Silva Development of a localized drug delivery system with chitosan and ferromagnetic nanoparticles produced by wetspinning for cancer treatment Master's Dissertation Integrated Master in Biomedical Engineering Biomaterials, Rehabilition and Biomechanics Work developed under supervision of Doctor Diana Sara Pereira Ferreira Professor Raul Manuel Esteves Sousa Fangueiro Universidade do Minho Escola de Engenharia october 2022 i COPYRIGHT AND CONDITIONS OF USE OF THE WORK BY THIRD PARTIES This is an academic work that may be used by third parties provided that internationally accepted rules and good practices regarding copyright and related rights are respected. Therefore, this work may be used under the terms of the license set out below. If the user needs permission to use the work under conditions not foreseen in the license indicated, he/she should contact the author, through RepositóriUM of the University of Minho. Licence granted to users of this work Attribution-NonCommercial-ShareAlike CC BY-NC-SA https://creativecommons.org/licenses/by-nc-sa/4.0/ iii 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. iv AGRADECIMENTOS Assim chega ao fim uma das etapas mais difíceis da minha vida e sem dúvida o ano mais difícil do meu percurso académico. Um ano repleto de muito trabalho e esforço e onde foi preciso ter enorme resiliência. Com isto, quero agradecer às pessoas que contribuíram para que este ano ainda assim contribuísse para todo o meu crescimento e conhecimento. Antes de tudo quero agradecer aos meus orientadores, ao Professor Raul Fangueiro por disponibilizar todas as condições que fui tendo para a realização do meu trabalho e à Doutora Diana Ferreira pela orientação, disponibilidade e por me ter ajudado a levar sempre este trabalho com o devido rigor, motivação e empenho. Agradecer também a todas as pessoas do 2C2T, Fibrenamics e à Sónia que sempre estiveram lá quando precisei de algo, mostrando total disponibilidade. Para além disto, queria deixar também os meus agradecimentos à Joana, à Sofia e à Marta por todo o apoio, incentivo e ajuda que me foram prestando também ao longo deste ano que foi fundamental para que pudesse concluir o meu trabalho. Sem esquecer, claro, os meus “amigos do lab”, do “binde pá festa”, o Milhazes e o Gil com quem tive oportunidade de partilhar mais uma vez memórias, risos e desabafos que só nós sabemos o quanto nos tornam mais felizes e o quanto podem melhorar o nosso ano. Por fim, mas nunca menos importante, à minha família em especial aos meus pais e irmãos e à minha namorada Mariana, por saberem sempre a melhor forma de dizer e fazer as coisas, por mostrarem que independentemente das dificuldades nunca me vão deixar para trás sozinho, por me fazerem ver que nestes momentos precisamos sempre de alguém que mostre que se preocupa, de verdade, e que essas pessoas independentemente da altura do ano ou hora, serão sempre eles, por todo o carinho e incentivo incondicional e por me conhecerem como ninguém, tornando a minha continuidade nos estudos sempre mais fácil de suportar e acabar. Obrigada por tudo o que passou e por tudo o que aí vem! É apenas o começo. v RESUMO O cancro é uma das principais causas de mortalidade no mundo, depois das doenças cardiovasculares. Assim, há uma necessidade urgente de desenvolver novas estratégias para melhorar a eficácia da prevenção, diagnóstico e tratamento desta patologia. Uma das terapias estudadas para tal na atualidade é a hipertermia. Este procedimento baseia- -se no aumento da temperatura para 40-43 °C do tecido corporal para danificar e destruir as células cancerígenas ou torná-las mais suscetíveis aos efeitos de outras terapias como a radio ou a quimioterapia. Recentemente, e devido aos avanços científicos na área da nanotecnologia, surge o conceito de hipertermia magnética que se baseia na utilização de nanopartículas magnéticas. Apesar das diversas vantagens de utilizar nanopartículas, estas podem apresentar alguma dificuldade em alcançar os tecidos específicos a tratar e, devido ao comum processo de formação de agregados, a sua aplicabilidade pode tornar-se difícil. No sentido de ultrapassar estas limitações, as nanopartículas podem ser incorporadas em sistemas de entrega localizada, nomeadamente compostos por fibras, capazes de atuar como agentes terapêuticos localizados. Sendo assim, este trabalho teve como objetivo desenvolver novos filamentos por wetspinning capazes de serem utilizados na construção e design de estruturas fibrosas de entrega localizada de agentes bioativos. Vários parâmetros da técnica de wetspinning foram otimizados para obter os melhores filamentos, sendo que os melhores foram obtidos utilizando uma taxa de fluxo de 1 mL/min, uma agulha de 0.41 mm de diâmetro e um banho de coagulação de 1M de hidróxido de sódio. As fibras obtidas com melhores resultados mecânicos foram produzidas com uma formulação otimizada de 3 %(m/v) de óxido de polietileno com 3 %(m/v) de quitosano em 12 %(v/v) de ácido acético. O banho de crosslinking também foi otimizado, sendo que se utilizou um banho de trifosfato de sódio a 1 %(v/v) durante 4 horas. As nanopartículas ferromagnéticas foram então sintetizadas usando o método de coprecipitação e foram introduzidas nas fibras em diferentes percentagens. Os filamentos funcionalizados com 2 %(m/v) de nanopartículas mostraram o melhor desempenho mecânico, melhorando também a estabilidade estrutural do sistema ao longo do tempo. Palavras-chave: Fibras, Hipertermia, Nanopartículas Ferromagnéticas, Sistema de Entrega de Fármacos Localizado, Wetspinning vi ABSTRACT Cancer is one of the leading causes of mortality in the world, after cardiovascular diseases. Thus, there is an urgent need to develop new strategies to improve the effectiveness of prevention, diagnosis and treatment of this disease. One of the therapies currently being studied for this is hyperthermia. This procedure is based on increasing the temperature to 40-43 °C of the body tissue to damage and destroy cancer cells or make them more susceptible to the effects of other therapies such as radio or chemotherapy. Recently, and due to scientific advances in nanotechnology, the concept of magnetic hyperthermia has emerged, which is based on the use of magnetic nanoparticles. Despite the several advantages of using nanoparticles, these may present some difficulty in reaching the specific tissues to be treated and, due to the common process of aggregate formation, their applicability may become difficult. In order to overcome these limitations, nanoparticles can be incorporated into localized delivery systems, namely composed of fibres, capable of acting as localized therapeutic agents. Therefore, this work aimed to develop new filaments by wetspinning that can be used in the construction and design of fibrous structures for localized delivery of bioactive agents. Several parameters of the wetspinning technique were optimized to obtain the best filaments, and the best ones were obtained using a flow rate of 1 mL/min, a 0.41 mm diameter needle, and a 1M sodium hydroxide coagulation bath. The fibres obtained with the best mechanical results were produced with an optimized formulation of 3 %(w/v) polyethylene oxide with 3 %(w/v) chitosan in a 12 %(v/v) acetic acid aqueous solution. The crosslinking bath was also optimized, and a 1 %(v/v) sodium triphosphate bath was used for 4 hours. Ferromagnetic nanoparticles were then synthesized using the co-precipitation method and were introduced into the fibres in different percentages. The filaments functionalized with 2 %(w/v) nanoparticles showed the best mechanical performance, also improving the structural stability of the system over time. Keywords: Drug Delivery System, Ferromagnetic Nanoparticles, Fibres, Hyperthermia, Wetspinning. vii Table of Contents 1 Introduction .............................................................................................................. 1 1.1 Context and Motivation ...................................................................................... 2 1.2 Objectives .......................................................................................................... 4 1.3 Structure of the Dissertation ............................................................................. 5 2 State Of Art ............................................................................................................... 6 2.1 Cancer Therapies .............................................................................................. 7 2.1.1 Hyperthermia Therapy and Nanotechnology Advancements .......................................... 10 2.1.2 Localized Drug Delivery Systems .................................................................................. 17 2.2 Fibrous Structures as localized drug delivery systems ..................................... 19 2.2.1 Fibrous Structures with Ferromagnetic NPs .................................................................. 19 2.3 Wetspinning - Technology and Process operation ............................................ 22 2.3.1 Operating Parameters .................................................................................................. 22 2.3.2 Fibrous systems based on Chitosan, PEO and PVA ....................................................... 23 3 Materials and Methods ........................................................................................... 30 3.1 Materials ......................................................................................................... 31 3.1 Methods .......................................................................................................... 33 3.1.1 Formulations of Chitosan ............................................................................................. 33 3.1.1 Crosslinking ................................................................................................................. 35 3.1.1 Chitosan/PEO Formulations ......................................................................................... 36 3.1.1 Chitosan/PVA Formulations ......................................................................................... 37 3.1.1 Synthesis of Ferromagnetic NPs ................................................................................... 38 3.1.2 Development of the Fibrous Structures ......................................................................... 38 3.2 Characterization Methods ............................................................................... 39 3.2.1 Optical Microscopy ...................................................................................................... 