Bionanostructures for intracellular temperature sensing during photothermal cancer treatment

julho de 2021 Ânia Barata Micaelo Bionanostructures for intracellular temperature sensing during photothermal cancer treatment UMinho | 2021 Ânia Barata Micaelo Bionanostructures for intracellular temperature sensing during photothermal cancer treatment julho de 2021 julho de 2021 Ânia Barata Micaelo Bionanostructures for intracellular temperature sensing during photothermal cancer treatment Master thesis Master in Biophysics and Bionanosystems Work developed under the supervision of Prof. Dr. Maria Elisabete da Cunha Dias Real Oliveira Universidade do Minho Dr. rer. nat. Jana Berit Nieder International Iberian Nanotechnology Laboratory iv DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ v Acknowledgments This work benefitted from the input of numerous people, either by theoretical, practical or personal support, for which I would like to give a word of appreciation, as this would have not been the same experience without them. To my supervisor at University of Minho, Professor Maria Elisabete C. D. R. Oliveira, I appreciated immensely your theoretical classes, and I couldn’t have asked for a better thesis’ supervisor. I am grateful for all the availability and support you have given throughout this work, and has been a pleasure being your student. I would like to thank my supervisor, Dr. rer. nat. Jana B. Nieder, for welcoming me into her group and giving me this wonderful learning opportunity. The support, guidance and teaching will forever be a model for me to what a supervisor truly should be. You are an inspiration for the kind of professional I want to be in the future, for which you have my full admiration and respect. To Dr. Juan Luis Paris, thank you for all the support during these years I have spent at INL. You are a wonderful professional that taught me not only the way to do many experiments, but how to think about everything that surrounds it. I particularly thank you for the support in nanohybrid preparation and characterization, as well as biocompatibility assays, and so many fruitful discussions and patience for all of my questions. To Dr. Ima Ghaeli, all of your support throughout this work has been immense, and I highly regard you as a professional and as a friend. I would like to particularly thank you for all of the support in FLIM and in MATLAB. To Filipe Camarneiro, I mostly want to thank you for all the patience that you had with me, my endless questions for which you always had an answer for, all the discussions, laser alignments, set-up building, cell culture lessons and confocal imaging help, in all truth, all of the support throughout my time at INL. To Ricardo Adão, thank you for all of the support in MATLAB and fruitful discussions, as while confronted with something that for me always looked so challenging and impossible, I managed to understand and succeed, so many times only due to your outlooks and never-ending patience for teaching me. To Dr. Christian Maibohm I thank all the help with set-up and laser alignments and to Dr. Oleksandr Savchuk I thank for helping me with starting parameters and experiments, as well as introducing me into intracellular temperature field of research. A special thanks to Dr. Sebastian Thompson for including me in a wonderful project, teaching me vi further on intracellular temperature due to such exceptional collaboration. To the rest of UBNP group, as well as NAPS group, thank you all for welcoming me into the world of Photonics, I have appreciated all of the discussions and advices, but mostly all the friendship you have given me and made me regard you as a wonderful group, not only in a professional setting, but at a personal level as well. To my family, thank you for all of your immeasurable support, especially to my parents and brother, who keep on being an inspiration every day. These years have not been short of challenges, but the strength and resilience you have shown me has not only made me extremely proud of you, but served as an example on how I should face every trial life puts ahead. Thank you for everything, I love you all more than words could ever express. To Madalena, thank you for every day of our lifelong friendship, may there many years of us being family as well be ahead. The force has truly always been with you, and I am happy you manage to make with be with me too. To my friends from Aveiro, none of what I present here would be possible without all of the words, time and wonderful memories you have given me throughout these years. To Ana, thank you for all the patience, advices and different perspectives you give me, I am beyond grateful for everything you have done for me. To Tânia, you have given me some of the kindest words that have really got to me, and your dedication is something that has inspired me very much throughout this work. To Rita, all of the support, laughs and memories you have given me these years is something that I can only define as being a true sisterly bond. I also want to thank Alexandre, Sheri, Luz and Lucía for all of the amazing, meaningful and crucial long-distance encouragement. A special thank you to Ana Sofia, Dina, Maria Inês, Ânia and Catarina as well, for proving that we always have support, care and laughs to offer each other. To Micaela, Patricia, Armando and Pedro, thank you for making these years in Braga so wonderful. To Dra. Mafalda, thank you for all the patience, support and words that have so many times been crucial into guiding me throughout the challenges that life holds prepared for me. To my dearest colleague J. P., the perseverance, strength and willpower that takes to face the challenge that is a Master’s thesis, I could really only have learned it from you. An endless thank you to your family, who have been a wonderful source of support and a reference for me in resilience, something that has made all the difference throughout these years. Although this journey has been tough, I am proud of how far I’ve come and I hope you are too. I love you. vii 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. viii Bionanoestruturas para medição de temperatura intracelular durante terapia fototermal contra o cancro Resumo O cancro é uma das causas de morte mais comuns em países desenvolvidos, com tratamentos agressivos e de fraca eficácia, agravando-se pela inexistência de uma cura universal para todos os tipos de tumores. A hipertermia surge como um potencial tratamento complementar, ao debilitar as células cancerígenas e aumentando a sua suscetibilidade a outros tratamentos como quimioterapia e radioterapia, permitindo uma maior eficácia na sua aplicação, reduzindo a exposição a tais tratamentos, causadores de inúmeros indesejados e danosos efeitos secundários. O objetivo deste estudo foi desenvolver e caracterizar nanohíbridos multifuncionais para terapias fototermais, com capacidades simultâneas de aquecimento e medição de temperatura celular. A geração de calor é efetuada pela excitação de plasmões de superfície localizados de nanobastões de ouro integrados numa capa mesoporosa de sílica, enquanto a leitura da temperatura é executada pelas moléculas fluorescentes conjugadas de Rodamina B, que apresentam um tempo de vida de fluorescência dependente da temperatura, que decresce à medida que a temperatura sobe. Ao aplicar estes nanohíbridos a células tumorais e irradiar os