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(1)WYDZIAŁ INŻYNIERII MATERIAŁOWEJ I CERAMIKI Katedra Biomateriałów i Kompozytów. Rozprawa Doktorska. INHALABLE ANTICANCER DRUG DELIVERY SYSTEMS BASED ON FATTY ACIDS AND MAGNETIC NANOPARTICLES Wziewne nośniki leków przeciwnowotworowych oparte na kwasach tłuszczowych i nanocząstkach magnetycznych. mgr inż. Katarzyna Reczyńska Promotor: prof. dr hab. inż. Elżbieta Pamuła. Kraków, 2018.

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(3) Acknowledgements I would like to express my gratitude to my supervisor prof. dr hab. inż. Elżbieta Pamuła for her guidance and mentorship, the help in completing my thesis, patience and all good advice through all those years. Thank you for your trust and faith in me. My sincere thanks goes to mgr inż. Krzysztof Pietryga and dr inż. Łucja Rumian for their support and lots of stimulating discussions we had together. I would like to thank dr inż. Małgorzata Krok-Borkowicz and dr inż. Urszula Cibor for their help, advice in the laboratory and introducing me to the cell cultures. I thank also my fellow members of prof. Pamuła group: mgr inż. Agata Łapa, mgr inż. Bartosz Mielan, mgr inż. Anna-Maria Tryba and mgr inż. Agata Flis. This work would not have been possible without the support from Professor Wojciech Chrzanowski, PhD, DSc from the Faculty of Pharmacy at the University of Sydney. Thank you for the opportunity to stay in you laboratory at the University of Sydney, for your guidance, inspiration, support and whole help. Those months spent with you and your group were a special experience for me and I will never forget them. I would also like to acknowledge the amazing team of Professor Chrzanowski at the University of Sydney. My special thanks goes to Dipesh Khanal, PhD and Sally Yunsun Kim, PhD for their invaluable help, suggestions and support. I would also extend my gratitude to Priyanka Tharkar, M.Phil and Kamini Divakarla, M.Phil for their warmth and hospitality. I would not be able to finish my PhD thesis without the support from prof. dr hab. inż. Marek Langner and dr inż. Tomasz Borowik who helped me with HPLC analyses. I also thank Dr Aleksandr Mironov, MD, PhD, dr hab. Marta Wolny-Marszałek, prof. IFJ, dr Witold Reczyński and mgr inż. Kamil Kornaus for all help with characterisation of modified SPION. I would also like to acknowledge dr hab. inż. Kinga Pielichowska for the help with thermal analyses. I am indebted to my family and friends who supported me during those years. Thank you for your faith, patience and encouragement.. This study was supported from the National Science Centre, Poland (Harmonia Grant no. 2014/14/M/ST5/00649) in cooperation with the University of Sydney, Australia and Wrocław University of Science and Technology, Poland.. 3.

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(5) List of Abbreviations A549 AAS-ET AFM ALK Am ATP BEAS-2B BET CAP CCK-8 CT CTAB CVD DAPI DLS DMEM DMSO DNA DPI DSC EDTA EGFR Et FA FBS FDA FTIR HCL HPLC IARC LAU LDH MP MRI MWCO MYR MYR/PAL MYR/STE NP NSCLC PAL PAX PBS PCM PEG. human lung epithelial cells of lung carcinoma origin atomic absorption spectrometry, electrothermic method atomic force microscopy anaplastic lymphoma kinases ammonia adenosine triphosphate human lung epithelial cells of normal lung/bronchus origin Brunauer–Emmett–Teller capric acid cell counting kit-8 computed tomography hexadecyltrimethylammonium bromide chemical vapour deposition 4′,6-diamidine-2′-phenylindole dihydrochloride dynamic light scattering Dulbecco’s modified Eagle medium dimethyl sulfoxide deoxyribonucleic acid dry powder inhaler differential scanning calorimetry ethylenediaminetetraacetic acid epidermal growth factor receptor ethanol fatty acid foetal bovine serum Food and Drug Administration Fourier-transform infrared spectroscopy hollow-cathode lamp high performance liquid chromatography International Agency for Research on Cancer lauric acid lactate dehydrogenase microparticle magnetic resonance imaging molecular weight cut off myristic acid a mixture of myristic and palmitic acids a mixture of myristic and stearic acids nanoparticles non-small cell lung cancer palmitic acid paclitaxel phosphate buffered saline phase change material poly(ethylene glycol) 5.

(6) PET PLGA PTMS PVA PVD PVP RNA ROS SCLC SLM SPION SPION@SiO2 SPION@mSiO2 SPION@SiO2@mSiO2 STE TEM TEOS Tm TMOS UHQ-water UV VEGF WHO. positron emission tomography poly(lactic-co-glycolic acid) phenyl trimethoxysilane poly(vinyl alcohol) physical vapour deposition poly(vinyl pyrrolidine) ribonucleic acid reactive oxygen species small cell lung cancer solid lipid microparticle superparamagnetic iron oxide nanoparticles superparamagnetic iron oxide nanoparticles coated with non-porous silica superparamagnetic iron oxide nanoparticles coated with mesoporous silica superparamagnetic iron oxide nanoparticles coated with non-porous silica and mesoporous silica stearic acid transmission electron microscopy tetraethyl orthoslicate melting temperature tetramethyl ortosilcate ultra-high quality water ultraviolet vascular endothelial growth factor World Health Organisation. 6.

(7) Abstract Lung cancer is one of the most lethal types of cancer. Since it is believed that conventional methods of treatment reached their maximal therapeutic efficiency, there is an urgent need for novel and more effective treatment options. This thesis was aimed at the development of novel, inhalable anticancer drug delivery system based on fatty acid microparticles loaded with magnetic nanoparticles (superparamagnetic iron oxide nanoparticles – SPION) and anti-cancer drug (paclitaxel, PAX). The microparticles are intended to be delivered directly to the patient’s lungs via inhalation and guided to the tumour using external magnetic field. Upon application of alternating electromagnetic field, magnetic nanoparticles embedded in the microparticles will heat up, melt the lipid matrix of the microparticles and release the drug straight to the tumour. For inhalation purposes, the microparticles should have the diameters ranging from 1 to 5 µm, while for hyperthermia conditions they should melt at the temperatures between 42 – 47°C. At the beginning of the study, several saturated fatty acids were tested in order to determine their physico-chemical properties and the influence on lung epithelial cells (viability, cell membrane stiffness and uptake). Among tested materials, only lauric acid (LAU) and an eutectic mixture of myristic acid and palmitic acid (MYR/PAL, mass ratio: 58:42) had suitable melting temperature and showed no negative influence on lung epithelial cells of normal origin (BEAS-2B). Surprisingly, MYR and PAL decreased the viability and cell membrane stiffness of lung epithelial cells of normal origin. The studies on surface modification of SPION were performed simultaneously. SPION were coated with different silica layers deposited via sol-gel method. All types of silica coating significantly inhibited iron release and were cytocompatible with lung epithelial cells. SPION coated with mesoporous silica (SPION@mSiO2) were selected to be incorporated into fatty acid-based microparticles. Further studies were aimed at optimisation of the manufacturing process in order to obtain spherical microparticles with desired size and uniform size distribution. Finally, the microparticles loaded with modified SPION and paclitaxel were fabricated using LAU and MYR/PAL. It was possible to obtain homogenous batch of microparticles with average particle size ranging from 2.0 µm to 3.2 µm, with high loading efficiency of both magnetic nanoparticles and paclitaxel. Drug loaded microparticles effectively supressed the growth of malignant lung epithelial cells. In simulated hyperthermia conditions, the decrease in cell viability was more pronounced for LAU-based MPs due to more appropriate melting temperature of the MPs and facilitated PAX release. Presented results proved that the developed anti-cancer drug delivery system based on fatty acids and magnetic nanoparticles can increase the efficacy of lung cancer treatment and significantly improve the quality of life of thousands patients with lung cancer.. 7.