39 3.2.2 Scanning Electron Microscopy (SEM) ........................................................................... 39 3.2.3 Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy (ATR-FTIR) ....... 39 3.2.4 Scanning Transmission Electron Microscopy (STEM) .................................................... 40 3.2.5 Energy dispersive X-ray spectroscopy (EDS) .................................................................. 40 3.2.6 Thermogravimetric Analysis (TGA) ................................................................................ 40 2 1.1 CONTEXT AND MOTIVATION Cancer incidence and mortality is increasing worldwide, with estimated 19.3 million new cases and 10 million cancer deaths in 2020, according to the International Agency for Research on Cancer (IARC) [1]. Malignant tumors become more heterogeneous as they develop, resulting in a mixed population of cells with varying molecular characteristics and receptivity to therapy. This variability may be seen at both the geographical and temporal levels, and it is the driving force behind the formation of resistant phenotypes induced by a selection pressure during cancer treatment [2]. Thus, finding novel strategies to improve the therapy efficiency while minimizing the side effects is crucial [3]. Hyperthermia is a cancer therapy and is regarded to be an artificial means of increasing body tissue temperature by administering heat from external sources to destroy malignant cells or inhibit their further growth. One advantage of employing hyperthermia for cancer treatment is that the use of radiation or chemotherapy medications can be reduced. Furthermore, certain versions of the treatment do not involve surgery and have less side effects [4]. Although, a key drawback of traditional hyperthermia is that both malignant and non-malignant cells are equally susceptible to heating, which can cause substantial issues for healthy cells [5][6]. As a consequence, there has been a great deal of interest in the concept of biologically targeted magnetic hyperthermia, in which targeted magnetic iron oxide nanoparticles (MIONs) are used to increase the temperature in tumors ‘site under an alternating magnetic field [7].These nanoparticles (NPs) are used for various biomedical applications and a have multifunctional role in therapeutics, diagnostics, imaging, and drug delivery due to their excellent properties such as chemical stability, nontoxicity, biocompatibility, high saturation magnetization and high magnetic susceptibility [8]. In order to target the required area, drug delivery systems have been used. A drug delivery system is defined as a formulation or a device that enables a therapeutic substance to selectively reach its site of action without reaching the nontarget cells, organs, or tissues [9]. To design intelligent drug delivery the use of biocompatible materials and the design of stimuli-responsive systems are required [8][9]. Wetspinning (WS) is a relatively simple and scalable method to manufacture drug delivery systems. This technique also allows the selection of suitable materials from non-biodegradable to 3 biodegradable polymers, with the purpose of achieving control over the release profile through diffusion or diffusion and fibre degradation. From the different available materials, chitosan (CH) is preferred as it is a biopolymer that may be used to create unique features, functions, and applications in the biomedical field mostly employed because of its biocompatibility, biodegradability, and non-toxicity. Polyethylene oxide (PEO) is also an exceptionally biocompatible and biodegradable polymer that has the ability to interact with polarized surfaces and is frequently affordable, making it an intriguing biomaterial [10][11]. Poly(vinyl alcohol) (PVA) can also be used, since it is a polymer that has good film forming and physical properties, high hydrophilicity, processability, biocompatibility and good chemical resistance [12][13]. So, in this case, the localized drug delivery system will be based on fibrous structures produced by WS with biocompatible polymers, like CH, PEO and PVA [14][15]. Iron (II,III) oxide (Fe3O4) NPs will be synthesized and incorporated into the fibres in different percentages. Therefore, the development of localized drug delivery systems based on fibrous structures produced by WS, using biodegradable polymers and ferromagnetic NPs, can be a promising strategy to treat cancers by hyperthermia. 4 1.2 OBJECTIVES The main objective of this work was to develop a localized drug delivery fibrous system with ferromagnetic NPs using the WS process with possible application in the treatment of cancer by magnetic hyperthermia. Firstly, an optimization process was performed in order to obtain defect-free CH, CH/PEO and CH/PVA fibres by WS. For this, diverse formulations based on CH, CH/PEO and CH/PVA as well as different WS parameters were evaluated. The produced fibres were characterized using different techniques, such as Optical Microscopy, Field Emission Scanning Electron Microscopy (FESEM), Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy (ATR-FTIR), Thermogravimetric Analysis (TGA), Scanning Transmitted Electron Microscopy Detector (STEM) and Energy Dispersive X-Ray spectroscopy (EDS). After optimization of the polymeric formulations to obtain the fibres and their characterization, the NPs were synthesized and incorporated into the optimized polymeric formulations to produce fibres with ferromagnetic NPs. These results were characterized using techniques such as ATR-FTIR, TGA, Optical Microscopy, FESEM. Mechanical properties and fibres swelling and degradation were also analyzed. Finally, the fibres that presented better properties were used for the development of a fibrous structure with magnetic properties that was also fully characterized with the techniques enunciated before as a possible application as a localized therapeutic system for cancer by magnetic hyperthermia. 5 1.3 STRUCTURE OF THE DISSERTATION This dissertation is divided into six sections: section 1 contains a brief introduction to the subject under study; section 2 covers the state of the art of the topic under consideration; section 3 contains all the materials and methods used in the development of the dissertation; section 4 presents the results obtained as well as their discussion; section 5 illustrates the main conclusions of the developed work and section 6 contains proposals for future work. In the first section, a topic framework was created, and the key inspirations that led to this effort, as well as the main objectives of the proposed work, are given. This chapter also includes a synopsis of the content of each part. Section 2 includes a thorough literature review that takes into account: cancer therapies, with a primary focus on hyperthermia; localized drug delivery systems; the wetspinning technique; the biodegradable polymers used in the production of fibres by this technique and a survey of the work done using the polymers under study and framed in tissue of hyperthermia treatment applications. Section 3 describes the materials and methodologies that served as the foundation for the experimental execution of this dissertation, as well as the various approaches employed to optimize the five polymeric systems. This chapter also examines and describes the many methodologies utilized for system characterization established during this effort. Section 4 presents and discusses the findings achieved for each of the created materials. The various optimization phases are correctly presented, both in terms of formulation parameters and wetspinning parameters, demonstrating their effect on the morphology of the generated fibres. Finally, the characterization studies done on the various samples developed in this work are provided. Section 5 summarizes the study's general results and section 6 outlines possible future possibilities that take into consideration the potential of this application. 6 2 STATE OF ART 7 2.1 CANCER THERAPIES Cancer is a vast category of illnesses that can begin in practically any organ or tissue of the body when abnormal cells develop uncontrolled, invade neighboring tissues, and/or spread to other organs. The latter phase is known as metastasizing, and it is a primary cause of cancer mortality. Cancer is also known as a neoplasm and a malignant tumor. Normally, human cells grow and multiply to form new cells as the body needs them. In also normal cases, when cells grow old or become damaged, they die, and new cells take their place [16]. However, sometimes this orderly process breaks down and abnormal or damaged cells (cancer cells) grow and multiply, raising the opportunity for tumors to appear. These cells develop because of multiple changes in their genes, which can have many possible causes like lifestyle habits, genes or being exposed to cancer-causing agents. Once these cells tumors appear, they can expand, owing to their increased vascularization, and spread to other parts of the body, resulting in metastasis and death [17]. In order to fight these diseases, some therapies for cancer are being used. These treatments change depending on the type of cancer being treated and whether it is at an advanced stage or not. Some cancer patients will only receive one therapy to fight this disease, however, the majority of patients will receive a combination of therapies, searching for immediate results, such as surgery along with chemotherapy and radiation therapy [15]. Table 1 shows the mainly strategies in use today and a brief description of them: 8 Table 1: Most used therapies for cancer treatment Type of treatment Description Photodynamic Therapy This therapy involves the use of a photoactive molecule, light and molecular oxygen present in tissues. When combined, these three compounds are able to produce reactive oxygen species (ROS), which will induce the death of target cells [18]–[20]; Immunotherapy This therapy is made up of white blood cells as well as lymphatic organs and tissues. As known, the immune system helps the body fight infections and diseases, and, therefore, this type of cancer treatment helps immune system fight cancer [21]; Endocrine therapy Cancer treatment that slows or prevents the proliferation of cancer cells that need hormones to proliferate [22]; Surgery Procedure in which a surgeon tries to remove tumors from a patients’ body [23]; Chemotherapy Treatment that employs strong chemicals to destroy rapidly developing cells in the body [24]; Radiation therapy Therapy where strong doses of radiation are used to destroy cancer cells and reduce tumors [25]; Hyperthermia therapy Treatment in which body tissue is heated to