mesmos com um laser de 808 nm (capaz de atravessar tecidos), o intuito será gerar calor e debilitar as células, enquanto a leitura da temperatura atingida dentro das células nas localizações dos nanohíbridos marcados é feita através de microscopia de imagens de tempo de vida de fluorescência. A leitura da temperatura permitirá a otimização das condições para atingir a temperatura intracelular ideal sem causar danos nos tecidos circundantes. Neste trabalho, os nanohíbridos foram sintetizados e caracterizados com êxito, enquanto a sua biocompatibilidade foi estudada em duas linhas celulares (células A431 e HeLa). Além disso, o aquecimento fototermal com nanotermómetro integrado foi estabelecido, ao medir a eficácia de aquecimento e a curva de calibração do tempo de vida de fluorescência dependente da temperatura. Adicionalmente, o nanohíbrido foi aplicado no estudo de mapas de temperatura resolvidos no espaço, em células vivas sujeitas a tratamento fototermal, exibindo uma sensibilidade de cerca de 0.23% ºC-1. Porém, a robustez dos nanohíbridos sob mudanças noutros fatores ambientais (ex.: pH) é desconhecida, precisando de estudos e aperfeiçoamentos. Palavras-chave: Nanohíbrido; Ouro; Rodamina B; Temperatura; Tempo de vida de fluorescência. ix Bionanostructures for intracellular temperature sensing during photothermal cancer treatment Abstract Cancer is one of the most common causes of death in developed countries, with aggressive and often poor treatment efficacy, while also lacking a universal cure suited for every type of tumor. Hyperthermia is presented as a potential complementary treatment option, by debilitating the cancer cells and increasing their susceptibility to other treatments, such as chemotherapy and radiotherapy, allowing for improved effectiveness on their application, by reducing exposure to such treatments that cause numerous undesired and harmful side-effects. The aim of this study was to develop and characterize multifunctional nanohybrids for photothermal therapies, with simultaneous heating and intracellular temperature-sensing capabilities. The heat generation shall be achieved by excitation of localized surface plasmons of gold nanorods embedded in a mesoporous silica shell, while the temperature readout shall be attained by conjugated fluorescent Rhodamine B molecules, which present a temperaturedependent fluorescence lifetime that decreases as the temperature increases. By applying these nanohybrids to tumor cells and irradiating them with a 808 nm laser (capable of tissue penetration), it is intended to generate heat and weaken cells, while fluorescence lifetime imaging microscopy is used to assess the temperature achieved inside of the cells in the locations of the labeled nanohybrids. The temperature reading shall allow optimization of the conditions to achieve the desired level of intracellular temperature without damaging surrounding tissues. In this work, these nanohybrids were successfully synthetized and characterized, as well as assessing their biocompatibility for two cell lines (A431 and HeLa cells). Furthermore, the photothermal heating with integrated nanothermometer was established by recording heating efficacy and the temperature dependent fluorescence lifetime calibration curve. Additionally, the nanohybrid was applied to study the spatially resolved temperature maps in photothermally treated live cells, with a sensitivity shown to be around 0.23% ºC-1. However, the nanohybrids’ robustness when facing changes in other environmental factors (e.g. pH) is unknown, requiring further studies and improvements. Keywords: Fluorescence Lifetime; Gold; Nanohybrid; Rhodamine B; Temperature. xvi excitation at 560 nm in aqueous solution at room temperature. Au@MSN-RhBITC hybrids show fluorescence within RhBITC’s emission range (around 590 nm), while Au@MSN particles do not show any fluorescence. ........................................................................................................... 45 Figure 21 – Graph representing the data obtained on the photothermal conversion assay. The sample represented refers to Au@MSN-RhBITC nanohybrids at a concentration of 500 µg/mL, irradiated by two conditions of laser power (1.3 and 1.8 W). The sample reaches its highest temperatures at 1.8 W 808 nm laser power irradiation, taking around 20 minutes to reach their maximum temperature. .......................................................................................................... 46 Figure 22 – Comparison of ΔT depending on different sample’ concentrations and different laser power irradiation in A – Au@MSN nanohybrids and B – Au@MSN-RhBITC nanohybrids. In both samples, at higher laser power irradiation (1.8 W), higher ΔT is reached. ................................ 48 Figure 23 – Comparison of ΔT depending on different sample’ concentrations and different nanohybrid composition in A – 1.3 W laser irradiation power and B – 1.8 W laser irradiation power. The samples containing RhBITC are shown to reach higher ΔT. ............................................... 48 Figure 24 – Frequency of fluorescence lifetimes recorded for each temperature condition, in percentage. The lower temperatures show higher occurrences’ percentage in higher fluorescence lifetimes values, while at higher temperatures, the occurrences befall in lower fluorescence lifetime values. ................................................................................................................................... 50 Figure 25 – Calibration curve of the Au@MSN-RhBITC nanohybrids. In red is represented the measured points and in blue the predicted fluorescence lifetimes when considering the calibration curve presented. The fluorescence lifetimes of the sample decrease as the temperature increases. .............................................................................................................................................. 51 Figure 26 – A – Number of HeLa cells per condition; B – Cell viability (percentage of live cells). The cells retain cellular viability up to at least 72 hours, as they present percentages of living cells over 89 % for every condition of exposure time and concentration of Au@MSN-RhBITC nanohybrids tested. .................................................................................................................................... 55 Figure 27 – A – Number of A431 cells per condition; B – Cell viability (percentage of alive cells). The cells retain cellular viability up to at least 72 hours, as they present percentages of living cells over 94 % for every condition of exposure time and concentration of Au@MSN-RhBITC nanohybrids tested. .................................................................................................................................... 55 xvii Figure 28 – HeLa cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL, exposed in A – 12 hours and B – 24 hours. In red is represented LysoHunt Blue DND-22 dye, while in blue are represented Au@MSN-RhBITC nanohybrids. ............................................................................ 57 Figure 29 – A431 cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL, exposed in A – 12 hours and B – 24 hours. In red is represented LysoHunt Blue DND-22 dye, while in blue are represented Au@MSN-RhBITC nanohybrids. ............................................................................ 