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(9) Streszczenie Nowotwór płuc jest jedną z najbardziej śmiertelnych odmian nowotworów. Uważa się, że dostępne metody leczenia nowotworów osiągnęły swoje maksymalne możliwości, dlatego konieczne jest opracowanie nowych metod leczenia. Celem niniejszej pracy było opracowanie wziewnego preparatu farmakologicznego w postaci mikrocząstek zbudowanych z kwasów tłuszczowych, z dodatkiem nanocząstek magnetycznych (SPION, ang. superparamagnetic iron oxide nanoparticles) i leku przeciwnowotworowego (paklitakselu). Mikrocząstki będą dostarczone do płuc pacjenta poprzez inhalację, a przy użyciu zewnętrznego pola magnetycznego nakierowywane w miejsce występowania nowotworu. Następnie, przyłożone zmienne pole elektromagnetyczne doprowadzi do ogrzania się nanocząstek magnetycznych znajdujących się wewnątrz mikrocząstek tłuszczowych, co z kolei spowoduje stopienie się kwasów tłuszczowych i uwolnienie leku bezpośrednio do tkanki nowotworowej. Mikrocząstki powinny mieć średnicę w zakresie 1 – 5 µm, optymalną dla inhalacji oraz charakteryzować się temperaturą topnienia w granicy 42 – 47°C, która z kolei jest stosowana w hipertermii Pierwszym etapem badań była ocena właściwości fizyko-chemicznych różnych nasyconych kwasów tłuszczowych oraz określenie ich wpływu na komórki nabłonkowe płuc. Spośród badanych materiałów tylko kwas laurynowy (LAU) oraz eutektyczna mieszanina kwasu mirystynowego i palmitynowego (MYR/PAL, w stosunku 58:42) charakteryzowały się optymalnymi właściwościami termicznymi i nie wykazywały negatywnego wpływu na prawidłowe komórki nabłonkowe płuc. Co ciekawe, MYR i PAL znacząco zmniejszyły żywotność komórek nabłonkowych pochodzenia nowotworowego oraz sztywność ich błony komórkowej. Równocześnie prowadzone były badania nad modyfikacją SPION przy pomocy powłok krzemionkowych osadzanych metodą zol-żel. Wszystkie zastosowane powłoki skutecznie zapobiegały uwalnianiu się jonów żelaza z SPION, jednak najbardziej optymalne właściwości wykazywały SPION pokryte mezoporowatą krzemionką (SPION@mSiO 2). Nie wykazywały one też negatywnego wpływu na komórki nabłonkowe płuc, dlatego zostały wyselekcjonowane do dalszych badań. Następnie dobrana została metoda wytwarzania jednorodnych mikrocząstek o wymaganych średnicach. Otrzymane mikrocząstki z dodatkiem modyfikowanych SPION i paklitakselu na bazie LAU lub MYR/PAL mieściły się w zakresie od 2.0 µm do 3.2 µm oraz charakteryzowały się wysokim stopniem załadowania zarówno nanocząstek magnetycznych jak i paklitakselu. Mikrocząstki z dodatkiem leku przeciwnowotworowego skutecznie hamowały żywotność komórek nabłonkowych płuc pochodzenia nowotworowego. Dodatkowo udowodniono, że w temperaturze 45°C żywotność komórek inkubowanych z mikrocząstkami opartymi na LAU była znacznie niższa niż w 37°C, co świadczy o zwiększonym uwalnianiu paklitakselu. 9.

(10) Opracowany wziewny system dostarczania leków przeciwnowotworowych przeznaczony do leczenia nowotworów płuc spełnia wszystkie stawiane mu wymogi. Może on znaleźć zastosowanie w leczeniu nowotworów płuc, zmniejszyć śmiertelność wśród pacjentów oraz znacząco poprawić jakość ich życia.. 10.

(11) Contents. Acknowledgements ............................................................................................................................. 3 List of Abbreviations ........................................................................................................................... 5 Abstract ............................................................................................................................................... 7 Streszczenie ......................................................................................................................................... 9 Contents ............................................................................................................................................. 11 Chapter 1. Introduction .................................................................................................................. 15 1.1.. Lung cancer ....................................................................................................................... 15. 1.2.. Causes of lung cancer ........................................................................................................ 15. 1.3.. Histological types of lung cancer ....................................................................................... 16. 1.4.. Conventional methods of lung cancer treatment ................................................................ 18. 1.5.. Novel methods of lung cancer treatment ............................................................................ 21. 1.6.. Causes of high mortality in lung cancer patients ................................................................ 23. Chapter 2. Aims and general outline of the thesis ......................................................................... 25 Chapter 3. Analysis of physico-chemical properties of saturated fatty acids and their influence on human lung epithelial cells ......................................................................................................... 29 3.1.. Fatty acids (FAs) ................................................................................................................ 29. 3.1.1.. Chemical structure and properties of FAs .................................................................. 29. 3.1.2.. Biological role and metabolism of FAs ...................................................................... 31. 3.2.. Materials and methods ....................................................................................................... 33. 3.2.1.. Physico-chemical characterization of FAs ................................................................. 34. 3.2.2.. In vitro characterization of FAs .................................................................................. 34. 3.2.3.. Statistics ..................................................................................................................... 36. 3.3.. Results ............................................................................................................................... 37. 3.3.1.. Thermal properties of FAs ......................................................................................... 37. 3.3.2.. Chemical composition of FAs .................................................................................... 38. 3.3.3.. In vitro influence of FAs on lung epithelial cells ....................................................... 39. 3.4.. Discussion .......................................................................................................................... 47. 3.5.. Conclusions........................................................................................................................ 50. Chapter 4. Surface modification of superparamagnetic iron oxide nanoparticles (SPION) and evaluation of their physico-chemical and biological properties ................................................... 51 4.1.. Superparamagnetic iron oxide nanoparticles (SPION) ....................................................... 51. 4.1.1. Synthesis of SPION ................................................................................................... 52. 4.1.2. Modifications of SPION ............................................................................................ 54. 4.2.. Materials and methods ....................................................................................................... 56. 11.

(12) 4.2.1. Materials .................................................................................................................... 56. 4.2.2. Deposition of silica coatings ...................................................................................... 56. 4.2.3. Physico-chemical characterization of modified SPION ............................................. 57. 4.2.4. In vitro cytotoxicity ................................................................................................... 58. 4.2.5. Statistics .......................................................................................................................... 62 4.3.. Results ............................................................................................................................... 62. 4.3.1. Chemical composition, morphology and magnetic properties of modified SPION.... 62. 4.3.2. Surface properties and iron release ............................................................................ 65. 4.3.3. In vitro influence of modified SPION on lung epithelial cells ................................... 66. 4.4.. Discussion ......................................................................................................................... 71. 4.5.. Conclusions ....................................................................................................................... 74. Chapter 5. Design of fabrication method for manufacturing of fatty acid-based microparticles (MPs) ................................................................................................................................................ 75 5.1.. Methods for fabrication of solid lipid microparticles (SLM) ............................................. 75. 5.2.. Materials and methods ....................................................................................................... 78. 5.2.1.. Materials .................................................................................................................... 78. 5.2.2.. Emulsification using magnetic stirrer ........................................................................ 78. 5.2.3.. Emulsification using vortex mixer ............................................................................. 79. 5.2.4.. Microparticle size and size distribution ..................................................................... 80. 5.2.5.. Statistics .................................................................................................................... 80. 5.3.. Results ............................................................................................................................... 80. 5.3.1.. Manufacturing of MPs using magnetic stirrer ............................................................ 80. 5.3.2.. Manufacturing of MPs using vortex .......................................................................... 83. 5.4.. Discussion ......................................................................................................................... 86. 5.5.. Conclusions ....................................................................................................................... 88. Chapter 6. Fatty acid based microparticles loaded with magnetic nanoparticles and paclitaxel: manufacturing, physico-chemical properties and efficacy in vitro .............................................. 89 6.1.. Introduction ....................................................................................................................... 89. 6.2.. Materials and methods ....................................................................................................... 90. 6.2.1. Materials .................................................................................................................... 90. 6.2.2. Fabrication of FA-based MPs .................................................................................... 90. 6.2.3. Determination of MPs morphology, size and surface properties ................................ 91. 6.2.4. Composition of MPs .................................................................................................. 92. 6.2.5. Magnetic mobility of MPs ......................................................................................... 92. 6.2.6. Thermal Analyses of MPs.......................................................................................... 93. 6.2.7. In vitro release of PAX from MPs ............................................................................. 93. 6.2.8. In vitro efficacy of MPs ............................................................................................. 94. 6.2.9.. Statistics .................................................................................................................... 97 12.