temperatures between 40 - 43°C [26] in order to harm and destroy cancer cells [27]; 9 Although certain cancer therapies are still available, most of them have significant drawbacks that make the treatment dangerous. The photodynamic therapy, although a promising treatment, has been performed in only a reduced number of patients, so many clinics don't adopt this type of treatment. Furthermore, with systemically administered photosensitizers, skin photosensitivity is one of the most common adverse events. Patients must avoid sunlight and strong artificial light for weeks, which is usually highly undesirable. Others adverse events often reported are pain and the decreasing efficacy of photodynamic therapy for larger lesions. Besides that, due to inadequate tissue penetration of light, bulky or deep seated tumors are difficult to treat, showing the limitations on this therapy [18]. Regarding immunotherapy, current anticancer immunotherapies have increased overall survival in several cancers at various stages of development, including metastatic disease. In contrast, certain malignancies, create immunosuppressive microenvironments marked by elevated expression of immune checkpoint molecules, reduced tumor antigen expression, and restricted infiltration of circulating immune effector cells. These non-immunogenic, non-inflamed ("cold") tumors respond poorly to immunotherapies and successfully avoid anticancer immune responses which can lead to problems. Additionally, immunotherapy can harm organs and systems due to some of the drugs used on it and also could take longer to work than other treatments [28]. Although there are reports of successful cancer treatment with hormone therapy, this therapy is usually never used without the combination of another, as hormone therapy only attempts to extend "control" over the stage of cancer. Allied to the use of this therapy are side effects that can disrupt the daily life of the patient in question. Some organs may be damaged and the patient may suffer side effects of the administered hormones such as: hot flashes, weight gain and muscle loss, breast swelling and tenderness, fatigue, irritability and also anemia, increased risk of cardiovascular disease (infarction) and increased risk of a metabolic syndrome which may cause concern for those involved [29]. Regarding surgery, the method is never easy and there are cases where it cannot be performed. In situations when it can be used, there is a chance that the cancer will not be entirely eradicated and for that same reason there is a risk that it will spread from its original position to other parts of the body, leading to metastasis. Furthermore, there are secondary dangers like as bleeding, tissue and organ damage, pain, and poor recovery of other body functions that are not well perceived by the patients in question [30]. It is known that chemotherapy may shrink the cancer enough or slow down its growth to make surgery to remove the cancer possible. However, for this to be functional, there are parameters which must be taken into account and which must be studied, such as: the drug uptake and the intracellular 10 activation of the effective drug. As it is a systemic therapy, not localized, this therapy induces the death of cancerous cells and also the death of healthy cells, which induce many side effects, including fatigue, sickness, loss of appetite and hair and even blood clots [31]. Regarding radiation therapy, the radiation used is called ionizing radiation because it forms ions (electrically charged particles) and deposits energy in the cells of the tissues it passes through. This deposited energy can kill cancer cells or cause genetic changes resulting in cancer cell death. Highenergy radiation damages genetic material (deoxyribonucleic acidDNA) of cells and thus blocking their ability to divide and proliferate further. Nevertheless, this radiation does not kill cancer cells right away, it can take days or weeks of treatment before DNA is damaged enough for cancer cells to die and there’s the possibility of also damaging normal cells as well. Beyond that, the equipment is expensive, which leads to a high cost of the treatment not always supported by the patients [32]. Despite this, various investigations on these treatments are still being conducted to overcome the obstacles they imply. One of the treatments studied today portrays the hyperthermia therapy, which will be explored and discussed in the following section [33]. 2.1.1 HYPERTHERMIA THERAPY AND NANOTECHNOLOGY ADVANCEMENTS For a long time, the ability of heat creation to heal has been widely understood and employed in the treatment of numerous diseases [34]. As previously stated, hyperthermia procedure is based on the notion of subjecting body tissue to high temperatures, in order to harm and kill cancer cells (by apoptosis) or to render cancer cells more susceptible to the effects of radiation and specific anticancer drugs [35]. Different approaches have been used to apply hyperthermia in tumor regions such as radiofrequency, microwave, water-filtered infra-red-A, ultrasound and capacitive heating techniques, depending on the tumor location, but with harmful secondary effects in the healthy tissues [4]. Although this therapy can increase the intracellular temperature up to the cellular death, one of the main limitations is that both cancerous and non-cancerous cells are in general equally sensitive to heat. Therefore, one of the most challenging aspects of hyperthermia is to keep a sufficiently high temperature on the tumour, while keeping the normal tissues at a lower temperature, so that normal cells aren’t damaged [36]. This encouraged the research and development of new strategies capable of increasing the temperature of damaged areas, while keeping the rest of tissues healthy. Nanotechnology has been 11 brought into biomedical applications in the hopes of changing present diagnostic and treatment approaches with the use of NPs [4][37][38]. NPs can absorb energy from an external source and so improve the effects of hyperthermia. In reality, NPs serve as the principal heat source and reverse the direction of heat loss, using direct energy from an external source onto the tumor, causing localized thermal death while reducing the impact in collateral tissues. Based on this, nanotechnology advancements have recently permitted, by the use of magnetic NPs (MNPs), the application of a localized hyperthermia therapy [39]. This magnetic hyperthermia allows to remotely induce local therapy due to the capacity of certain MNPs to convert electromagnetic radiation into heat, which could rise the temperature in well-defined locations of the human body containing tumor cells [40][41]. 2.1.1.1 FERROMAGNETIC NPS AND APPLICATIONS MNPs have a huge potential in biomedical applications for controlled drug release [42]–[44], hyperthermia [45], magnetic resonance imaging diagnosis (MRI) [46][47], gene therapy and regenerative medicine [48] (Figure 1). Figure 1: Illustration of MNPs applicability [43]. These NPs are typically classified into pure metals, metal oxides, and magnetic nanocomposites. The most popular MNPs in the biomedical field are Cobalt (Co), Iron (Fe), Nickel (Ni), Titanium (Ti), Iron Oxide and some ferrites [49]. 18 particularly with anticancer and new biotechnological treatments. Another downside is the patient's low compliance with repeated drug delivery [67]. By the use of localized drug delivery systems some of the described drawbacks may be overcome. Drug delivery systems are designed to provide the delivery of therapeutic drugs in a targeted and/or regulated manner. These systems evolved into a viable and practical technique of resolving many of the challenges associated with traditional pharmaceutic compound delivery. The optimal drug delivery system should be biocompatible, inert, mechanically strong, capable of high drug loading and safe from inadvertent release. The benefit of such a system is that the therapeutic concentration of a drug can be maintained in the body for longer periods of time without recurrent administration, boosting patient compliance, reducing drug under/overdosage concerns and delivering it to the specifically required location [67]. Current localized drug delivery systems are in the form of drug-eluting films, hydrogels, fibrous structures, wafers, rods, microspheres between others [68]–[71]. In this work, the drug delivery systems used will be fibrous structures. Numerous studies have already been done in order to be able to conclude on the potential of this whole procedure [71], [72], [81], [82], [73]–[80]. 19 2.2 FIBROUS STRUCTURES AS LOCALIZED DRUG DELIVERY SYSTEMS Fibrous structures are very promising materials to be used as localized drug delivery systems for cancer treatment and so they have emerged as an appealing option. The main justifications for the growth of this approach are based on the fact that they present ease on their fabrication, generally high mechanical properties, desirable drug release profile and high surface area to volume ratio [71]. In regard to their processing, these structures could be based on biodegradable polymers with also low immunogenicity, meaning that, with the application of these structures, there won’t be strong immune responses from the patient's immune system. Additionally, these structures can be adjusted by changing the polymers used to make them, as well as their length and cross-sectional radius, to find the best composition and morphology for the application required [71]. For all these reasons, a combination of fibrous structures and NPs has the potential to maximise the effective functional output from NPs in the desired target, which will result in the enhancement of cancer therapy efficiency and the reduction of side effects, improving the patient’s life quality [83][84][85]. 