57 Figure 30 – General scheme on how the FLIM results were processed. In A is represented the intensity image, obtained directly from the APD detector. In B is shown the curve fitting, to understand if the algorithm used was adequate for the sample, obtaining C, which represents the distribution of fluorescence lifetimes. After obtaining the FLIM image, two analysis could be done: the first is visible in C, which is selecting the cell region (in black), and in D is the image obtained by converting the FLIM image into temperature by using the calibration curve. Example represented by HeLa cells without 808 nm laser irradiation at 12 hours exposure time to nanohybrids. ...... 59 Figure 31 – Fluorescence intensity and FLIM characterization of Au@MSN-RhBITC nanohybrids: A – Intensity image, B - FLIM image that represents fluorescence lifetimes found in the measurement as well as cell region selection, C - Histogram of fluorescence lifetime values in highlighted region, which matches the extension of a cells - in accordance with the intensity distributions observed in A). The samples are both 12 h nanohybrid incubation with HeLa cells, and in Condition 1 (top row) is represented the absence of 808 nm laser irradiation, while Condition 2 (bottom row) undergoes 808 nm laser irradiation, 1.3 W for 3 minutes. ....................................................... 61 Figure 32 – Fluorescence intensity and FLIM characterization of Au@MSN-RhBITC nanohybrids: A – Intensity image, B - FLIM image that represents fluorescence lifetimes found in the measurement as well as cell region selection, C - Histogram of fluorescence lifetime values in highlighted region, which matches the extension of a cells - in accordance with the intensity distributions observed in A). The samples are both 24 h nanohybrid incubation with HeLa cells, and in Condition 1 (top row) is represented the absence of 808 nm laser irradiation, while Condition 2 (bottom row) undergoes 808 nm laser irradiation, 1.3 W for 3 minutes. ....................................................... 62 Figure 33 – Fluorescence intensity and FLIM characterization of Au@MSN-RhBITC nanohybrids: A – Intensity image, B - FLIM image that represents fluorescence lifetimes found in the measurement as well as cell region selection, C - Histogram of fluorescence lifetime values in highlighted region, which matches the extension of a cells - in accordance with the intensity distributions observed in A). The samples are both 12 h nanohybrid incubation with A431 cells, and in Condition 1 (top xviii row) is represented the absence of 808 nm laser irradiation, while Condition 2 (bottom row) undergoes 808 nm laser irradiation, 1.3 W for 3 minutes. ....................................................... 63 Figure 34 – Fluorescence intensity and FLIM characterization of Au@MSN-RhBITC nanohybrids: A – Intensity image, B - FLIM image that represents fluorescence lifetimes found in the measurement as well as cell region selection, C - Histogram of fluorescence lifetime values in highlighted region, which matches the extension of a cells - in accordance with the intensity distributions observed in A). The samples are both 24 h nanohybrid incubation with A431 cells, and in Condition 1 (top row) is represented the absence of 808 nm laser irradiation, while Condition 2 (bottom row) undergoes 808 nm laser irradiation, 1.3 W for 3 minutes. ....................................................... 64 Figure 35 – Temperature maps resulting from converting FLIM images using the calibration curve determined in Equation 6 for Au@MSN-RhBITC nanohybrids incubated for A - 12 h with HeLa cells, without 808 nm laser irradiation; B - 12 h with HeLa cells, with 808 nm laser irradiation for 3 minutes at 1.3 W laser power; C - 24 h with HeLa cells, without 808 nm laser irradiation, D - 24 h with HeLa cells, with 808 nm laser irradiation for 3 minutes at 1.3 W laser power. ............... 66 Figure 36 – Temperature maps resulting from converting FLIM images using the calibration curve determined in Equation 6 for Au@MSN-RhBITC nanohybrids incubated for A - 12 h with A431 cells, without 808 nm laser irradiation; B - 12 h with A431 cells, with 808 nm laser irradiation for 3 minutes at 1.3 W laser power; C - 24 h with A431 cells, without 808 nm laser irradiation, D - 24 h with A431 cells, with 808 nm laser irradiation for 3 minutes at 1.3 W laser power. ............... 67 xix Table of Supplementary Figures Supplementary figure 1 – Absorption spectrum of RhB (Adapted from Houde et al. (158)). ...... 84 Supplementary figure 2 – Detailed distribution of sizes for each sample of Au@MSN in A – Linear and B – Logarithmic scale. ..................................................................................................... 85 Supplementary figure 3 – Detailed distribution of sizes for each sample of Au@MSN-RhBITC in A – Linear and B – Logarithmic scale. ....................................................................................... 86 Supplementary figure 4 – Average frequency of sizes of Au@MSN and Au@MSN-RhBITC particles in linear scale. ........................................................................................................................ 86 Supplementary figure 5 – Photothermal conversion over time in dependence of nanohybrids’ concentration at 1.3 W laser power irradiation in A – Au@MSN nanohybrids and B – Au@MSNRhBITC nanohybrids. .............................................................................................................. 90 Supplementary figure 6 – Photothermal conversion over time in dependence of nanohybrids’ concentration at 1.8 W laser power irradiation in A – Au@MSN nanohybrids and B – Au@MSNRhBITC nanohybrids. .............................................................................................................. 91 Supplementary figure 7 – FLIM and histogram image acquired in nanohybrids samples used to obtain the calibration curve. Sample at 35 ºC. ......................................................................... 92 Supplementary figure 8 – FLIM and histogram image acquired in nanohybrids samples used to obtain the calibration curve. Sample at 40 ºC. ......................................................................... 93 Supplementary figure 9 – FLIM and histogram image acquired in nanohybrids samples used to obtain the calibration curve. Sample at 45 ºC. ......................................................................... 93 Supplementary figure 10 – FLIM and histogram image acquired in nanohybrids samples used to obtain the calibration curve. Sample at 50 ºC. ......................................................................... 94 Supplementary figure 11 – Au@MSN-RhBITC nanohybrids fluorescence lifetime’ dependence on pH. ......................................................................................................................................... 94 Supplementary figure 12 – HeLa cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL concentration – 63x Objective. A – Camera view at 12 hours exposure to Au@MSN-RhBITC nanohybrids; B – 405 nm laser irradiation at 12 hours exposure to Au@MSN-RhBITC nanohybrids; C – 561 nm laser irradiation at 12 hours exposure to Au@MSN-RhBITC nanohybrids. ............. 