(13) 6.3.. Results ............................................................................................................................... 97. 6.3.1. Morphology, size and composition of MPs ................................................................ 97. 6.3.2. Magnetic mobility of MPs........................................................................................ 101. 6.3.3. Thermal properties of MPs....................................................................................... 102. 6.3.4. In vitro release of PAX from MPs............................................................................ 102. 6.3.5. In vitro efficacy of MPs ........................................................................................... 102. 6.4.. Discussion ........................................................................................................................ 107. 6.5.. Conclusions...................................................................................................................... 111. Chapter 7. Summary and final conclusions ................................................................................. 113 References ....................................................................................................................................... 115 List of Figures.................................................................................................................................. 127 List of Tables ................................................................................................................................... 131 Appendix ......................................................................................................................................... 133. 13.

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(15) Chapter 1. Introduction. 1.1. Lung cancer According to the World Health Organization (WHO) and the International Agency for Research on Cancer (IARC), in 2015 cancer was the leading cause of death among people below age of 70 in Europe, Asia, North and South Americas and Australia. WHO estimated that in 2018 there will be over 18 million new cases of cancer and almost 10 million cancer deaths [1, 2]. Worldwide, lung cancer is the most commonly diagnosed type of cancer in men and the third most common type of cancer in women (14.5% and 8.4% of all cases, respectively). What is more, it is the leading cause of cancer related death being responsible for 22.0% of deaths in men and 13.8% deaths in women [1, 3]. Considering only cancer patients in Europe, 183 400 males and 92 300 females died of lung cancer in 2017 alone, which corresponds to 24.1% and 13.9% of all cancer related deaths within each subpopulation [4]. At the beginning of 20th century lung cancer was regarded as a rare disease. A rapid increase in the number of lung cancer patients was observed in Europe and North America around 1920 – 1930. Only in the United Kingdom, in 1970 there was 50 times more lung cancer male patients than in 1911 [3]. Interestingly, the frequency of lung cancer in men started to decline after 1990, while it is steadily growing in women (especially between 45 and 54 years old) [2-4].. 1.2. Causes of lung cancer It is now well known that lung cancer is predominantly caused by tobacco smoking. However, at the beginning of the 20th century it was not so obvious. At that time, the majority of men with higher socioeconomic status (also scientists and medics) smoked cigarettes or pipes. The first claims on smoking being a cause of lung cancer were based on the autopsies performed in Germany and the Netherlands in 1929 – 1932 [5]. More advanced research on the relationship between smoking and increased lung cancer morbidity were done in 1950 – 1960 [6-9]. Mills et al. [8] found out that over 90% of the patients suffering from respiratory tract cancer (from the larynx downward) were cigarette or pipe 15.

(16) smokers. Doll et al. [6] proved that the mortality from lung cancer was significantly higher in cigarette smokers compared to non-smokers (0.07% for non-smokers and between 0.47% to 16.6% for smokers). What is more, it was evidenced that the risk of lung cancer was growing with the increase in amount of tobacco smoked daily. Similar observations were done in 1964 by Kissen et al. [10]. Yet, it was already 1964, when the US Surgeon General 1 published the report stating that one of every 10 men smoking more than two packs of cigarettes per day died of lung cancer and that the occurrence of lung cancer is 5 to 15 times higher in tobacco smokers than in non-smokers [11]. This publication is still regarded as a turning point in the recognition of injuriousness of smoking. Now, after over 50 years from the first Surgeon General’s report, at least 16 cancers are confirmed to be coincided with tobacco smoking (including oral cavity, nasopharynx, larynx, lung, stomach, liver, pancreas etc.) [35]. Second-hand (passive) smoking was recognized as a causative factor in lung cancer only at the end of 20th century. One of the first studies on passive smoking were done by Correa et al. [12] in 1983. The research focused on the effects of passive exposure to cigarette smoke in the household (including smoking spouses or parents). Although the findings of this study were puzzling and rather inconclusive, it indicated that smokers not only harm themselves, but also compromise the health of other people in their surroundings. In 1990 Janerich et al. [13] confirmed that approximately 17% of lung cancers in non-smokers can be caused by passive smoking. Although active or passive smoking is responsible for 85 – 90% of lung cancers, other factors leading to lung cancer were also identified [3]. Darby et al. [14] proved that there was a strong association between radon2 concentration at home and lung cancer morbidity. It was estimated that the exposure to radon is responsible for 9% of lung cancer related deaths. Occupational exposure to asbestos [15], aromatic hydrocarbons [16], heavy metals (e.g. chromium, cadmium, lead, mercury) [17], crystalline silica [18] or wood dust [19] can also increase the risk of lung cancer. Other risk factors of lung cancer include outdoor (e.g. particulate matter, diesel engine exhaust) and indoor (emissions from household combustion of coal, oil heating etc.) air pollution or genetic factors [3, 5, 20].. 1.3. Histological types of lung cancer Similarly to other cancer types, lung cancer is composed of populations of cells or clones differing in morphology and molecular features. However, some distinct differences can be found in different tumour types, thus two main types of lung cancer can be distinguished: small cell lung cancer (SCLC). 1. Surgeon General is a senior physician (commissioned by the government of the US) responsible for public health affairs and the leading spokesperson on the issues connected with public health. 2 Radon is a colourless and odourless radioactive gas, being produced during a decay of radioactive elements (e.g. uranium) present in soil and rocks. Radon concentration is low outdoors, however its concentration builds up indoors (in houses or other buildings). The highest levels of radon are found underground (e.g. in uranium mines).. 16.

(17) and non-small cell lung cancer (NSCLC) [21-23]. This classification is based on the histological appearance of the tumour cells. Proper diagnosis of lung cancer type is necessary for selection of the most appropriate treatment method, since SCLC and NSCLC differ in biology, responses to different treatment modes and prognosis [21, 24]. SCLC accounts for almost 15% of all lung cancer cases and is predominantly caused by smoking (almost all SCLC patients are/were heavy smokers) [25]. It was first identified in 1926, but only in 1959 it was recognized as a distinct form of lung cancer by Azzopardi [26]. SCLC is regarded as a highly aggressive type of lung cancer, giving early and distant metastases. Only 6% of the patients diagnosed with SCLC survive five years after diagnosis3 [27]. SCLC produces vast number of paraneoplastic syndromes4 at the locations remote from the primary tumour site [24, 25]. SCLC tumours are usually highly vascularized and undergo multiple genetic mutations in the course of time [25]. Usually SCLC can be found in areas near the bronchi [28]. The precursor cells for SCLC are neuroendocrine (NE) cells, which are present in the basal layers of the epithelial lining of large airways. Once activated, NE cells start to grow (hyperplasia) and form in situ SCLC. The overexpression of several nuclear factors and signalling molecules result in progression of the tumour growth and formation of widespread metastases in other organs [25]. Histological images showing formation of SCLC are shown in Figure 1.1.. Figure 1.1 – Multistage pathogenesis of SCLC. Adapted from [25].. In the limited stage SCLC tumour cells can be found only on one side of the chest (one lung lobe and nearby lymph nodes), while in the extensive stage cancer is spreading to other organs and parts of the body (outside of the chest) [22]. NSCLC is diagnosed in 85% lung cancer patients [28]. NSCLC can be caused by active or passive smoking but it also occurs in non-smokers, most likely due to the exposure to radon, asbestos or air pollution [29]. NSCLC is regarded as less aggressive than SCLC (21% of the patients survive five years after diagnosis), however the prognosis strongly depends on NSCLC subtype and stage [27, 28].. 3 4. Data for the US only. A paraneoplastic syndrome is mediated by the molecules secreted by tumour cells (hormones or cytokines).. 17.

(18) Three subtypes differing in histological appearance and biological behaviour of NSCLC can be distinguished: a. Adenocarcinoma – accounting for 40% of all lung cancer cases, one of the most common form of lung cancer in non-smokers. It is predominantly found in the alveoli, often in the peripheral parts of the lungs [24, 27]; b. Squamous cell carcinoma – found in 25 – 30% of lung cancer patients. Usually it is located near the bronchi. The cells are flat and wide spread, they also grow relatively slowly [27, 30]; c. Large cell carcinoma (undifferentiated) – diagnosed in 10 – 15% of lung cancer patients. It can be found in different parts of the lungs (including alveoli). The cells grow quickly and spread rapidly [27, 31]. The exemplary images of histological appearance of NSCLC are shown in Figure 1.2.. Figure 1.2 – Histological appearance of adenocarcinoma (A), squamous cell carcinoma (B) and large cell carcinoma (C). Adapted from [27].. Regardless of the type, the typical symptoms of lung cancer include coughing (may be with blood), chest pain, shortness of breath, weight loss and general weakness [21, 22, 29]. The diagnosis of lung cancer is based on computed tomography (CT) or positron emission tomography (PET) imaging coupled with sputum cytology, bronchoscopy and biopsy (in patients having a centrally located tumour) or transthoracic needle aspiration (in patients with peripheral tumours, which cannot be examined using a bronchoscope) [29, 32].. 1.4. Conventional methods of lung cancer treatment Selection of the treatment mode depends on the histological type of lung cancer, its stage (advancement) and general condition of the patient. Surgical resection Surgical resection is the most consistent and effective treatment option for patients with NSCLC, on condition that the tumour is fully resectable and that the patient can withstand the surgery [33]. It is used only in 15 – 20% of the patients [34]. In the US, surgery alone is no longer used in SCLC patients. 18.