2.2.1 FIBROUS STRUCTURES WITH FERROMAGNETIC NPS As stated before, fibres containing magnetic NPs reveal a huge potential for many applications, that could be: magnetic filters, sensors, magnetic shielding devices and magnetic induction devices and even protective multifunctional systems [86][20]. For this very reason, some studies have been developed in order to comprehend the behaviour of this systems with NPs. Wang et al., reported the production and characterization of superparamagnetic composite polymer/magnetite nanofibres, searching for their mechanical properties and behavior. As it is stated, using magnetite nanoparticle suspensions in PEO and PVA, they obtained polymer nanofibres ranging in diameter from 140 to 400 nm and verified that both sets of fibres responded to an externally-applied magnetic field by deflecting in the direction of increasing field gradient, showing also that magnetite NPs reinforced the mechanical properties of the nanofibres, as expected [87]. In another study, made by Miyauchi et al., polyvinylpyrrolidone fibres with high concentrations of ferromagnetic and superparamagnetic NPs were also developed and the magnetic properties of these 20 fibres were then explored, using a superconducting quantum interference device. They concluded that mixed magnetic composites may offer an important platform for better understanding the magnetic properties of materials and provide a scalable process for the fabrication of nanocomposites with novel magnetic properties [88]. Nevertheless, it is important to mention some articles where the use of this combination is verified for the desired application: cancer treatment. Thus, in a study made by Kim et al. , a magnetic composite nanofibre mesh that could achieve mutual synergy of hyperthermia, chemotherapy, and thermo-molecularly targeted therapy for highly potent therapeutic effects was produced. The nanofibre was composed of biodegradable poly(εcaprolactone) with DOX, magnetic NPs and 17-allylamino-17-demethoxygeldanamycin. The developed nanofibres mesh exhibited hyperthermia, good biocompatibility, a sustained and pH-sensitive release behavior that was favorable for the long-term maintenance of effective drug concentration in tumor tissue. In MCF-7 cells (a breast cancer cell line), this nanofibre mesh efficiently induced apoptosis, reveling its potential as a new tumor therapy and as an effective locally implantable system for enhancing the efficacy of combination cancer treatments [89]. In another study, by Tiwari et al., it was developed a magnetically actuated smart textured fibrous system based on polycaprolactone with MIONs, anti-cancer drug (DOX) and fluorescent carbogenic nanodots. This system demonstrated enhanced heating with the application of an alternating magnetic field that led to an increase in drug release and enhanced the efficacy of the therapy, also showing the ability to navigate in the fluid with the application of gradient magnetic field. Further, this system was nontoxic to cells and do not leach out any toxic material to them during incubation, proving that it is a potential candidate in the field of cancer therapy [90]. Sasikala et al., also reported on this subject, a smart nanoplatform responsive to a magnetic field to administer both hyperthermia and pH-dependent anticancer drug release for cancer treatment. To achieve this, MIONs were incorporated onto the nanofibres matrix. These nanofibres were developed by electrospinning (ES) with a biocompatible polymer, poly(D,L-lactide-co-glycolide). In regard to the anticancer drug delivery, this step was performed by surface functionalization using dopamine to conjugate the bortezomib through a catechol metal binding in a pH-sensitive manner. The in vitro studies confirmed that this device exhibited a synergistic anticancer efficacy due to the simultaneous application of hyperthermia and drug delivery, providing a secure pathway for delivering the anticancer drug specifically towards the tumor, as well as the retention of the magnetic NPs in the tumor region in a sufficient concentration for hyperthermia treatment [91]. 21 Kim et al. , reported another example of smart hyperthermia nanofibres with simultaneous heat generation and drug release in response to ‘on-off’ switching of alternating magnetic field. The nanofibres were composed of a chemically-crosslinkable temperature-responsive polymer (Poly(NIPAAm-co-HMAAm) with an anticancer drug (DOX) and MNPs. In the results shown, 70 % of human melanoma cells died in after only 5 minutes of alternating magnetic field application in the presence of this device, by double effects of heat and drug, showing again the immense potential on this area [92]. Finally, Lin et al., used Fe3O4 NPs incorporated onto crosslinked electrospun CH nanofibres using chemical coprecipitation. Such system could be delivered to the treatment site precisely by surgical or endoscopic method. They also grafted iminodiacetic acid onto the CH with an aim to increase the amount of magnetic NPs formed in the magnetic nanofibre composite. The results of this study revealed that this incorporation led to more magnetic NPs formed in the matrix of the nanofibre. In addition, the magnetic iminodiacetic acid-grafted CH nanofibre composite showed that could reduce the proliferation/growth rate of malignant cells of the tumor under the application of magnetic field [93]. Based on the good results obtained in the previously mentioned papers using fibrous structures as drug delivery systems, it was necessary to choose the method of processing them for this work. Therefore, in order to do so, it’s important to understand that over the past few decades, various methods have been used for the fabrication of micro and nanofibres. WS, rotary spinning, ES, microfluidic fibre fabrication, and self-assembly are the most common methods [71]. However, in this thesis, the main focus will be WS once this process offers the advantage of producing a wide variety of fibre cross-sectional shapes and sizes and the possibility to work at lower temperatures, while also allowing to use different polymers and not being too expensive. Hence, it’s important to fully understand the concept behind this technique and some of the most used polymers [94]. 22 2.3 WETSPINNING - TECHNOLOGY AND PROCESS OPERATION WS consists in a non-solvent induced phase inversion that enables the creation of polymeric microfibres with a homogeneous shape using the precipitation concept. In fact, in many studies developed with this technique, various medicinal agents such as chemotherapeutics [82][76], antibiotics [95][96] and nonsteroidal anti-inflammatory medications [97][98] have been loaded into these fibrous structures, showing the possibilities opened by it. Generally, a WS equipment consists of four key components: the first is a syringe pump or a gravitational pull that will be responsible for pumping the polymeric solution, which is the second component, in the third component, that is the coagulation bath. Sometimes WS set-up also comprises a spinneret [94]. So, in general, the solution is extruded directly into the coagulation bath and solidifies upon contact with the non-solvent throughout the spinning process, creating the fibres. The shape and diameter of these fibres is affected by thermodynamic conditions of the polymeric solution and the coagulation bath and also by factors like the viscosity of the polymeric solution, the injection rate and the size of the spinneret [71]. Aside from the ability to control the diameter of the resulting structures, fibres produced by WS typically have large pore sizes, resulting in beneficial improvements in cell adhesion and penetration within scaffolds and films. Another advantage is the possibility to create fibres with low processing temperature [99]. 2.3.1 OPERATING PARAMETERS A collection of processing factors that can influence the morphology, shape, diameter, and mechanical performance of the produced fibres has a considerable impact on the WS process. This collection of parameters is organized into three major groups in Table 3, which comprise solution, process, and environmental factors: 23 Table 3: Operating parameters of WS Process Parameters Solution Parameters Environmental factors Coagulation Bath Polymers molecular weight Humidity Coagulation Bath Temperature Polymers concentration Drying temperature Flow Rate Viscosity Needle Diameter Solvent 2.3.2 FIBROUS SYSTEMS BASED ON CHITOSAN, PEO AND PVA Typically, synthetic and natural polymers are blended to increase the performance of the created biomaterial, overcoming the limits of each polymer when employed alone. For the production of fibres by WS, many different polymers have been studied, as well as mixtures of polymers, always based on the final objective and the characteristics needed. Therefore, in this part of the study, the polymers chosen will be addressed in order to understand their usefulness and importance [100]. 2.3.2.1 CH CH is a copolymer made up of multiple monomeric units of glucosamine and N-acetyl-D glucosamine with β(1→4) bonding (Figure 2) and its solubility is proportional to the number of protonated amine groups in the polymer chain. When CH is dissolved in acid, the amino groups in the chain protonate and the polymer becomes cationic, allowing it to interact with a wide range of molecules. This positive charge is assumed to be responsible for its antibacterial function since it interacts with microbes' negatively charged cell membranes [101]. 24 Figure 2: Chemical structure of CH [78]. Because CH is soluble in acidic environments, acids must be used [102][103]. CH conjugates can be generated depending on the acid used, resulting in the production of a complex that combines the characteristics of CH and the acid utilized. Because of its solubility in weak acid solutions, chitosan may be synthesized into multiple forms under considerably gentler conditions than its parent polymer chitin, making chitosan a more appealing biopolymer for a number of applications. For this and for the fact that CH is biocompatible, biodegradable, nontoxic and has wound healing and antibacterial capabilities, this polymer is receiving a lot of attention in the pharmaceutical and biomedical industries, which leads to the continuing study of this polymer for a wide range of biological applications [104]. East and Qin reported the first application of chitosan in WS in 1993. They concluded that chitosan fibres could be created by the wetspinning of its solution in dilute aqueous acetic acid and that the fibres properties were affected by the spinning conditions, such as spin stretch ratio, coagulation bath concentration and drying conditions [105]. In another article written by Shigerino Hitano et al. , a study of chitin and chitosan fibres obtained through WS was conducted, where the characteristics required for their applicability in different areas such as textiles, industrial materials, and medical and biotechnological materials was verified [106]. In other study made by School of Biological Sciences, it was demonstrated that drug-loaded hydrogel fibres made with CH could be used as a device capable of delivering sustained high concentrations of gemcitabine locally, achieving tumor control, with minimal toxicity for localized therapy of pancreatic cancer. This device was also produced by WS [82]. Vega et al ., also prepared and studied the properties of CH–PVA fibres produced by WS. In this article, they studied and analyzed different properties such as the polymers concentration, the coagulation bath and the crosslinking solution. Finally, they concluded that they could create fibres with mechanical characteristics and morphologies that are acceptable for a variety of specific applications [107]. 