96 Supplementary figure 13 – HeLa cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL concentration – 63x Objective. A – Camera view at 24 hours exposure to Au@MSN-RhBITC nanohybrids; B – 405 nm laser irradiation at 24 hours exposure to Au@MSN-RhBITC nanohybrids; C – 561 nm laser irradiation at 24 hours exposure to Au@MSN-RhBITC nanohybrids. ............. 97 xx Supplementary figure 14 – A431 cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL concentration – 63x Objective. A – Camera view at 12 hours exposure to Au@MSN-RhBITC nanohybrids; B – 405 nm laser irradiation at 12 hours exposure to Au@MSN-RhBITC nanohybrids; C – 561 nm laser irradiation at 12 hours exposure to Au@MSN-RhBITC nanohybrids. ............. 98 Supplementary figure 15 – A431 cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL concentration – 63x Objective.; A – Camera view at 24 hours exposure to Au@MSN-RhBITC nanohybrids; B – 405 nm laser irradiation at 24 hours exposure to Au@MSN-RhBITC nanohybrids; C – 561 nm laser irradiation at 24 hours exposure to Au@MSN-RhBITC nanohybrids. ............. 99 Supplementary figure 16 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. ......... 100 Supplementary figure 17 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 100 Supplementary figure 18 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. ......... 101 Supplementary figure 19 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 3 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 101 Supplementary figure 20 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 102 Supplementary figure 21 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 102 xxi Supplementary figure 22 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 103 Supplementary figure 23 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 103 Supplementary figure 24 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 104 Supplementary figure 25 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 104 Supplementary figure 26 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 3 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 105 Supplementary figure 27 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 105 Supplementary figure 28 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 106 Supplementary figure 29 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 106 xxii Supplementary figure 30 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.3 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 107 Supplementary figure 31 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.3 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 107 Supplementary figure 32 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.3 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve (* indicates the existence of an outlier pixel reporting a temperature below 0 degrees, with a fluorescence lifetime above 5 ns), D – Histogram of fluorescence lifetime values in cell region selected. .......................................................................................................... 108 Supplementary figure 33 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.8 W, 3 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 108 Supplementary figure 34 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.8 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 109 Supplementary figure 35 – Temperature map study on FLIM characterization of Au@MSN-RhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.8 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 109 Supplementary figure 36 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.8 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 110 Supplementary figure 37 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.3 W, 6 minutes. xxiii A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 110 Supplementary figure 38 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.3 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 111 Supplementary figure 39 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.3 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 111 Supplementary figure 40 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.8 W, 3 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 112 Supplementary figure 41 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.8 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. . 112 xxiv Abbreviations APD – Avalanche Photodiode C12EDMAB – Dodecylethyldimethylammonium Bromide CTAB – Cetyltrimethylammonium Bromide DLS – Dynamic Light Scattering DMEM – Dulbecco’s Modified Eagle Medium DNA – Deoxyribonucleic Acid EGFR – Epithelial Growth Factor Receptor EPR – Enhanced Permeability and Retention FBS – Fetal Bovine Serum FLIM – Fluorescence-Lifetime Imaging Microscopy FPA – Fluorescence Polarization Anisotropy FWHM – Full Width Half Maximum GFPs – Green Fluorescent Proteins HPVs – Human Papillomaviruses HTLV-1 – Human T-lymphotropic virus 1 INL – International Iberian Nanotechnology Laboratory IRF – Instrument Responsese Function KSHV – Kaposi Sarcoma-Associated Herpesvirus LSPR – Localized Surface Plasmon Resonance MCPyV – Merkel Cell Polyomavirus MEM – Minimum Essential Medium MSN – Mesoporous Silica Nanoshell xxv N. A. – Numerical Aperture NBD – Nitrobenzoxadiazole NIR – Near infra-red PBS – Phosphate Buffer Solution PEG – Polyethylene Glycol PTAs – Photothermal Conversion Agents PTT – Photothermal therapy RhB – Rhodamine B RhBITC – Rhodamine B isothiocyanate RNA – Ribonucleic Acid SPC – Single Photon Counting TCSPC – Time Correlated Single Photon Counting TEM – Transmission Electron Microscopy TEOS – Tetraethyl Orthosilicate UBNP – Ultrafast and BioNanophotonics W. D. – Working Distance 7 2.2. Hyperthermia-based therapies 2.2.1. Hyperthermia as a cancer therapy Hyperthermia is characterized as being the condition in which the body temperature has been raised above 38.2ºC, as the normal temperature has been defined as being in the 37ºC - 38ºC range (29,30). Differences between a healthy person’s body temperature and a diseased individual had been reported (31) prior to Coley’s experimentations (32), where hyperthermia was used as a cancer therapy for the first time, to promote the regression of sarcoma after inoculation with Streptococcus erysipelatis , provoking fever and thus releasing an anti-tumor response. While hyperthermia may have positive effects in many diseases, including cancer, as per instance provoking apoptosis in cancer cells (by proteins denaturation and/or formation of oxygen reactive species), acting alongside chemotherapy (increasing blood flow to enhance drug delivery and immune cell migration) or radiotherapy (increasing sensibility to X-rays) (33), among others, it can also have some harmful consequences. Hyperthermia may be the cause of neurological, cognitive and physical disorders, such as seizures, unconsciousness, memory, comprehension issues (34), arrhythmias, sweating, hypertension (35) and many others. Hyperthermia may be of natural body response, in other words, fever (associated with inflammation, sepsis, drug-induced, endocrine factors or derived from brain injury (29)), or promoted by external elements. In the scope of this project, only external factors-mediated and localized hyperthermia is of interest, instead of the natural occurring type. This form of hyperthermia may then be directed to the tumor by radiation, only reaching the most superficial tumors, or reach the most deep located ones by inserting