(19) [35]. Lobectomy5 is the most commonly used treatment method in patients diagnosed with early stage NSCLC, when cancer is localized only in one lobe and there is no evidence of severe lymph node spread [33]. It has been reported that over 80% of the patients who underwent complete lobectomy, survived five years after the diagnosis and surgery [36]. Several trials aimed at evaluation whether complete lobectomy is necessary in patients with small tumours (below 2 cm in diameter) had been done recently [37, 38]. However, since the trials were done on relatively small group of patients, the results were inconclusive. The standard surgical removal of NSCLC is based on an extensive thoracotomy, thereby some patients are not allowed to undergo such a procedure [34]. Since the median age at diagnosis for lung cancer patients is 70 years, the majority of the patients suffer from cardiovascular diseases or other co-morbidities and are not able to tolerate such a procedure [39]. Thus, novel minimal access surgical procedures for removal of lung tumours (e.g. video-assisted lobectomy) are being developed. Those methods are less invasive and safer for the patients, while providing the same efficacy as standard procedures [33, 40, 41]. Chemotherapy Chemotherapy is used in over 70% of patients diagnosed with advanced or metastatic lung cancer [33]. It is the main treatment option for patients diagnosed with SCLC (both in limited and extensive stage), but it is also used in NSCLC patients that underwent surgical removal of the tumour (adjuvant chemotherapy) [34, 39]. Chemotherapeutic drugs are supposed to inhibit the growth and proliferation of cancer cells by means of inhibiting cell mitosis, damaging cellular DNA or stressing the cells leading to their apoptosis. Chemotherapeutic drugs can be divided regarding their mode of action [42]: a) antimetabolites – molecules with chemical composition similar to DNA or RNA. They prevent cell mitosis, because they block the enzymes responsible for DNA synthesis or are incorporated into DNA or RNA strands. Occurring DNA damage leads to cell apoptosis. The examples of antimetabolites used in cancer treatment are: capecitabine (Xeloda®), floxuridine or methotrexate [43-45]; b) alkylating agents – one of the oldest chemotherapeutics that are still in use. Those are the molecules that are able to covalently bind to e.g. proteins, DNA or RNA. Alkylated DNA cannot replicate properly and in consequence the cells undergo apoptosis. The most popular alkylating agents used in chemotherapy are: cisplatin and its derivatives (e.g. carboplatin), mechlorethamine, cyclophosphamide or mitomycin C [46-48];. 5. Lobectomy is a surgical removal of a lobe. It may refer to a lung lobe, thyroid lobe or brain lobe.. 19.

(20) c) antimicrotubule agents – usually plant derived substances interfering with microtubules that are indispensable for cell division. Two groups of opposite mechanism of action can be distinguished within antimicrotubule agents: • vinca alkaloids – prevent the formation of microtubules, e.g. vincristine, vinblastine [49], • taxans – prevent the disassembly of microtubules, e.g. paclitaxel, docetaxel [50]; d) topoisomerase inhibitors – the molecules that affect enzymatic activity of topoisomerases (I and II) – the enzymes responsible for splitting and merging DNA strands during transcription or replication, thus preventing both of those processes. The example of topoisomerase I inhibitor is camptothecin and its derivatives [51]. Chemotherapeutics blocking topoisomerase II are etoposide, doxorubicin, mitoxantrone or novobiocin [52-55]. In the majority of patients, the drugs are administered systematically (intravenously or orally), leading to severe side effects as they are delivered not only to the tumour but also to other healthy tissues and organs. Among the most commonly occurring side effects of chemotherapy are: immunosuppression6, myelosuppression7, anaemia8, infertility, loss of weight, nausea and vomiting, pain and peripheral neuropathy, heart palpitations, loss of hair 9, dry skin, loss of taste or hearing10 etc. [56, 57]. Radiotherapy Another method of lung cancer treatment is radiotherapy that utilizes ionizing. This method is used for both SCLC and NSCLG, usually as a complimentary method for previously described chemotherapy or surgical removal of the tumour [34]. Radiation therapy delivers high energy X-rays damaging DNA of the cancer cells and leading to their apoptosis [58]. Standard radiation therapy is based on administration of total 45 to 66 Gy (in 1.8 – 2.0 Gy single doses). In this approach the control over Xray beam orientation is limited and it is estimated that 55 – 70% of the patients experience cancer recurrence [33, 59]. External beam radiation therapy is the most commonly used technique of radiotherapy. In this method, patients are immobilized in exactly the same position each time and irradiated from different sites with multiple X-ray beams focused directly at the tumour [33, 34, 60]. Recent advancements. 6. Immunosuppression is a depression of the immune system, decrease in concentration of white blood cells, red blood cells and platelets. 7 Myelosuppression is caused by the damage to the bone marrow stem cells. 8 Anaemia in patients treated with chemotherapy results from myelosuppressive effects of chemotherapy combined with increased bleeding, destruction of blood cells, nutritional problems or dysfunctions of other organs such as kidneys. 9 Chemotherapeutic drugs destroy rapidly dividing cells including those in hair follicles, nails etc. 10 Especially in the case of cisplatin.. 20.

(21) in stereo-radiation planning allowed for more precise control over beam trajectory and focusing, increasing the therapeutic effects of radiation therapy and diminishing its negative side effects and toxicity [61, 62]. Another option of radiation therapy for the treatment of lung cancer is brachytherapy (internal radiation therapy). In this method, radioactive sources are placed in close proximity to the tumour or directly inside the tumour. Radioactive material is usually in a form of small pellets that can be delivered to the tumour via a bronchoscope or during a surgery. The pellets are either removed from the implantation site or, in the case of smaller or weaker radioactive sources, they can be left permanently in patient’s body [34, 63]. In comparison to the standard radiation therapy, brachytherapy is more accurate and more effective. It is also less toxic for the patient, decreases severity of the side effects and is safer for the caregivers and personnel [63, 64].. 1.5. Novel methods of lung cancer treatment It is believed that conventional therapies alone or in combination are close to achieving their therapeutic limits [65]. Thus, novel methods with potentially increased efficacy and reduced side effects are being developed. Hyperthermia The concept of hyperthermia treatment is based on the fact, that cancer cells are more sensitive to elevated temperatures (above 41°C) than normal cells [66]. Hyperthermia is accomplished using magnetic nanoparticles that can be delivered directly to the tumour through blood circulation or via inhalation. Their distribution can be easily controlled using magnetic resonance imaging (MRI) [67]. One of the properties of magnetic nanoparticles is the fact that, when exposed to an external high frequency alternating magnetic field, the nanoparticles heat up to the temperature above 42°C. The increase in temperature is related to losses during reverse magnetization of the nanoparticles. Two modalities of the hyperthermia treatment are used in clinics: application of 42 – 45°C for up to few hours (usually coupled with other treatment methods such as chemo- or radiotherapy) or application of at least 50°C for few minutes [68]. Targeted therapy Recent advancements in genetic testing and the understanding of biochemical and molecular mechanisms of cancer progression allowed for development of novel targeted therapies for the treatment of lung cancer. Unlike the previously described methods, targeted therapies are aimed at suppression of cancer cell growth via altering of intracellular signalling [34]. Selection of the most optimal targeted treatment requires detailed screening of biomarkers allowing for prediction of tumour susceptibility and more accurate estimation of survival prognosis [69].. 21.