25 As can be seen CH nanofibres have a high potential for biomedical applications, however sometimes their characteristics are not in the ideal values, showing limited water resistance, and a poor capacity to absorb bodily fluids. For hence, combining CH with other polymers, such as PEO or PVA, and using a crosslinking agent are common techniques for improving some of these properties. CH Crosslinking The mechanical integrity of natural polymers, specifically chitosan, must be improved before implantation into the body [108][109]. For this reason, crosslinking fibres would be a viable option. To reinforce the chitosan nanofibrous structure in solid/aqueous environments, various crosslinking methods have been used. Physical crosslinking is one of the two types of crosslinking that has been used the most for chitosan. This type of crosslinking results from the dehydration of the chitosan acetate salt via amide bond formation [110]. In the other hand, the other crosslinking used is chemical crosslinking, that results from covalent bonds formed between the amino (–NH2) or hydroxyl groups (–OH) of chitosan with the chemical of interest [111][112]. As said, these crosslinking strategies may improve mechanical strength of the fibres and conduction in conductive material that will be essential in their development, as well as drying, which will also contribute to these properties [109]. To produce a crosslinking there must be one or more crosslinking agents. These crosslinkers are molecules that contain two or more reactive ends capable or chemically attaching to specific functional groups (primary amines, sulfhydryl’s, etc.) on proteins or other molecules [113]. In this case, since the work is directed to develop a structure based on chitosan it is important understand what agents can crosslink this polymer and what work has been done in this field, giving special attention to the chemical crosslinking, once it’s the one being used in this thesis. Hence, in the following Table 4 are some types of crosslinking done in some articles. 26 Table 4: Crosslinking systems applied on fibres Fibres produced Crosslinking system Reference Chitosan/poly(ethylene oxide) nanofibres Glutaraldehyde (GTA) [114] Chitosan Fibres Sodium triphosphate (TPP) [113] Chitosan/poly(ethylene oxide) nanofibres GTA [109] Chitosan Fibres Epichlorohydrin [115] Chitosan-PVA Fibres GTA/TPP [105] Based on this table, three crosslinkers have especial attention in the articles where fibres from CS are produced. Knowing by literature that epichlorohydrin has been reported as toxic to cells sometimes, in this thesis the other two options will be explored [115]. As mentioned earlier, in addition to crosslinking, the conjugation of polymers such as PEO and PVA could also improve the properties of CH. 2.3.2.2 PEO Poly(Ethylene Oxide) is an extremely biocompatible and biodegradable polymer, since it has the capacity to interact with polarized surfaces and is often inexpensive, making it an appealing biomaterial. In addition, it is hydrophilic, linear and readily soluble in both aqueous and organic solvents [116][117]. The repetitive structural unit of polyethylene oxide, PEO, is shown on Figure 3. Figure 3: Chemical structure of PEO [87]. 27 PEO may be used with CH and other polymers to improve mechanical properties and the spinning process. This polymer has been studied in many different areas, not excluding the one that is it most important for this thesis: the production of fibres. However, there are not many articles that reported the use of these two polymers for the production of wet-spun fibres, reinforcing the innovative character of this work. Still, some conclusions can be draw about the use of these polymers together from articles that reported the use of another technique (ES). As a matter of fact, in a study made by Fatemeh Kalalinia et al. , the authors focused on developing a new effective topical drug delivery system. Nanofibres of CH/PEO containing vancomycin (VCM) were successfully fabricated. The results showed that the CH/PEO/VCM fibres had very good mechanical, biomechanical, antimicrobial properties and an effective healing effect [114]. Also, S. Abib et al . created CH/PEO nanofibres with high antibacterial capabilities, good thermal stability, regulated antibacterial agent release and no toxicity in cutaneous and keratin fibroblast cell lines, allowing them to be used in the prevention and treatment of wounds and burns [118]. In another one, Guiping Ma et al . made the characterization of CH/hyaluronic acid nanofibres. In this article, they obtained a highly porous CH nanofibres membrane by using CH/PEO and then removing PEO with water. Then, the porous nanofibres were soaked in 0.1 wt % paclitaxel solution to load the cancer drug and a polyanion nature macromolecular hyaluronic acid was encapsulated on the chitosan polycation porous nanofibres. The nanofibres' activities in vitro on DU145 prostate cancer cells were investigated and cell culture findings revealed that the nanofibres mats were effective in preventing cell attachment and growth [119]. Finally, Jafari A et al. conducted a study in order to recognize CH/PEO/Berberine (BBR) nanofibres effect on cancer cell lines. An inverted microscope was used to examine the development and proliferation of human breast cancer cell lines, human HeLa cervical cancer cells, and fibroblast cells in cultured media. When compared to the control group cell lines, nanofibres containing 0.5-20 wt % BBR concentrations reduced cell proliferation. Cancer cell lines’ viability was drastically reduced after being exposed to CH/PEO/BBR [120]. 34 WS Parameters After having the desired solutions, they were transferred to syringes and WS tests were performed with two different needle diameters - Ø 0.61 mm and Ø 0.41 mm - and three different baths, also varying the flow rate. A summary of the parameters that were used, both in relation to the WS parameters and to the concentrations of the solutions is described in the following Table 6. Table 6: Parameters changed to obtain different CH fibres Solution Bath Conditions 2 % CH with 50/50 water and AcOH 1 M NaOH Room temperature, 0.41 and 0.61 mm needles 2 M NaOH Room temperature, 0.41 and 0.61 mm needles 2 % CH with 2 % AcOH aqueous solution 1 M NaOH Room temperature or after fridge, 0.41 and 0.61 mm needles 2 % CH with 6 % AcOH aqueous solution 1 M NaOH Room temperature or after fridge, 0.41 and 0.61 mm needles 2 M NaOH Room temperature or after fridge, 0.41 and 0.61 mm needles 3 % CH with 12 % AcOH aqueous solution 1 M NaOH Room temperature or after fridge, 0.41 and 0.61 mm needles 2 M NaOH Room temperature or after fridge, 0.41 and 0.61 mm needles 4 % CH with 12 % AcOH aqueous solution 1 M NaOH Room temperature or after fridge, 0.41 and 0.61 mm needles 2 M NaOH Room temperature or after fridge, 0.41 and 0.61 mm needles 35 3.1.1 CROSSLINKING Crosslinking techniques were employed after WS. In order to do these, three different methods were tested to see in which of them resulted in the best fibers. Two of these methods were based on the steam deposition of the crosslinking agent and another one based on a bath of the same. In the first procedure tested, Figure 7A, the fibres were collected and placed on a net over a beaker containing different quantities of GTA in a dissector. After that, a part of the fibres was placed inside the oven at different temperatures and others at room temperature, to study the effect of different temperatures for 24 hours. In the second procedure, the same idea was kept in mind but this time putting the fibres directly suspended in the beaker, leaving the net used before aside as shown in Figure 7B. Once again, GTA and different temperatures were used. The last procedure consisted of a bath resulting from a solution with the selected crosslinking agent where the fibres were immersed (Figure 7C). In this procedure, two different crosslinking agents and mixtures of both were tested: GTA and TPP. Different temperatures of drying of the fibres after the bath were tested afterwards. A B C Figure 7: A) First procedure tested with fibres collected and placed on a net over a beaker; B) Second procedure with fibres directly suspending in the beaker; C) Third procedure with a bath where the fibres were immersed. 36 3.1.1 CHITOSAN/PEO FORMULATIONS Preparation of the solution After optimizing the solution of CH and AcOH, PEO was added in order to be able to benefit the mechanical properties and thermal stability of the fibres. Different PEO concentrations were then tested: 1 %, 2 % and 3 %. To prepare these solutions, CH (sieved) and PEO were dissolved in water under mechanical stirring, individually. After achieving the correct dissolution for both polymeric formulations, they were mixed and acetic acid was slowly added and left in stir overnight. The final solution was centrifuged at 4000 rpm for 20 minutes and was then placed in the fridge for further tests. WS Parameters After obtaining the final solution, the production of fibres by WS was performed using the needle with the best results so far (0.41 mm diameter), a flow rate of 1 mL/min. and a 1 M NaOH bath. In summary, the parameters used are shown in Table 7 below. Table 7: Parameters used to obtain different CH/PEO fibres CH + AcOH PEO% Bath Conditions 3 % CH with 12 % AcOH aqueous solution 1 % 1 M NaOH Temperature after being in the fridge and 0.41 mm needle 2 % 3 % 37 3.1.1 CHITOSAN/PVA FORMULATIONS Preparation of the solution A CH and PVA mixture was also tested. In this case, four different PVA concentrations — 2 %, 5 %, and 10 % — were evaluated. Both polymers were separately dissolved in water while being mechanically stirred to create these solutions. It should be noted that the PVA solution needed a temperature of around 30 °C to dissolve properly. After the proper dissolution of both polymeric formulations, they were mixture. The solution was stirred overnight while acetic acid was gradually added. The final solution was centrifuged at 4000 rpm for 20 minutes before being stored in the fridge. WS Parameters The synthesis of fibres by WS was carried out using again the parameters optimized from the last results: the needle with 0.41mm diameter, a flow rate of 1 mL/min and a bath of 1 M NaOH. Table 8 below summarizes the parameters that were used. Table 8: Parameters used to obtain different CH/PEO fibres CH + AcOH PVA% Bath Conditions 3 % CH with 12 % AcOH aqueous solution 1 % 1 M NaOH Temperature after being in the fridge and 0.41 mm needle 3 % 5 % 38 3.1.1 SYNTHESIS OF FERROMAGNETIC NPS For synthesizing the ferromagnetic NPs, a protocol previously described was adapted [126]. In the first step of the NPs production, two solutions were made: an acidic solution of Iron II Chloride (2 M) and an aqueous solution of Iron III Chloride (1 M). These solutions were subsequently mixed and an ammonium hydroxide solution (1 M) was added. The solution obtained was placed under stirring for 30 minutes. After this, the solution was washed with water using a magnet attached to remove the material of interest. This material was subsequently dispersed in 250 mL of water, to which was added 2 mL of oleic acid and 5 mL of acetone. Again, the solution was placed under stirring for 30 minutes and the material of interest was separated using the magnet and washed with acetone. After this process, the leftover material was dispersed in 15 mL of cyclohexane and centrifuged for 30 minutes at 2000 rpm. In order to obtain the NPs in solid state, the solution was placed in the oven at 150 °C for 3 hours. Following its synthesis, different percentages of synthesized NPs were added to the optimized solutions studied in order to reach the better results. 