an electrode (36). When conjugated, the heat generated from the electrode forms a barrier between healthy and burnt tissue, which can prevent the propagation of the radio frequency (37). Besides these two most common methods, innovative solutions are being tested to induce hyperthermia, like ultrasounds, magnetic fields, radiofrequency and light-based processes, being the latter the most important for this project, as it is safer, less invasive, more profitable and with least side effects (38). As a cancer approach, hyperthermia has many advantages, starting with the fact that abnormal cells cannot tolerate higher temperatures for as long as healthy cells, which makes them a good target for therapy (39). Next, hyperthermia can also be used to increase oxygenation, pH variation, 8 tissue elasticity and ion diffusion, which can help in pain relief (33). Finally, hyperthermia does show apoptotic properties, but is not recommended to be used for that sole purpose, as it may affect surrounding healthy cells, if the temperature reached becomes excessively high (33,40), and it is best to combine it with more conventional therapies. Hyperthermia becomes then a secondary solution, with an effort to increase the efficiency of the primary approach. In terms of nanotechnology applied to hyperthermia treatments, it can make use of magnetothermal or photothermal nanoparticles (33), the latter being the focus of this project. 2.2.2. Photothermal nanotechnology-based therapies Photothermal therapies (PTT) are based on the use of a photoabsorbing agent, also known as photothermal conversion agents (PTAs) (41) capable of generating heat from light (42). This agent must absorb light in the near infra-red region (NIR), as it is the optimal wavelength absorption region by the human tissues (43), further explained in 2.2.3. This procedure is used predominantly in cancer research, where several types of PTAs have already been under study. These must be biocompatible, nontoxic, allow easy surface modification and have good photothermal conversion efficiency as well as stability (41). Gold is one of the most common materials for PTAs, due to its surface plasmon resonance at specific photo-frequencies (taking shapes as nanorods, nanocages, spheres, nanoshells, nanoflowers or nanorings) (44–46), but other commonly-used materials include carbon (in its graphite-related structures like graphene or nanotubes with single or multiwalls), rare earth doped nanocrystals (33) or organic-derived (e.g. sodium humate) (41), among many others. The PTAs are frequently used for cancer research, but they may have other types of applications. Hu et al. (47) studied PTT as a bactericidal approach, employing gold-covered nanocapsules to apply radiation and reduce the dosage of drug to eliminate E. coli BL21 in a combined and synergistic manner. This successful experiment may allow the decrease in use of antibiotics, decreasing the appearance of antibiotic-resistant organisms as well. Another study was performed by Beytollahi et al. (48), where PTT was used to destroy biofilm provoked by Streptococcus mutans . This bacterium is responsible for dental caries, and by using PTT mediated by indocyanine green under a diode laser, the appearance of colonies was significantly decreased, making it a suitable alternative to control caries. In a very different manner from the previous 9 examples, Maitland et al. (49) characterized the photothermal properties of shape-memory polymer micro-actuators in an effort to provide an alternative for treating stroke. This was eventually achieved by Small IV et al. (50), using a device based on Maitland et al. ’s work (49) that would retrieve the blood clot by changing its shape after heating. The polymer would suffer 810 nm laser irradiation and with the increase in temperature, change its shape to a corkscrew, capturing the blood clot and restoring blood flow. PTT presents many advantages to cancer treatment when compared to traditional approaches, being less invasive, and dealing a smaller amount of damage to the surrounding healthy cells, as it can have higher selectivity by doing specific targeting (51). PTT also does not require oxygen singlet formation (which is the case for photodynamic therapy), and by making use of a long and less energetic wavelength irradiation, it is safer for cell and tissue application (52). Many challenges still surround these approaches, such as low photothermal conversion efficiency, reduced stability of the photoabsorbing agents, poor cellular uptake, tumor resistance of the photothermal treatment or even metastasis (41), as well as over-heating, which can be prevented by conjugating PTT in a co-therapy system (53). 2.2.3. Near-Infrared (NIR) laser and its interaction with biological tissue Near-infrared laser radiation is used in photothermal therapy due to its tissue penetrating properties, but also for being absorbed less than other types of radiation (54). Human tissue has two biological windows for irradiation, due to several absorption bands provoked by the tissues’ different constituents. The first biological window goes from 700 – 980 nm, starting in the absorption band of hemoglobin and ending at the absorption band of water. The second biological window encompasses 1000 - 1400 nm, in which the limits both refer to water absorption bands (33). The use of NIR radiation at 808 nm falls into the first biological window absorption, which makes it ideal to be absorbed by the skin, as well as to generate heat from the gold nanorods when irradiated (55,56). 10 Figure 3 – Extinction coefficient of a representative tissue, along with indication of spectral extensions of the two biological windows (Adapted from Jaque et al. (33)). Boulnois has stated (57) four types of interaction between light and tissue: photochemical, photoablative, electromechanical and thermal. Photochemical interaction refers to long exposures of light at low power densities, absorbed by molecules in the tissue, which can be naturally present or posteriorly added, resulting on the foundation of photodynamic therapy (57,58). Photoablative interaction consists on cutting tissue using ultra-violet light irradiation, where photons cause dissociation of biopolymers without necrosis and minimal thermal interaction (57). Electromechanical light/tissue interaction is caused when a high energy short term light pulse creates a local high electric field, which causes dielectric breakdown of the tissue, as well as forming microplasma 1 , creating a shock wave when it expands (57,58). Lastly, Boulnois explains thermal light/tissue interaction, which is the most relevant in the scope of this project, and also the most frequent in surgical application of lasers. Attained by focusing a high energy density laser beam into a small area, the heat can remove tissue, injure or control bleeding. To comprehend how light is transformed into heat, Boulnois (57) summarizes it in the following reactions: Equation 1 that indicates the conversion of light into heat, denoting its absorption and Equation 2 that indicates the conversion of light into heat, denoting its deactivation. 𝐴𝑏𝑠𝑜𝑟𝑝𝑡𝑖𝑜𝑛: 𝐴 + ℎ𝑣 → 𝐴 ∗ Equation 1 1 Boulnois defines “microplasma” as an ionized volume with a very large free electron density (57). 