(22) The most commonly targeted genetic mutation is epidermal growth factor receptor (EGFR). Different studies found out that EGFR is overexpressed in 40 – 80% of the patients suffering from NSCLC [70]. Over the last years numerous EGFR inhibitors have been developed. Those include receptor tyrosine kinases inhibitors or monoclonal antibodies [33]. The first targeted therapy registered by the Food and Drug Administration (FDA) was Gefitinib, however up to date there is no solid clinical evidence of significant improvement in the survival rate of the patients treated with Gefitinib [71, 72]. Besides EGFR, other commonly targeted genetic mutations are anaplastic lymphoma kinases (ALK). Different genetic mutations in ALK (rearrangements, point mutations or amplifications) were found in several malignancies, including NSCLC. Although the detailed functions and mechanism of activation of ALK are still not fully understood numerous studies focused on the development of ALK tyrosine kinase inhibitors [73]. Several of those inhibitors (crizotinib, ceritinib, alectinib) are already approved for clinical use [74-76]. Although some of the studies evidenced that targeted therapies are effective in patients diagnosed with lung cancer, some barriers still limit the use of those therapies. Those include gaining of the tumour resistance (usually in up to 12 months from the beginning of the treatment), differences in patients’ susceptibility (e.g. Asians were more susceptible for the treatment with crizotinib than Caucasians) or negative side effects (nausea, vomiting, skin rash etc.) [34, 69, 74]. Anti-angiogenic therapy Anti-angiogenic therapies used in cancer treatment are based on the fact that tumours cannot increase their size over 2 mm without a vascular supply. Blood flow inside the tumour is necessary for the delivery of nutrients, but it also enables the migration of cancer cells and formation of metastases in other organs. Thus, limiting neovascularisation and formation of blood vessels should inhibit the growth of tumour [65]. The majority of the studies developing anti-angiogenic therapies, especially for NSCLC, focused on inhibition of vascular endothelial growth factor (VEGF), as it is one of the most potent factor in angiogenesis [77]. Serval II/III phase studies testing different anti-angiogenic agents (e.g. angiozyme, endostatin, thalidomide or bevacizumab) are ongoing [78]. Still, it must be remembered that VEGF inhibitors affect angiogenesis not only in the tumour, so that negative side effects such as increased blood pressure and delayed wound healing may occur [34]. Immunotherapy Immunotherapy is the newest option for the treatment of lung cancer. In this method, immunotherapy agents block specific proteins present on the surface of cancer cells and thus make them visible for the patients’ innate immune response cells11. Once the T cells in patients body can recognize cancer cells, they can respond property and neutralize those cells [34, 79]. Several agents, such as nivolumab [80],. 11. Surface proteins present in cancer cells are normally not recognized by the body’s immune system.. 22.

(23) pembrolizumab [81, 82] or atezolizumab [83], had already been proved effective in cancer suppression, however multiple trials are still ongoing.. 1.6. Causes of high mortality in lung cancer patients Selection of the most appropriate treatment of lung cancer depends on various factors, such as histological type of cancer, its stage and localization, but also on general condition of the patients and their co-morbidities. Despite recent advancements in medicine and multiple treatment methods (many of the previously described methods are used in combination, e.g. chemo- and radiotherapy, surgical removal of the tumour followed by chemo- or radiotherapy etc.), the survival rate of lung cancer patients is the lowest among other cancer types. According to WHO, the majority of patients is diagnosed with lung cancer at III/IV stage (77%). Fiveyear survival rate for the patients diagnosed with advanced NSCLC is below 6%, while for the patients with the same type of lung cancer, but diagnosed at early stages it is around 21 – 35% [1]. The reason for a late diagnosis of lung cancer is that at early stages the tumour occupies relatively small volume of the lung and does not cause a significant and observable deterioration in respiratory functions. The symptoms of bigger tumours – coughing, chest pain or general weakness – can be easily mistaken with a common cold or flu and ignored. Once the patient finally seeks doctors’ help, the tumour is usually quite advanced [3, 34]. On the other hand, lung cancer is more prevalent in elderly people, who suffer from other co-morbidities (e.g. cardiovascular diseases, diabetes). Such patients may not be able to withstand surgical removal of the tumour or systemic chemotherapy/radiotherapy and thus the treatment options are limited.. 23.

(24) 24.

(25) Chapter 2. Aims and general outline of the thesis. Despite recent advancements in medicine and the development of novel anti-cancer therapies, lung cancer is still among the leading causes of death. As mentioned before (Chapter 1.4), there are multiple anti-cancer drugs available on the market, however their use is limited due to severe toxicity associated with systemic administration. Therefore, the main aim of this thesis is to develop novel, stimulisensitive drug carriers that enhance the efficacy of lung cancer therapy through guided accumulation directly at the tumour site and controlled drug delivery. Such drug delivery carriers will be in a form of solid microparticles composed of fatty acids, loaded with magnetic nanoparticles and anticancer drug (paclitaxel). Schematic representation of such microparticles is shown in Figure 2.1.. Figure 2.1 – Schematic representation of the composition of fatty acid-based microparticles (MP) loaded with magnetic nanoparticles (NP) and anticancer drug (paclitaxel – PAX).. Prepared microparticles will be in a form of dry powder for inhalation (DPI). The microparticles will be delivered to the patient via inhalation using a standard DPI inhaler. Upon inhalation, the microparticles will be guided to the tumour site using external magnetic field. Once the microparticles will be accumulated in the tumour, alternating electromagnetic field will be applied. Magnetic nanoparticles embedded in the microparticles will heat up to around 42 – 47°C resulting in melting of the fatty acid matrix of the microparticles. Anticancer drug will be thus released directly to the tumour, while fatty acids will be metabolized in surrounding cells. Remaining magnetic nanoparticles will be removed from the lung via natural clearance mechanisms. Schematic representation of the proposed treatment is shown in Figure 2.2.. 25.

(26) Figure 2.2 – Schematic representation of the proposed method for the treatment of lung cancer.. 26.

(27) The following criteria must be met in order for such an inhalable formulation to be effective: •. the microparticles must be spherical in shape and have diameters in range of 1 – 5 µm for the most efficient accumulation in lower respiratory tract and alveoli (the particles with diameters >5 µm are predominantly accumulated in upper respiratory tract, while the particles with diameters <1 µm can be easily exhaled);. •. the microparticles must be efficiently loaded with magnetic nanoparticles (i.e. superparamagnetic iron oxide nanoparticles) to enable their guidance to the tumour site and triggered release; the microparticles should be responsive to external magnetic field;. •. fatty acids and magnetic nanoparticles must not be toxic for cells; additionally magnetic nanoparticles cannot release significant amounts of iron ions, which can stimulate the growth of cancer cells;. •. fatty acid matrix of the microparticles should melt at 42 – 47°C so that the microparticles will melt in hyperthermia conditions releasing the drug directly to the tumour.. The development of such drug delivery carriers is a complex problem, thus the following work was divided into several research tasks (Figure 2.3).. Figure 2.3 – Schematic representation of the specific aims of the thesis.. The study started with the evaluation of different saturated fatty acids and their mixtures in terms of their thermal properties and in vitro influence on lung epithelial cells (of malignant and normal origin). This allowed for selection of the most appropriate fatty acids for further fabrication of the microparticles. The results of this study are presented in Chapter 3 together with a brief introduction presenting general characteristics of fatty acids. The studies on magnetic nanoparticles and their surface modification with silica coatings were performed simultaneously. The main goal of surface modification of the nanoparticles was to prevent undesired iron release. Chemical composition, magnetic properties and morphology of the modified. 27.

(28) nanoparticles were characterized. Detailed in vitro studies in contact with lung epithelial cells (of malignant and normal origin) were also performed. General information on magnetic nanoparticles and the results of abovementioned studies are presented in Chapter 4. The third task was related to fabrication of unloaded fatty acid based microparticles in order to optimise manufacturing conditions to obtain spherical microparticles with uniform diameters in 1 – 5 µm range. The results of this study are presented in Chapter 5 preceded by a brief review of the manufacturing methods used for production of solid lipid microparticles. The final task was aimed at fabrication of the microparticles based on selected fatty acids and loaded with modified magnetic nanoparticles and anticancer drug. Obtained microparticles were characterized in terms of their physio-chemical properties and efficacy in vitro. The results of characterization of the microparticles are presented in Chapter 6. Chapter 7 summarizes all of the results and presents final conclusions based on previously performed analyses.. 28.