3.1.2 DEVELOPMENT OF THE FIBROUS STRUCTURES For the production of the localized drug delivery structure composed by the optimized fibres produced by WS, three different approaches were tested. The first was the production by weaving: process of producing fabric by interlacing two sets of strands at right angles. The second was based on set the structure directly in the crosslinking bath, hoping that after it the fibres would dry and produce a well-structured form. The last procedure comprised in leaving the fibres in the crosslinking bath and after it making the structure by laying the fibres on themselves. In all of them, a Teflon sheet was used as platform to dry the structures. 39 3.2 CHARACTERIZATION METHODS 3.2.1 OPTICAL MICROSCOPY In the initial step, the optical microscope was utilized to analyze the arrangement and morphology of the produced fibres. This microscope employs visible light and an ocular lens system to create a magnified picture of the sample under examination, allowing the study of features that are not apparent to the human eye [106]. In this work, the fibres produced by the WS method were examined using a Leica DM750 M (bright field) Microscope equipped with two eyepieces and four objectives (5x, 10x, 20x and 40x). 3.2.2 SCANNING ELECTRON MICROSCOPY (SEM) SEM was also used to produce images of a sample by scanning the surface with a focused beam of electrons. The one used in this was NOVA Nano SEM 200, FEI Company (Hillsboro, OR, USA), instrument at 5000 and 50.000 × magnifications. All the fibres samples were added to aluminum pin stubs with an electrically conductive carbon adhesive tape (PELCO Tabs™). 3.2.3 ATTENUATED TOTAL REFLECTANCE - FOURIER TRANSFORM INFRARED SPECTROSCOPY (ATR-FTIR) The ATR-FTIR method was employed to investigate the interaction between the different polymers used and the chemical composition of the wetspun fibres produced. This method of vibrational spectroscopy vibrates the molecular bonds of the sample that absorbs infrared light and given that various samples have distinct molecular bonds or configurations, this approach may be used to learn the chemical makeup of the molecules found in each sample under study [109]. The chosen samples were subjected to an ATR-FTIR spectroscopic study using an IRAffinity-1S instrument from SHIMADZU (Kyoto, Japan) that has an ATR attachment. Each spectrum was recorded in a diamond ATR cell in transmittance mode using an accumulation of 40 45 scan cycles and an 8 cm-1 resolution. The machinery was set up to measure the samples' transmittance throughout a spectrum spanning 400 to 4000 cm-1. 3.2.4 SCANNING TRANSMISSION ELECTRON MICROSCOPY (STEM) The Scanning Transmission Electron Microscopy (STEM) was used to study the morphology and size of the produced NPs. The microscope lenses are changed in scanning transmission electron microscopy mode to provide a focused convergent electron beam or probe at the sample surface. This focused probe is then traversed across the sample, collecting signals point by point to generate a two-dimensional picture. The morphology of the synthetized NPs was studied using a NOVA 200 Nano SEM from FEI Company (Hillsboro, OR, USA) with a scanning transmitted electron microscopy detector. 3.2.5 ENERGY DISPERSIVE X-RAY SPECTROSCOPY (EDS) The samples were characterized using a desktop scanning electron microscope coupled with EDS analysis (Phenom ProX with EDS detector; Phenom-World BV, The Netherlands). All results for the quantification of the concentration of the elements present in the fibres were acquired using ProSuite software integrated with Phenom Element Identification software. 3.2.6 THERMOGRAVIMETRIC ANALYSIS (TGA) To confirm the thermal stability of the generated fibres, a TGA analysis was carried out [127]. In a STA 700 SCANSCI apparatus, the thermal behavior of the generated fibres was studied. The TGA curve was produced using nitrogen atmosphere, a temperature range of 30– 600 °C, and a constant heating rate of 10 °C/min. The samples were put in a crucible that was attached to a microbalance for the analysis, and the mass loss was tracked while the crucible was gradually heated. 41 3.2.7 MECHANICAL TESTS The equipment used for these testing was a Hounsfield Tinius Olsen model H100KPS with a 250 N load cell. The samples were divided into strips that were 2.5 mm wide and 7.5 mm long. 50 mm was used as the first grasp separation value. The stress-strain curves were then used to compute the Young's modulus, elongation at break, and breaking point. Fibres and structures of fibres were analyzed. 3.2.8 SWELLING DEGREE The weight difference between the samples that were dry and those that were swelled was used to gauge the degree of swelling. For 24 hours, the dried fibres were submerged in water heated to 37 °C. The wet weight of the fibres was calculated after extra liquid was removed. Assuming that Ws is the mass of the swollen material and Wd is the original dry mass, the swelling degree was determined as follows and represented as a percentage of the dry sample: 3.2.9 IN VITRO DEGRADATION The fibres were cultured in water for 24 hours at 37 °C. Following full drying in an oven set at 50 °C for 24 hours, they were weighed to determine the degree of degradation using the equation below, where Mi refers for initial dry mass and Mf represents for final dry mass: 42 4 RESULTS AND DISCUSSION 43 As previously mentioned, the main purpose of this work is to develop polymeric formulations based on CH, PEO and PVA to produce microfibres by wetspinning. For this, several WS and solution parameters were optimized as could be seen in the following sections. After the optimization of the polymeric formulations, several fibres were produced and some of them were selected. In these selected formulations for the fibres production, NPs were added in different percentages to analyze their effect in the final fibres. After complete characterization and analysis of some properties, the best fibres were utilized to develop fibrous structures with possible application as localized drug delivery systems for magnetic hyperthermia. 4.1 PRODUCTION AND OPTIMIZATION OF CHITOSAN WETSPUN FIBRES The first polymer tested for the production of fibres by wetspinning was chitosan. CH is a polymer that is widely utilized in the biomedical field and its solubility is related to the quantity of protonated amine groups in the polymer chain. When CH is dissolved in acid, the amino groups in the chain protonate, causing the polymer to become cationic and capable of interacting with a wide spectrum of molecules. AcOH was utilized in the preparation of the CH solutions for this purpose [128]. Several parameters, including pre-processing, percentage and proportion of the polymers, percentage of the solvent, needle diameter, temperature of the solution and postprocessing of the solution mixture (centrifugation and homogenization), were evaluated and optimized to produce CH fibres. Also, three different baths were tested: 1 M NaOH, 2 M NaOH and 4 M NaOH. The 4 M NaOH was directly excluded once it was impossible to use, due to its adverse reaction with the material thanks to its high concentration. The formulations used are now described in Table 9. 50 As a result of the poor results obtained previously, a new method was also tested. In this method, the fibres were immersed in a crosslinking bath. Three different baths were tested, one with GTA, another with TPP and another with a mixture of both. Only in the TPP bath it was possible to acquire results suitable for analysis, since in the other baths the fibres ended up breaking very easily, escaping the expectations of the same ones. Through a new microscopic analysis illustrated in Figure 13, it was possible to verify that the results obtained by this method were much better than the ones obtained before, with less defects in the fibres. Besides, through the analysis of the average diameters of the fibres presented in Table 12, it was possible to corroborate the previous idea and to choose the best formulation of the bath that would be used from now on. Figure 13: Optical microscopy image (10x magnification) of fibres obtained with bath method: A) and B) 4 h of 1 % TPP bath dried at room temperature; C) and D) 4 h of 1 % TPP bath dried at 50 °C. 51 Table 12: Mean and standard deviation values of the diameters of the fibres obtained Fibre diameters at Mean (µm) S. Deviation (µm) Room temperature 114 3.87 50 °C 95 9.34 Thus, as it is possible to analyze through the table above, the smaller standard deviation present in the average of the fibres diameters would be the one resulting from the TPP bath for four hours and subsequent drying at room temperature, leading to the conclusion that it is with this formulation that the fibres with less defects and closer to the best results are obtained. 