11 𝐷𝑒𝑎𝑐𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛: 𝐴 ∗ + 𝑀(𝐸)→ 𝐴 + 𝑀′(𝐸 + ∆𝐸) Equation 2 After light irradiation, a molecule that Boulnois designated as “A” absorbs a photon of energy, being promoted to a vibrational excited state, labeled as “A*”. After colliding with a collisional partner that belongs to the surrounding medium (designated “M”), the internal energy of a “A*” is transferred to “M”, increasing its kinetic energy (transforming “M” into “M’”), which is released under the form of heat, increasing the temperature. Boulnois also refers that the penetration depth depends on the wavelength applied and different type of tissue that will undergo irradiation, being essential to differentiate and select the outcome intended (57). Though seemingly simple at the molecular scale, tissue irradiation with light is in fact a much more complex process, as multiple light scattering must always be considered as well (58). There are a few models that study the distribution of optical radiation through the tissues, such as the Kubelka-Munk, the Monte-Carlo and the diffusion model (59). In terms of cellular optics, Gazdaru et al. (60) describe cells as having an anisotropic absorbing media, with a refractive index greater than that of air, which is responsible for partial radiation reflection and for radiation refraction into tissues. Inside cells, the organelles like mitochondria or collagen fibers are the main scatterers of laser radiations, although other organelles (Golgi apparatus, lysosomes and ribosomes, among others) may also be responsible for some scattering. Furthermore, Gazdaru et al . (60) refer different kinds of scattering made by cells components, being either elastic or inelastic. The former may be submitted to either Mie’s (when the dimension of the scattering particles is comparable to the wavelength of the radiations e.g. mitochondria) or Rayleigh’s (dependent on: incident wavelength; dimension of scattering centers; and on the differences between the refractive indexes/cytosol. It’s present in collagen fibers) theory, while the latter is much weaker in cells and tissues, being either of Raman or Brillouin scattering. 2.2.4. Fluorescence-based nanothermosensing Fluorescence-based techniques were among the pioneers for intracellular temperature measurement, with organic commercial probes like Nitrobenzoxadiazole (NBD) and Laurdan 12 attaching to cell membrane and detecting temperature changes in a 2ºC resolution for the former and 0.1ºC – 1ºC for the latter (61,62). Beyond the work with these compounds, further formulations were developed, mostly concerning the coupling of organic ligands onto metal based nanoparticle, due to the fact that organic compounds alone can be affected by environmental changes like pH, swelling, protein denaturation and/or cell membrane rupture (63). For temperature based studies involving luminescence, one can use several parameters, indicated by Jaque and Vetrone (43), such as intensity, band-shape, spectral, polarization, bandwidth and fluorescence lifetime, illustrated in Figure 4. Spectral luminescence thermometry depends on the energy that separates the electronic states responsible for emission, as they will be responsible for the position of the bands. This measurement is independent of concentration, therefore not affected by phenomena like photobleaching of the fluorophores, producing the best results in systems where even small temperatures would generate higher spectral shifts (43). These may be used in peak fraction approaches, as per used by Savchuk et al. (64), who showed a redshift of the emission spectra as the temperature of wt-GFPs rose. Figure 4 – Different cases of luminescence changes depending on temperature (Adapted from Jaque et al. (43)). 13 Regarding temperature-induced spectral band-shape changes, these occur when the electronic states responsible for emission are so close in energy that they are thermally coupled as well, which usually happens in samples with two emitting centers that quench or transfer energy between each other, or samples with one emitting center, where the energy levels get re-distributed (44). In the work of Kulinich et al. (65), the effect of temperature on the fluorescent properties of merocyanines was verified to reflect on their position and shape of the absorption and fluorescence bands, mainly by changing the polarity and viscosity of the medium in which they are inserted. The width of fluorescence emission peak typically increases with temperature, as the density of phonons increases as well, leading to a broadening of the emission peak (43). In the work of Zhang et al. (66), the temperature sensing properties of a Ce3+-doped yttrium aluminum garnet by fluorescence properties was demonstrated by i) shifts in the emission band, ii) spectral bandwidth and intensity ratio. As for intensity changes related to temperature, the number of counts per second changes, which can cause the emission spectrum to change in intensity as well, which is caused by thermal activation of luminescence quenching mechanisms and/or increases in non-radiative decay probabilities (43). Intensity may also change due to factors unrelated to temperature (e.g. photobleaching), in which may be used one of the alternative fluorescence-based methods mentioned above. In terms of fluorescence lifetime thermometry, this is based on the time that it takes to decay to 1/e of the initial value of intensity after a pulsed excitation. This decay depends on many factors, including temperature, like phonon-assisted energy transfer processes and multiphoton decays (43). Intensity measurements are considered simpler for a laboratory setting (67), while fluorescence lifetime is considered more robust (68). The intensity signal presents dependence on intensity of laser excitation (69), which prevents a quantitative sensing of analytes without wavelength-ratiometric probes (67). Whilst fluorescence lifetime has no laser power dependency, it is dependent however on other parameters, such as concentration, having longer fluorescence lifetimes as the concentration decreases until it plateaus (70). Another fluorescence parameter that can show temperature dependency is the fluorescence polarization, often called fluorescence polarization anisotropy (FPA), which will happen when a fluorescent molecule, excited with a linearly polarized laser, re-emits the absorbed photon via the fluorescence emission process, not rotating before said emission. As a process dependent on the viscosity of the solvent, as well as the temperature molecular movement and rotation, this will lead 14 to an overall change in polarization fluorescence emission. Since an increase in Brownian motion induces molecular rotation, in dependence of increase in temperature, the polarization anisotropy will decrease, while it reaches its peak in absence of molecular motion (43). Pikulik et al. (71) demonstrated the dependence of fluorescence polarization to temperature in phthalimides, where the polarization decreased, as the temperature increased. Thompson et al. (72) reported the first use of a protein-dye hybrid as a FPA based thermoprobe, by conjugating small fluorescent dyes to larger proteins. This leads to a slower molecular rotation of the dye, increasing the dynamic range of FPA temperature sensitivity, which has an optimal ratio between fluorescence lifetime of the emitter and the size of the fluorescence emitting system. 