(29) Chapter 3. Analysis of physico-chemical properties of saturated fatty acids and their influence on human lung epithelial cells. Fatty acids (FAs) were selected among other lipids for fabrication of the microparticles. Chapter 3.1 provides general characteristics of FAs in regards to their chemical structure, properties and biological role. Further sections present the results of physico-chemical analyses of several saturated FAs and their in vitro evaluation in contact with human lung epithelial cells.. 3.1. Fatty acids (FAs) A single fatty acid (FA) molecule consists of one carboxylic group (‒COOH) and an aliphatic chain (usually unbranched, with even number of carbon atoms) (Figure 3.1). The physical properties of FAs are determined mostly by the length and degree of saturation of the aliphatic chain [84-87].. Figure 3.1 – Examples of saturated, monounsaturated and polyunsaturated FAs. Adapted from [88].. 3.1.1. Chemical structure and properties of FAs Saturated FAs contain only single bonds between carbon atoms in the aliphatic chain. The majority of saturated FAs have straight chains with an even number of carbon atoms, although FAs with odd number of carbon atoms can also be found in some natural fats (e.g. in oil from shark’s liver) [88, 89]. 29.

(30) The properties of saturated FAs are determined by the length of the aliphatic chain. Short chain FAs (up to 6 carbon atoms) are soluble both in water and in organic solvents. Their melting temperature is below 0°C and those FAs are liquid at room temperature. Medium chain FAs contain between 7 to 13 carbon atoms. Their melting temperatures are higher than in the case of short chain FAs (16 – 54°C) and therefore the majority of them are solid at room temperature. The increase in the number of carbon atoms and molecular mass of medium chain FAs make them poorly soluble or insoluble in water, but they are still soluble in the majority of organic solvents. Long chain FAs having 14 or more carbon atoms are completely insoluble in water. Also their solubility in organic solvents decreases with the increase in molecular mass. The melting temperatures of long chain FAs exceed 55°C [88, 90]. The examples of several saturated FAs and their properties are given in Table 3.1. Table 3.1 – Examples of saturated FAs, their melting temperatures and solubility in water and organic solvents [88, 90].. Systematic name. Common name. Solubility (g/l at 20°C). Designation. Melting temperature (°C). Water. Chloroform. Ethanol (96%). Butanoic acid. Butyric acid. C4:0. (-7) – (-8). >60. Soluble. Soluble. Octanoic acid. Caprylic acid. C8:0. 16 – 18. 0.7. Soluble. Soluble. Dodecanoic acid. Lauric acid. C12:0. 42 – 45. 0.055. 830. 912. Octadecanoic acid. Stearic acid. C18:0. 54 – 71. 0.003. 60. 11. Hexacosanoic acid. Cerotic acid. C26:0. 88 – 90. <0.0001. No data. No data. Monounsaturated FAs contain one double bond between neighbouring carbon atoms. If there is more than one double bond in the molecule of FAs, then the FA is referred to as polyunsaturated FA. The number and positions of double bonds differ between unsaturated FAs. In the majority of naturally occurring unsaturated FAs the double bond is in cis-configuration, meaning that the hydrogen atoms on both sides of the double bond are arranged in the same direction resulting in a kink of the molecule. The cis fatty acids are thus thermodynamically unstable and their melting temperatures are lower compared to saturated FA containing the same number of carbon atoms (Table 3.2) [88, 91]. Saturated FAs gained much attention as phase change materials (PCMs). PCMs are materials which are capable of storing and releasing large amount of energy (in a form of latent heat) during their phase changes. The energy is absorbed during melting (solid → liquid) and released during solidification (liquid → solid). PCMs are used in solar heating or air-conditioning systems [92-95]. Saturated FAs offer several advantages as PCMs, such as wide range of melting temperatures, high heat capacity, non-toxicity, non-corrosiveness to metals, chemical and thermal stability, low price, small volume change.. 30.

(31) Table 3.2 – Examples of saturated and unsaturated FAs containing the same number of carbon atoms and their melting temperatures [88].. Common name. Designation. Group. Melting temperature (°C). Stearic acid. C18:0. Saturated FAs. 54 – 71. Oleic acid. C18:1. Monounsaturated FAs. 13 – 14. Linoleic acid. C18:2. Polyunsaturated FAs. (-5) – (-12). The application of FAs as PCMs was distinctively broadened as soon as it was realized that FAs can form eutectic mixtures [90, 96]. The eutectic mixture is defined as a homogenous mixture of two or more different substances in such proportions that the melting temperature (Tm) of the mixture is lower than the melting temperature of its single components [90, 95]. Different eutectic mixtures based on saturated FAs have already been developed tailoring the use of FAs as PCMs. Sari et al. [97] tested different binary mixtures of lauric, myristic, palmitic and stearic acids. It was found out that FAs form eutectic mixtures only at strictly defined ratios. Any deviations from the proper proportions of the components resulted in increased discrepancies in melting temperatures measured for those mixtures using differential scanning calorimetry (DSC). For example, lauric and stearic acids mixed at 75.5:24.5 (wt.%) form the eutectic mixture with Tm equal to 37.0°C. However, Tm of the same FAs mixture but at ratio of 75.1:24.9 varied from 36.9 to 37.6°C [97]. Nevertheless, by varying the composition of FAs mixtures it is possible to adjust melting temperature of the material for specified purposes.. 3.1.2. Biological role and metabolism of FAs Free FAs rarely occur in nature, however they play an important role in cell metabolism, proliferation and regulation of intracellular/extracellular signalling since they are the main components of various lipids such as triglycerides, phospholipids and other complex lipids [86, 98]. Although FAs can be synthesized de novo in the endoplasmic reticulum from other FAs or from the organic precursors (e.g. glucose), the majority of FAs is delivered with food, mainly as triacyclglycerols [99]. FAs present in vegetable and fish oils, diary and meat products or grains provide 30 – 35% of total energy intake in many developed countries [91]. In animals (including humans), FAs are metabolized during oxidation process yielding high amount of adenosine triphosphate (ATP) molecules and in the case of low availability of glucose they are an alternative energy source [86]. The turnover of FAs present in human body starts with two forms of FAs: non-esterified (usually anionic form) or complexed (e.g. with albumin). After dissociation, FAs enter the cell by spontaneous diffusion or protein-mediated transport. Once inside the cell, FAs desorb from the cell membrane, bind to cytosolic fatty acid binding proteins and diffuse into the. 31.

(32) cytoplasm. In the end, FAs can be esterified by long chain fatty acyl-CoA synthetase and are metabolized in mitochondrial oxidation or can be further processed into glycerides, such as phospholipids, triacylglycerols and cholesterol esters (Figure 3.2) [91, 100-102].. Figure 3.2 – Metabolism of FAs. Free FAs (FFA) are transported through the cell membrane via protein carriers. Once inside the cell, FAs are transported intracellularly via FA-binding proteins (FABP). FAs activated with acyl-CoA are either β-oxidised in mitochondria or peroxisomes or are transported to endoplasmic reticulum, where FAs are esterified and become constituents of various lipids. Certain activated FAs may also regulate gene expression or become signalling molecules (eicosanoids). Adapted from [91].. The effects of both saturated and unsaturated FAs on human health were studied in detail. The majority of studies was focused on mono- or polyunsaturated FAs [103-105]. The most interest in the effects of FAs on human health was so far related to cardiovascular diseases, however recent studies indicate that FAs can also influence other diseases, such as diabetes, inflammations or neurological problems [86, 103]. Unsaturated FAs are in general regarded as healthier, however their influence on human health strongly depends on their chemical composition [106]. Oleic acid (C18:1), the most prevalent monounsaturated FA occurring in human diet, is present in plant oil and various fats of animal origin. In comparison with polyunsaturated FAs, its beneficial effects are moderate. On the contrary, increased intake of polyunsaturated FAs can significantly reduce the risk of cardiovascular diseases, regulate inflammatory responses or have a positive impact on cognitive functions, especially in the elderly [107-109]. Saturated FAs gained less attention so far, since usually they are considered to be unhealthy and responsible for increased cholesterol levels or liver diseases [110-112]. Several studies were aimed at saturated FAs, however the majority of them focused on palmitic acid (PAL), as this is the most 32.