52 4.2 CHITOSAN/PEO WETSPUN FIBRES CH and PEO are two biodegradable polymers that have been widely used for the production of fibres. In fact, several authors have combined these two biodegradable polymers in order to take advantage of the inherent advantages of each one of them, i.e., to combine the bioactivity of CH with the mechanical strength of PEO [118]. Therefore, in this section, the CH/PEO system was optimized. Similarly, to the previous system, at an early stage, the different parameters at the solution and WS process levels were evaluated. Fibres with a better diameter distribution and apparent morphology than the previous ones were obtained by using 3 %(w/v) CH/PEO (1:1) dissolved in an aqueous solution with 12 % AcOH aqueous solution. A flow rate value of 1 mL/min, a 0.41 mm diameter needle and a coagulation bath of 1 M of NaOH were used. The posterior crosslinking bath used was the one optimized as well: four hours of a TPP bath with drying at room temperature. 4.2.1 PRODUCTION AND OPTIMIZATION OF CHITOSAN/PEO WETSPUN FIBRES In order to produce CH/PEO fibres several parameters were evaluated and optimized, including pre-processing, percentage and proportion of the polymers, percentage of the solvent, post-processing of the solution mixture (centrifugation and homogenization), order of the addition of the reagents and polymers in the solution. For this, three solutions were obtained and then centrifuged for 20 minutes at 4000 rpm and cooled after 24 h in the fridge. With these solutions the WS was carried out, maintaining the previously optimized conditions. First formulation: 1 %(w/v) PEO with 3 %(w/v) CH in a 12 %(v/v) AcOH aqueous solution Second formulation: 2 %(w/v) PEO with 3 %(w/v) CH in a 12 %(v/v) AcOH aqueous solution Third formulation: 2 %(w/v) PEO with 3 %(w/v) CH in a 12 %(v/v) AcOH aqueous solution 53 A B C The microscopic images of the fibres obtained allowed conclusions to be drawn about them, once again helping to verify their diameter variation and surfaces. Thus, in Figure 14 the fibres obtained are represented with 1 %, 2 % and 3 % PEO, respectively. Their mean diameters and standard deviation values are exhibited in Table 13. Table 13: Mean and standard deviation values of the diameters of the fibres obtained With this, it can be seen that, regardless of the value of the percentage of PEO, there is a reduction in the mean value of the fibres’ diameter, in comparison to the ones without PEO. For higher percentages of PEO, it can be observed a higher reduction in fibre diameter, as well as a tendency for the standard deviation to be also lower. Regarding their appearance, it can also be seen a surface that seems to be less rough, as well as less brittle and more elastic than the previous ones, confirming the improvement of the physical properties with the addition of PEO. Fibre Diameters of Mean (µm) S. Deviation (µm) 1 % PEO 3 % CH 112 7.75 2 % PEO 3 % CH 108 6.32 3 % PEO 3 % CH 105 4.39 Figure 14: Optical microscopy image of fibres (5x magnification) with: A) 1 % PEO; B) 2 % PEO; C) 3 % PEO. 54 4.3 CHITOSAN/PVA WETSPUN FIBRES The PVA polymer may be used in an almost endless number of industries, especially as attention turns away from synthetic materials and toward biodegradable ones. This phenomenon has opened up a wide range of opportunities for such materials because of their environmentally friendly and user-friendly qualities [12]. When combined with polymers like CH, they can provide significant benefits for a variety of applications, so they will also be studied in this article and in this section. The evaluation of many parameters was done at both the solution and WS process levels. Using 5 %(w/v) PVA with 3 %(w/v) CH mixed in an aqueous solution with a 12 % AcOH aqueous solution, fibres were produced with a better diameter distribution and apparent morphology than the prior ones. A 0.41 mm diameter needle, a 1 M NaOH coagulation bath, and a flow rate of 1 mL/min were employed as well as a posterior crosslinking bath: four hours in a TPP bath, followed by drying at room temperature. 4.3.1 PRODUCTION AND OPTIMIZATION OF CHITOSAN/PVA WETSPUN FIBRES The pre-processing, percentage and proportion of the polymers, percentage of the solvent, post-processing of the solution mixture (centrifugation and homogenization), and order of the addition of the reagents and polymers in the solution were evaluated and optimized in order to produce CH/PEO fibres. Once again, three solutions were produced, centrifuged for this at 4000 rpm for 20 minutes, and then chilled in the refrigerator for 24 hours. The solutions produced were the following: First formulation: 1 %(w/v) PVA with 3 %(w/v) CH in a 12 %(v/v) AcOH aqueous solution Second formulation: 3 %(w/v) PVA with 3 %(w/v) CH in a 12 %(v/v) AcOH aqueous solution Third formulation: 5 %(w/v) PVA with 3 %(w/v) CH in a 12 %(v/v) AcOH aqueous solution 55 Figure 15: Optical microscopy image of fibres (5x magnification) with: A) 1 % PVA; B) 3 % PVA; C) 5 % PVA. The inferences that might be taken about the fibres from their microscopic images helped confirm their surface and diameter variation yet again. As a result, in Figure 15, the produced fibres are shown with, respectively, 1 %, 3 %, and 5 % PVA. Table 14 shows their mean diameters and standard deviation values. Table 14: Mean and standard deviation values of the diameters of the fibres obtained As previously seen in CH/PEO system, regardless of the value of the percentage of PVA, there is a reduction in the mean of the diameter of the fibres, in comparison to the ones without PVA, being noticed as well that once again, for higher percentages of polymer added it can be observed a higher reduction in fibre diameter, as well as a tendency for the standard deviation to be also lower. In comparison with the CH/PEO results, it can be seen that value for the standard deviation is better once it is lower. By looking at the images it can also be seen some less defects, showing that in this chapter the system of CH/PVA might be superior. However, these conclusions were not decisive Fibre Diameters of Mean (µm) S. Deviation (µm) 1 % PVA 3 % CH 123 5.24 3 % PVA 3 % CH 117 5.14 5 % PVA 3 % CH 103 3.94 56 for choosing what system should be used, once the most important parameters are the mechanical properties of this system and the incorporation of MNPs in these fibres. 4.4 FERROMAGNETIC NPS As stated before, ferromagnetic NPs are really promising NPs especially due to their biocompatibility and stability, biodegradability, ease of surface modification and functionalization [55][56]. In this work, ferromagnetic NPs were produced in order to be incorporated onto the fibres. After this, a fibrous structure with these NPs was developed, making it possible to act as a drug delivery system for hyperthermia therapy for cancer. In order to produce these NPs, as already highlighted, methods previously written in an article done by César A. Henriques et al. were followed [126]. 4.4.1 FERROMAGNETIC NPS PRODUCED - MAGNETIC CHARACTERISTICS AND STEM ANALYSIS After following the mentioned protocol, the final solution resulting from the coprecipitation process was obtained with the suspended NPs. Using a magnet, it was also possible to instantly verify the magnetic response capacity of the same solution as the magnet was moved forward as shown in the sequence of Figure 16. 57 In order to characterize the NPs obtained, they were analyzed via STEM, presenting the results in Figure 17. According to the analysis performed by TEM in the article previously followed for the production of these particles, these should have a size of approximately 8.3 nm [126]. However, since the equipment available for analysis in this case was a STEM with a minimum measurement until about 9 nm, it is possible that the NPs in question are also comprised between these sizes. As for the dispersion, it is possible to verify that there is not much agglomeration, which shows once again that it is possible to have a good stability, as also mentioned in the previous article. Related to the shape, it shows almost a spherical shape. These characteristics can be proven by the previously mentioned article and by studies related to the production of ferromagnetic NPs [113][129]–[131]. After checking its activity, this solution was placed in an oven for 3 hours at 150 °C so that all excess solvents could be evaporated. At the end of this time, the NPs were dried and once again they showed magnetic characteristics - Figure 18 -, proving that the drying process did not affect them, and that they can be used for the intended application. Figure 16: Magnetic response from the obtained NPs’ solution to the movement of a magnet. Figure 17: STEM images of ferromagnetic NPs. 58 4.5 FIBRES WITH FERROMAGNETIC NPS With the CH/PEO and CH/PVA systems optimized and after the desired MNPs were obtained a new step emerged. In this step the dissolution of the MNPs in the optimized solutions to make the NPs integrate the fibres was the goal. To reach this goal, combinations including the previously optimized solutions and various ferromagnetic nanoparticle concentrations were tested, trying to obtain the final solution. The combinations tried out are expressed in following Table 15: Table 15: Combination of different solutions and NPs percentages Solutions % NPS 3 % CH 3 % PEO 0.5 1 2 3 % CH 5 % PVA 0.5 1 Images of the different fibres with different percentages of NPs are shown in Figure 19. As it is visible, all fibres presented a good homogeneity in diameters, presenting few defects, as Figure 18: Magnetic response from the obtained dried ferromagnetic NPs to the movement of a magnet. 59 A B C D E expected, given the previous optimization. Since at this stage it was important to analyze the results obtained in more detail in order to choose the final solution that would serve as the basis for the final system, mechanical tests were performed, opting for the solution that stood out in that sense. 