2.2.4.1. Intracellular nanothermosensing Cells present some intrinsic temperature variation derived from their metabolic activities (73,74), and in cells where some pathology (e.g. tumor cells) is present, the metabolic activity is higher and temperature will be higher as well (74). Beyond their own temperature, cells can be heated from external sources, such as hyperthermia treatments, magnetic, or photothermal hyperthermia. In this case, knowing the temperature reached inside the cells will be useful to set the exposure parameters to light, in in vivo assays. While the generation of hyperthermia within cells is of high importance, the measurement of the reached temperature cannot be neglected either, as knowing the temperature will prevent overheating and damages on the healthy cells, as well as make possible the study of diseases where cellular temperature can be seen as an indicating factor of severity (e.g. localizing and monitoring the progression of tumors by their heat generation) (61,75). Temperature measurement is usually done by methods at a scale incompatible with an individual cell’s size (e.g. microcalorimetry) or by methods incompatible with living cells, but so has been done successfully when testing a cluster of cells (76). It is then necessary to create new strategies when referring to a single cell extent, making sure the method is accurate enough and at the same time, able to maintain the cells alive whilst undergoing thermal reading. To have a thermometer sensible enough for intracellular temperature measurement, it must possess several features: high spatial resolution and repeatability, measurements must be done in real time and in a user-friendly manner, as well as the thermometer itself must be independent from concentration 15 and any other environmental changes (75). The measurements can be then be done in one of two manners: contact or non-contact, with the latter making use of luminescence properties of compounds. When analyzing methods involving contact with the cells, one of the methods that has been most studied has been based on the variation of a typical thermocouple into a dimension compatible with cell size. Watanabe et al. (77) developed the first thermocouple-based approach, microfabricating a micropipette’ tip capable of performing Seebeck’s effect, the functioning base of thermocouples (75). The tip is capable of generating voltage according to the temperature of the sample, being converted into temperature by a digital thermometer or by comparing values on references tables. By Watanabe et al. (77) managing to place such thermocouple on a micropipette’s tip, it allowed for direct contact with cells, as the tip only had a diameter of 1 µm. Similar experiments involving size-adjusted thermocouples have been conducted by Wang et al. (78) and Shrestha et al. (79) as well. Another contact method include the development of a picocalorimeter, where temperature related changes alter the resonant frequency of the resonator, which Inomata et al. made of silicon (75,80). Relating to the non-contact temperature measurement techniques, these are mostly based on luminescence properties, but not exclusively, like the work of Gao et al. (81), where they utilized a method based on photoacoustic microscopy to measure the temperature of a single cell for the first time. In a most common way, non-contact temperature measurements are usually done by resorting to fluorescent probes, which can be one of the following: small and purely organic probes, metal complexes with organic ligands, thermoresponsive synthetic polymers, quantum dots, purely inorganic, or organic macromolecules like DNAor Green Fluorescent Proteins (GFPs)- based (61). The first report on resolving temperature maps and measuring from within biological cells using luminescence nanothermometry approaches were done in 2012 by Okabe et al. (73), while the pioneering work of Donner et al. (82), used GFPs as a temperature nanoprobe, based on fluorescence polarization anisotropy, which sensing mechanism was previously described (section 2.2.4), while using GNRs as a heating component. This temperature-assay method presented itself as biocompatible, with high spatial resolution and good temperature accuracy, presenting itself as less prone to artifacts, as per instance, ones caused by photobleaching. 16 2.3. Nanomaterials used for heating and nanothermometry 2.3.1. Gold in Medicine Gold has been used for medicinal applications (also known as chrysotherapy or aurotherapy) for millenniums, with evidence proving its use back in ancient China and Egypt, and keeping its presence in medieval medicinal practices. In late 19th century, breakthrough discoveries regarding gold salts influence in tuberculosis diagnosis and treatment allowed for gold to be used in a more vast and efficient array of diseases, such as rheumatoid arthritis and lupus erythematosus, well into the 20th century (83–85). Nowadays, gold is known to have many harmful side effects, and its uses in bulk as therapy are limited, as its long term effects are largely unknown (86). However, gold has gained new attention with a more secondary role in treatment, as nanoscience reinvents traditional methods of therapy, being the use of gold as a nanoparticle matrix one of the most interesting and studied, due to its attractive physical and chemical properties. In fact, gold nanoparticles may have a wide assortment of chemical, optical and electromagnetic characteristics, strongly depending on their shape and size (83), which may be controlled by rigorous regulation of experimental formation conditions (reducing agent, reaction time, temperature, capping agent 2 , among others) (87). These differences in size or chemical/physical characteristics may influence their cellular uptake, as well as their toxicity and biological interaction (83). Gold nanoparticles enter cells through a receptormediated clathrin-dependent endocytosis pathway, depending greatly on their size and shape, with rod-shaped nanoparticles having a slower uptake when compared to spherical nanoparticles (83,88). Studies have shown that gold toxicity also depends greatly on size, as smaller particles (1-2 nm) were observed to have higher cytotoxicity than larger particles (15 nm) in many diverse conditions, which is thought to be caused by differences in the uptake kinetics and/or cellular target specificities, as well as from interactions with the cell membrane (89). Currently, gold nanoparticles are being used in several medical approaches, such as diagnostics (bio-imaging and analytical methods) and many different kinds of 2 Capping agent is an amphiphilic molecule with a polar head group and a nonpolar hydrocarbon tail, responsible for providing colloidal stability, preventing agglomeration and stopping uncontrolled growth (166). 23 Chapter 3 - Materials and Methods 3.1. Au@MSN-RhBITC hybrids fabrication and nanocharacterization methods In the following section, the labelling procedure and several spectroscopic methods that were used to characterize the particles before applying them as heating sources and nanothermometers to cancer in vitro assays, will be described. 