(33) commonly occurring FA (up to 30% of total FAs in human body) [113]. Carta et al. [113] proved that excessive intake of palmitic acid disrupted lipid metabolism and increased fat accumulation presumably leading to health detriment. On the other hand, it was shown by Yao et al. [114] that the presence of exogenous palmitic acid increased the proliferation of fibroblasts, HeLa and H460 cells. The main objective of this part of the study was to determine the physico-chemical properties of various saturated FAs and to evaluate their in vitro influence on human lung epithelial cells of malignant and normal origin. Selection of FAs for further studies and manufacturing of the microparticles was done according to the following assumptions: 1. Thermal properties: a. Melting temperature (Tm) between 42 – 50°C, ideally around 42 – 47°C (FA-based matrix of the microparticles should melt at the temperatures reached during hyperthermia); b. Onset of melting at the temperatures higher than 40°C (the microparticles cannot melt before reaching their site of action and before application of hyperthermia); c. Single endothermic peak indicating melting of FA. 2. Cytocompatibility with human lung epithelial cells (at least those of normal origin).. 3.2. Materials and methods Saturated FAs (Table 3.3): capric acid – CAP (W236403), lauric acid – LAU (W261408), myristic acid – MYR (70082), palmitic acid – PAL (P0500) and stearic acid – STE (W303518) were purchased from Sigma-Aldrich and used without any purification. Table 3.3 – Saturated FAs tested in the present study: common name, designation, labelling, molar mass and chemical structure [115, 116].. Common name. Designation. Labelling in the present study. Molar mass (g/mol). Capric acid. C10:0. CAP. 172.3. Lauric acid. C12:0. LAU. 200.3. Myristic acid. C14:0. MYR. 228.4. Palmitic acid. C16:0. PAL. 256.4. Stearic acid. C18:0. STE. 284.4. Chemical structure. Two binary mixtures of FAs were prepared according to Yuan et al. [90] and Sari et al. [97]. For the preparation of MYR/PAL mixture (MYR:PAL mass ratio of 58:42), 5.8 g of myristic acid (MYR) was mixed with 4.2 g of palmitic acid (PAL) and placed in a falcon tube. The tube was heated up to 90°C in a water bath and dwelled for 30 min. Melted FAs were vigorously mixed using vortex mixer (VX200, Labnet International, 2 min) and left at room temperature to solidify. The MYR/STE mixture 33.

(34) (MYR:STE mass ratio of 64:36) was prepared in the analogue way using 6.4 g of MYR and 3.6 g of STE.. 3.2.1. Physico-chemical characterization of FAs Differential scanning calorimetry (DSC) Thermal properties of FAs and their mixtures were determined using DSC 1 (METTLER TOLEDO). 3.5-3.6 mg samples were weighted in aluminium crucibles (40 µl) and closed with pierced lids. The measurements were performed at temperature range 0 – 100°C, at 10°C/min heating rate and with 30 ml/min N2 flow rate. The characteristic melting temperatures (Tonset, Tpeak = Tm, Tendset) and normalized heat of phase transition were determined using STAR e software. Fourier-transform infrared spectroscopy (FTIR) Chemical composition of FAs was evaluated using FTIR. The measurements were performed using Bruker Tensor 27 spectrophotometer (Bruker) in attenuated total reflection (ATR) mode with the use of diamond/ZnSe crystal. The spectra were recorded between 4000 – 525 cm-1 with 4 cm-1 resolution. 64 single scans were collected for each sample. The obtained spectra were analysed using OPUS software provided by the manufacturer.. 3.2.2. In vitro characterization of FAs The influence of saturated FAs on cells was evaluated using human lung epithelial cells. Two cell lines: human lung epithelial cells of lung carcinoma origin (A459, ATCC® CCL-185TM, USA) and human lung epithelial cells of normal lung/bronchus origin (BEAS-2B, ATCC® CRL-9609TM, USA) were used. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM, PAN-Biotech, Germany) supplemented with 10% foetal bovine serum (FBS, South America origin, PAN-Biotech, Germany) and 1% penicillin/streptomycin (PAN-Biotech, Germany). In addition, cell culture medium for BEAS-2B cells was supplemented with 1% glutamine (Gibco® GlutaMAXTM, Life Technologies). The cells were cultured in humidified atmosphere enriched with 5% CO2 at 37°C. For all cell culture studies, the FAs were dissolved in ethanol (99.8%, Avantor Performance Materials, Poland S.A) at 10 mM and further diluted with cell culture medium to obtain final FA concentration of 25, 50, 75 and 100 µM (the concentration of ethanol in cell culture medium was ≤ 1%).. Cell proliferation. A549 and BEAS-2B cells were seeded in 96-well plates at the density of 2 000 cells/well in 200 µl of complete cell culture medium and allowed to attach to the bottom of the wells. After 24 h cell culture medium was replaced with fresh medium containing FAs at 25, 50, 75 and 100 µM concentration. The culture was continued for 3 days. The experiment was performed in triplicate for each type and concentration of FAs. The plates were kept inside IncuCyte ZOOM. 34.

(35) System (Essen BioScience). The system recorded phase contrast images of the cells every 2 h (4 images for each well) through the whole experiment. The images were then analysed using provided IncuCyte ZOOM System software to determine percentage confluency and growth profiles of the cells as a function of time. The percentage confluency was calculated as follows (Equation 3.1): 𝐶𝑜𝑛𝑓𝑙𝑢𝑒𝑛𝑐𝑦 (%) =. 𝐴𝑟𝑒𝑎 𝑜𝑐𝑐𝑢𝑝𝑖𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑐𝑒𝑙𝑙𝑠 𝑇𝑜𝑡𝑎𝑙 𝑎𝑟𝑒𝑎 𝑜𝑓 𝑡ℎ𝑒 𝑖𝑚𝑎𝑔𝑒. ∗ 100%. (Equation 3.1). The area occupied by the cells was detected automatically by the software, however some adjustments were done manually. Any objects smaller than 2 µm (precipitates, cellular debris etc.) were excluded from the calculation. Cell viability. A549 and BEAS-2B cells were seeded in 96-well plates at the density of 2 000 cells/well and treated with FAs as described above. The metabolic activity of the cells cultured with 25, 50, 75 and 100 µM FAs for 3 days was determined using Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Japan) according to manufacturer’s instructions. In brief, cell culture medium was removed from wells, the cells were washed twice with 100 µl PBS (PAN-Biotech, Germany) and 150 µl of fresh cell culture medium containing 10% of CCK-8 reagent was added to the cells. After 3 h of incubation at 37°C, 100 µl of cell culture medium was removed from wells and transferred into optically clear 96-well plate. The absorbance at 450 nm was measured using microplate reader (VICTOR Multilabel Plate Reader, Perkin Elmer). The viability of the cells incubated with FAs for 3 days was also analysed using live/dead fluorescent staining. A549 and BEAS-2B cells were seeded in 96-well plates at the density of 2 000 cells/well and treated with FAs as described above. The staining was performed as follows. Cell culture medium was removed from the wells and replaced with 200 µl of FluoroBriteTM DMEM (Gibco, Life Technologies) containing 0.1% calcein AM (Sigma-Aldrich) and 0.1% propidium iodide (Sigma-Aldrich). After 20 min of incubation in the dark, the images were taken using a fluorescent microscope (Axiovert 40 CFL with HXP 120 C Metal Halide Illuminator, Zeiss, Germany). Cell membrane stiffness. A549 and BEAS-2B cells were seeded in plasma treated polystyrene Petri dishes (diameter 6 cm, Corning® Gosselin™, USA) at density of 40 000 cells/dish in complete cell culture medium and allowed to adhere to the bottom of the dish. After 24 h, cell culture medium was replaced with 4 ml of fresh cell culture medium containing 25 µM FAs. After 24 h of incubation with FAs, the cells were washed twice with 2 ml of PBS, fixed in 3.8% formaldehyde (F8775, SigmaAldrich) for 20 min. Then the cells were washed twice with 2 ml of PBS and stored in PBS at 4°C until analysis (no longer than 48 h). The morphology and membrane stiffness of the cells were examined with atomic force microscope (AFM) (MFP-3D-Bio, Asylum Research, Santa Barbara, CA, USA). Topography imaging was performed in tapping mode with silicon nitride tip (nominal spring constant 64 – 76 pN/nm, scanning frequency 0.35 – 0.5 Hz, the calibration was done for all cantilevers). Next, the nanoindentation analysis was performed in order to determine the apparent Young’s 35.