4.5.1 MECHANICAL TESTS After the production of all the fibres resulting from all the different solutions by wetspinning, it was proceeded to the mechanical analysis of the fibres, since, as previously reported, after the optimization of the solutions, the microscopic analysis previously done is not very conclusive and it is not possible to detect such big differences as it was possible to do by comparing the diameters of the fibres. Therefore, and because it is also very important for the Figure 19: Optical microscopy image of fibres (5x magnification). PEO Fibres with: A) 0.5 % MNPs; B) 1 % MNPs; C) 2 % MNPs. PVA Fibres with: D) 0.5 % MNPs; E) 1 % MNPs. 66 By observation of Figure 22, it is visible that for both TGA curves there’s an initial mass loss occurs, between 40 °C and 150 °C, related to the evaporation of absorbed water and other volatile compounds [112]. The TGA and DTG curves also show two main mass loss steps: the first occurs between 250 °C and 300 °C, and corresponds to the degradation of CH, the second step is related to the degradation of PEO and occurs between 360 °C and 420 °C, confirming the presence of these two polymers in CH/PEO nanofibres. By observation of the DTG graph these previously mentioned peaks can also be identified and another one in the red band (with 2 % NPs) can also be identified. This peak, at around 400500 °C band is associated with the phase transformation of Fe3O4-Fe2O3 as already mentioned in an article by Nigam S. et al., showing once more the particles included on the fibres [131]. Thus, through this analysis, the success of the incorporation of NPs in CH/PEO nanofibres was proven and, at the same time, it was also verified that the incorporation of these NPs in CH/PEO nanofibres confers a higher resistance to thermal degradation, as their final value remains higher than the value presented by the fibres without MNPs. 4.5.5 SWELLING AND DEGRADATION TESTS Swelling and degradation tests were performed to the fibres with the best mechanical properties to study and understand how fibres would respond to being incubated for a long period of time in water. As seen in Table 18, the solution without NPs had the highest swelling degree (315 ± 5.49 %) and in vitro degradation (26.6 ± 1.69 %) after 24 h. The fibres with MNPs reached minor values. As is generally known, a more hydrophilic material may absorb more water, resulting in larger swelling degrees and consequently altering the degradation rates of the material's systems. For the fact that PEO and CH are two hydrophilic polymers and CH presents well deal of interests for hydrogen bonding with water molecules, the results found in fibres without NPs for swelling are according to the literature. As the swelling is higher in these fibres, when they are subjected to degradation tests their volume will be higher and therefore higher will be the chance of more mass loss and consequently higher degradation [140][141][142]. With the addition of NPs to the fibre matrix, the fibres tend to become more filled and, consequently, there's results like the reduction of porous that were present before and thus decreases the volume of fluid absorbed, favoring less swelling and less degradation [141]. 67 Having said this, it is possible to state that the incorporation of these NPs is satisfactory, as it allows the fibre structure to be more stable. Table 18: Swelling and degradation tests results for fibres Fibres Swelling degree (%) In vitro degradation (%) 3 % CH PEO 3 % 315 ± 5.49 26.6 ± 1.69 3 % CH PEO 3 % with 2 % Ferromagnetic NPs 243 ± 2.39 12.2 ± 1.28 68 4.6 PRODUCTION OF THE FIBROUS STRUCTURES MNPs can enter the body through systemic circulation and accumulate at tumor locations, or they can be injected directly into the tumor. However, particles injected intravenously are quickly covered by circulation components such as plasma proteins. The adsorption of proteins on the particle surface, termed opsonization, facilitates phagocytosis. Furthermore, most intravenously injected NPs are recognized by the body as "foreign bodies" and are quickly eliminated by circulating monocytes or fixed macrophages [143]. To avoid reticuloendothelial system activity, particle size must be controlled, else they would aggregate and have a short half-life in the blood circulation. The magnetic particles utilized are very tiny (dozens of nanometers) and, consequently, are easily taken away by blood flow and disseminated to different organs or tissues during hyperthermia therapy. To address these issues, a polymeric fibrous matrix is proposed for accurate administration of a magnetic material to the tumor site via a surgical or endoscopic technique for localized magnetic hyperthermia treatment. Diverse structures might lead to radically different consequences, ensuring or not, the success of the same [144]. In this case, the structure developed needed to be ready to cover up a tumor. In order to do so, and considering that tumors have irregular shape, the structure to be developed had to be like a net that would allow the whole tumor to be involved, and therefore flexibility and malleability were indispensable. These two conditions would also be important to define an easier means of administration such as a biopsy tube [82]. To meet these expectations and to produce the structures in question, three different methods were tested. 69 4.6.1 METHODS FOR FIBROUS STRUCTURES The methods used and the results obtained are expressed in the following Table 19 and Figure 24: Table 19: Results for every method used to create fibrous structures Method Description Results Weaving In this method, the fibres were used after dried. A technique called weaving was used, wich is based on interlacing two sets of yarns so that they cross each other, normally at right angles. This method didn’t work as the fibres obtained had not enough tension to stay crossed with each other. Directly in the crosslinking bath in this method, after obtaining the fibres by wetspinning, an attempt was made to make a net structure by overlapping the fibres one on top of the other directly in the crosslinking bath. This method didn’t work as the fibres were stable enough to create a well shaped structure. After the crosslinking bath In this method, after the obtaining the fibres and after diving them in the crosslinking bath for 4 h, the structure was made by overlaping the fibres and leave them to dry at room temperature. This method had the better results, being able to produce a good structure. 70 As the last method was the only method that presented satisfactory results, this was the method used for the production of the remaining structures used in this work. 4.6.2 SEM ANALYSIS AND MAGNETIC CHARACTERISTICS OF THE STRUCTURES PRODUCED Using the method previously described, different structures were then produced based on the previously selected formulation: CH/PEO with 2 % MNPs. Structures without the NPs were also made in order to serve as comparison to the previously described ones for mechanical, swelling and degradation tests. Figure 25 represents the structures obtained without NPs and Figure 26 the structures with 2 % MNPs. B A Figure 24: Methods used to produce fibrous structures: A) Directly in the crosslinking bath; B) After the crosslinking bath. 71 Figure 25: Structures produced with CH/PEO system without MNPs. It is possible to verify from the figures that well defined structures were obtained, without major defects to the naked eye. As for their color, it can be seen that the structures with NPs acquired an orange color resulting from the iron solutions used in the production of the NPs, unlike those without NPs, which present a white color. Figure 26: Structures produced with CH/PEO system with MNPs. 72 To be able to dimension and to really understand if there are enough defects and the appearance of the structure in the joints, the SEM analysis of the joints in the structures with MNPs was carried out. Thus, according to Figure 27 it can be seen that the structure of fibres with MNPs present a diameter always around 100 nm as previously mentioned with few defects and that the overlapping fibres are well bonded and even seem to have fused together after drying, proving a good optimization of the solution and an effective method of processing these structures. Also, the visual appearance is again similar to those obtained previously for the fibres with MNPs, and so surely if a new EDS analysis were performed it could be once again verified the presence of the iron ion in these structures. After this analysis, it was important to understand once again if, this time in structure, the magnetic properties of the MNPs were maintained, making the structure also responsive to magnetic stimulations and with a good and successful incorporation of the NPs. That said through exposure to a magnet again, the structures were quickly attracted by the magnet, showing magnetic field response behavior and potential for remote manipulation of these under a controlled magnetic field, as it is shown in Figure 28. In other articles, Kim et al. and Wang et al . also performed these tests, concluding that these structures were correctly functionalized with MNPs [87][92]. Figure 27: SEM images of the fibre structures with MPNs: joints and fibres present in it. 73 Figure 28: Illustration of the magnetic properties of the structures developed with MNPs: A) Magnet near the structure; B) and C) Structures attaches to the magnet; D) Structure in full contact with the magnet. 4.6.3 SWELLING AND DEGRADATION TESTS Swelling and degradation tests were performed again to the structures this time to see if the respond would corroborate the results obtained before. The results on Table 20 are essentially comparable, indicating that without NPs, there is a larger proportion of swelling, which corresponds to a higher percentage of degradation. It is also feasible to check an agreement in the values, with both being near to the previously acquired values. It should be noted that in the structure with NPs, there is still a reduction in the percentage of swelling and degradation, which, while tiny, may demonstrate a benefit of the junction of fibres, making its degradation more difficult. 74 Table 20: Swelling and degradation tests results 4.6.4 MECHANICAL TESTS As regards the mechanical tests performed on the structure, the expected improvements of all the characteristics in relation to those previously calculated with only fibres were proven. Thus, to compare the results obtained in structures, mechanical tests to structures with and without NPs with the optimized solution were performed. Table 21 shows the values obtained. The addition of NPs resulted in a 12 % increase in the maximum tension that the material can be subjected until rupture. The breaking point and Young's modulus also increased by 61 % and 30 % respectively, but the deformation at break was reduced by 25 %. Since the structures obtained were very similar in relation to its form, this reinforcement effect is due purely to the addition of the stronger and stiffer magnetite particles. These results are equally seen in another study, made by Miyauchi et al. , where Polyvinylpyrrolidone/PEO fibres with high concentrations of ferromagnetic and superparamagnetic NPs were developed. In this study, it was noticed that the inclusion of 10 % wt magnetite particles resulted in an increase in breaking point (+41 %) and Young's modulus (+33 %), but a decrease in elongation at break (-41 %). The decrease in elongation at break with higher particle loading was interpreted as being due to different abilities of the fibres to allow fibre necking to occur. This necking phenomenon is gradually restricted with increasing particle content which leads to these results [88]. 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