(36) modulus of the cell membrane. The cells were probed at constant speed (200 nm/min) and the threshold force was set to 10 nN. The probing was done in 900 points in the scanning area (30 x 30 points). The results were plotted as the force curves (the function of the applied force against the movement of piezo in z-direction). The apparent Young’s modulus was determined by adjusting the Hertz model to forward pull curve (the Poisson's ratio was set to 0.5 for cells). For each sample representing A549 and BEAS-2B cells treated with each type of FA, at least 3 different locations were probed. Uptake of FAs. A549 and BEAS-2B cells were seeded in plasma treated glass bottom Petri dishes (diameter 3.5 cm, Ibidi GmbH, Germany) at density of 20 000 cells/dish and allowed to attach to the bottom of the dish. After 24 h cell culture medium was removed and replaced with 2 ml of fresh cell culture medium containing 100 µM LAU or PAL. After 24 h of incubation with FAs, the cells were washed twice with 1 ml of PBS and visualized using optical tomography (Nanolive 3D Cell Explorer, Switzerland) without fixation in formaldehyde. 3D scans were analysed using Nanolive software (Steve, Nanolive, Switzerland). The differences in refractive index of cell structures allowed for identification of lipid vesicles inside the cells and for quantification of their volume. In order to confirm optical tomography observations, the fluorescent analogues of LAU and PAL were used (BODIPYTM FL C12 (D3822) and BODIPYTM FL C16 (D3821), Invitrogen, ThermoFisher Scientific). BODIPY stock solutions were prepared in dimethyl sulfoxide (DMSO) to obtain 10 mM concentration. A549 and BEAS-2B cells were seeded in 96-well plates at density of 2 000 cells/well in 200 µl of complete cell culture medium. After 24 h the cell culture medium was replaced with fresh cell culture medium containing LAU and PAL fluorescent analogues (LAU-FL, PAL-FL) at the concentration of 100 µM. After 24 h cell culture medium was removed, the cells were washed twice with 100 µl of PBS and fixed in 3.8% formaldehyde (Sigma-Aldrich) for 20 min. The cells were washed twice with 100 µl of PBS and the fluorescence intensity was measured at λex = 505 nm and λem = 512 nm using a multiplate reader (FluoroSTAR Omega, BMG Labtech). Cell nuclei were counterstained with DAPI (4′,6-diamidine-2′-phenylindole dihydrochloride, Sigma-Aldrich, 5 µg/ml in PBS) for 5 min and the cells were finally washed with 100 µl of PBS. The fluorescent microscopy images were taken afterwards (Axiovert 40 CFL with HXP 120 C Metal Halide Illuminator, Zeiss, Germany).. 3.2.3. Statistics Statistical analyses of obtained data were done using a one-way analysis of variance (one-way ANOVA) followed by Tuckey’s post hoc test. The assumptions of normal distribution and equal variance were verified using the Shapiro–Wilk and Levene median test, respectively (p-value < 0.05). The analyses were performed using SigmaPlot 12.3 software (Systat Software, Inc.). The results are presented as mean ± standard deviation (SD), unless stated otherwise.. 36.

(37) 3.3. Results 3.3.1. Thermal properties of FAs DSC curves of pure FAs are presented in Figure 3.3.A. The curves exhibited only one sharp endothermic peak corresponding to the melting of FAs. The peaks were relatively narrow, the difference between Tonset and Tedset ranged from 4.3°C for CAP to 5.6°C for PAL. Melting temperature Tm of pure FAs increased with the increase in molecular weight of FAs (Table 3.4). The correlation between those two parameters was of statistical significance (p<0.001) and the correlation coefficient was equal to 0.995. Normalized heat of fusion was similar for all tested FAs (178.0 J/g for CAP – 217.4 J/g for STE). The results of thermal analysis of binary mixtures of MYR/PAL and MYR/STE are depicted in Figure 3.3.B together with the DSC curves of their single components. In both cases, only single peaks originating from the melting of MYR/PAL and MYR/STE were present, indicating that both of the FAs in the mixture melted simultaneously. Melting temperature Tm of MYR/PAL (58:42) was 48.6°C and for MYR/STE (65:35) it was 51.7°C (Table 3.4); for both mixtures it was lower than Tm of single FAs.. Figure 3.3 – DSC curves of pure FAs (A) and their mixtures: myristic/palmitic acids (58:42) (B), myristic/stearic acids (65:35) (C). CAP – capric acid, LAU – lauric acid, MYR – myristic acid, PAL – palmitic acid, STE – stearic acid.. 37.

(38) Table 3.4 – Melting temperatures and normalized heat of fusion of pure FAs and their mixtures. CAP – capric acid, LAU – lauric acid, MYR – myristic acid, PAL – palmitic acid, STE – stearic acid.. Melting Sample. Normalized heat of fusion (J/g). Onset (°C). Peak = Tm (°C). Endset (°C). CAP. 31.3. 32.7. 35.6. 178.0. LAU. 43.7. 45.2. 48.6. 202.0. MYR. 53.7. 54.5. 58.1. 191.4. PAL. 62.2. 63.8. 67.8. 216.5. STE. 68.8. 70.6. 74.2. 217.4. MYR/PAL (58:42). 46.3. 48.6. 51.4. 178.1. MYR/STE (64:36). 49.5. 51.7. 54.1. 196.6. 3.3.2. Chemical composition of FAs The FTIR spectra of FAs are shown in Figure 3.4. All tested FAs exhibit the same position of all bands but differ in bands’ intensities. The presence of CH2 groups was confirmed by bands at: 2914 cm-1 and 2848 cm-1 (C-H asymmetrical and symmetrical stretching in CH 2 groups, respectively), 1471 cm-1 (CH2 deformation), multiple peaks between 1286 – 1190 cm-1 (CH2 twisting and wagging) and 719 cm-1 (CH2 rocking). The band at 1409 cm-1 was assigned to CH2 deformation at C2, i.e. carbon atom next to COOH group.. Figure 3.4 – FTIR spectra of FAs. CAP – capric acid, LAU – lauric acid, MYR – myristic acid, PAL – palmitic acid, STE – stearic acid.. 38.

(39) A single CH3 group could be identified based on bands at 2954 cm -1 and 2871 cm-1 (C-H asymmetrical and symmetrical stretching in CH3 groups, respectively), 1350 cm-1 (CH3 deformation) and 1122 cm-1 (CH3 rocking). The strong band at 1697 cm-1 originates from -COOH group (C=O stretching in hydrogen bonded FA dimers), together with the band at 1429 cm -1 (C-O stretching) and several peaks from -OH group (wide band in the range of 3500-2660 cm-1 from O-H stretching and a band at 930 cm-1 from OH out of plane bending) [26, 27]. With the increasing number of carbon atoms in FA chain, the intensity of CH2 related bands increased when compared with CH3 or COOH groups, indicating the presence of more CH 2 groups in the FA molecule, while the number of CH3 and C=O groups was constant (Table 3.5). This was also confirmed by the increase in ratio between signals from all CH 2 groups compared to a signal from single CH 2 group adjacent to COOH. The ratio between peaks related to CH 3 and COOH groups was similar for all tested FAs. Table 3.5 – Ratios between FTIR bands corresponding to CH3, CH2 and COOH groups in FAs. CAP – capric acid, LAU – lauric acid, MYR – myristic acid, PAL – palmitic acid, STE – stearic acid.. FA Compared bands. Trend. CAP. LAU. MYR. PAL. STE. (↑ C). CH2 (2914 cm-1) vs CH3 (2954 cm-1). 2.11. 2.76. 3.34. 4.30. 4.33. ↑. CH2 (2914 cm-1) vs C=O (1697 cm-1). 0.65. 0.88. 1.08. 1.39. 1.68. ↑. CH2 (2914 cm-1) vs CH2 (1409 cm-1). 2.39. 3.09. 3.75. 5.17. 5.20. ↑. CH3 (2954 cm-1) vs C=O (1697 cm-1). 0.31. 0.32. 0.32. 0.32. 0.38. ↔. 3.3.3. In vitro influence of FAs on lung epithelial cells Proliferation and viability The proliferation of A549 and BEAS-2B cells incubated with 25 – 100 µM FAs was evaluated using phase contrast imaging. The representative phase contrast images of the cells treated with 25 µM FAs for 4 days are presented in Figure 3.5.A. A549 cells treated with CAP and LAU exhibited normal morphology and they reached almost 96 – 98% confluency after 3 days of incubation with FAs. No differences were observed between CAP and LAU treaded cells compared to the control cells. Interestingly, the morphology of A549 cells was strongly affected by the addition of MYR, PAL and STE. The number of cells was lowered compared to control cells and many rounded or poorly spread cells could be found. As regards BEAS-2B cells, no significant changes in their morphology were observed for any of the FAs.. 39.