3.1.1. Au@MSN-RhBITC hybrids synthesis As per mentioned in 2.3.5, the formation of the gold nanorods and subsequent silanization were performed by Dr. Juan Luis Paris by the process described, while the removal of the silica from the mesopores and the adding of the RhB to the surface were performed at INL. After formation, the gold nanorods were covered with a mesoporous silica shell, using CTAB once more, but not as a stabilizer, being used as a template for silica instead. After adding the silica, the nanoparticles may be dried and stored (114), or continue the process, in order to extract the excess CTAB from the pores, hollowing and allowing them to be filled with other molecules. In this project, the Au@MSN nanorods were prepared in advance and were dried, so there was a need to re-disperse them in toluene and functionalizing with aminopropyl groups before progressing to the removal of CTAB. To achieve this, the prepared Au@MSNs were dispersed in dry toluene under a N2 atmosphere and placed at 85 ºC under magnetic stirring, adding APTES and leaving the sample to react overnight under an inert atmosphere. Afterwards, the Au@MSNs were washed with toluene and ethanol removing the surfactant by washing with an ethanolic solution of NH4NO3 (10 mg/mL NH4NO3 in 95 % ethanol). After several washes, RhBITC was added in a proportion of 1.38 mg to 20 mg of Au@MSN and some washes were needed to remove the molecules that didn’t adhere to the amine groups on the silica shell, thus becoming the final product denominated Au@MSN-RhBITC. The nanohybrids were dried and kept in powder form, to avoid silica shell degradation, as per explained in 2.3.4 section. 24 3.1.2. Hybrids nanocharacterization To better understand the physical and chemical properties of the Au@MSN-RhBITC nanohybrids, these must be characterized by several different techniques that will allow to know size, fluorescence and their correct assembly, among others. The methods used are comprised of Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), UV-Vis-NIR absorption spectroscopy and fluorescence spectroscopy 5 , which are described in more detail in the following sections. 3.1.2.1. Transmission Electron Microscopy TEM is a microscopy technique in which an electron beam reaches a sample, scattering electrons, generating contrast and producing an image (115). This technique is very useful in detecting very small objects, as they cannot be perceived by light microscopy. A distinctiveness in this method is the fact that samples must be placed under vacuum conditions, in order to allow the movement of electrons without influence of the electromagnetic fields produced by exterior currents and charges (116). This is a rather important feature, since these microscopes rely on solenoids to generate a magnetic field, which affects the electron beam’s focal length similarly to light and convex glass lens found in standard microscopes, focusing the electron beam (117). Experimental settings used: For this characterization, the equipment used was JEOL 2100 HT 200kV TEM (Cryo & Tomography). A drop of 200 µg/ml was pipetted onto a Formvar/Carbon Supported Copper Grids (size 400 mesh, thickness: 10 nm Formvar and 1 nm Carbon) grid, and for the imaging 200 kV electron beam was used. After focusing and some sample scanning, a characteristic area of the sample was selected for TEM image data acquisition. 5 Characterization experiments were performed by Dr. Juan Luis Paris and results analyzed by MSc. candidate. 25 3.1.2.2. Dynamic Light Scattering The Dynamic Light Scattering is a method used to determine the hydrodynamic size of particles dispersed in a solution (118). DLS is based on the scattering of the incident light by the particles, as the angle of scattering is dependent on the particle size, as smaller particles scatter less light than larger particles (119). This angle can be measured by a detector, usually at 90ᵒ or one at 173ᵒ (depending on its position in relation to the sample), since it’s the angles at which the dust contamination detection is lowest (120). Experimental settings used: A Horiba Dynamic Light Scattering System SZ-100Z instrument was used in the following parameters: 90ᵒ angle detection, five repetitions of measurements with 60 seconds integration time on 200 µg/ml Au@MSN and Au@MSN-RhBITC hybrids (in water) samples, of which 1 ml was placed in a disposable plastic cuvette. The results were compiled and represented using Excel. 3.1.2.3. UV-Visible-NIR absorption spectroscopy Spectroscopy is a group of techniques that comprise the study of the interactions between electromagnetic radiation and a matter of interest, and different kinds of techniques can be divided according to what they study, which can be the energies involved, the type of transitions that can occur between energy levels of a molecule subjected to incident radiation and whether the molecules assessed absorb or emit the energy (121). The absorption of electromagnetic radiation causes the molecule to transition from a lower-energy state to a higher-energy state, happening only when the incident radiation has equal energy to the one separating the molecule’s energy states (121). Kumar (121) also refers specifically that UV-Visible radiation promotes the molecule’s valence electrons to a higher-energy state, while infrared radiation changes the molecule’s vibrational energy. In Figure 7, an adapted Jablonski diagram (122) illustrates the energy levels of a phosphorescent molecule, where absorption is indicated by the arrow “A”. 26 In the case of UV-Visible-NIR absorption spectroscopy, this approach is based on the absorbance of the wavelength (in the range of 400 to 900 nm) by the samples, which can vary due to differences in composition and size of the particles in solution, although it can also be used to measure concentration and aggregation level with a few changes to the standard model used (123). Experimental settings used: The absorption for both Au@MSN and Au@MSN-RhBITC was assessed using a UV-Vis-NIR spectrometer (LAMBDA 950, Perkin-Elmer), in a quartz cuvette (1 cm per side and clear in all sides) and the results were compiled and represented using Excel. 3.1.2.4. Fluorescence spectroscopy Fluorescence spectroscopy is suited for the study of a different type of interaction between electromagnetic radiation and the molecules, when compared to the absorption spectroscopy. In this case, instead of studying the absorption of radiation by molecules of interest, it is studied the emission of radiation by said molecules, during their decay from the relaxed electronic excited to the electronic ground state manifold, indicated in Figure 7 by the arrow “B”. Regarding fluorescence, since there is always loss of energy by other processes other than radiative, the energy of the emitted light is lower than the absorbed light, meaning that the emission occurs at higher wavelengths than the absorption ones, since they are less energetic (121). Figure 7 – Diagram illustrating energy levels of a phosporescent molecule (F and N are stable energy levels, while M is a metastable energy level), with Anti-Stokes processes: A – Absorption, B – Fluorescence, C – Transition to metastable level, D – Thermal excitation, E and G – Phosphorescence and H – Absorbance of very small transition probability. Adapted from Jabło ń ski (122). 27 Experimental settings used: For this experiment, a microplate reader (Synergy H1, Biotek) was used. The excitation was set at 560 nm, and experiments performed at room temperature ( 25 ºC), while the fluorescence emission wavelengths measured for both Au@MSN and Au@MSN-RhBITC were between 590 – 700 nm using while the results were compiled and represented using Excel. 3.2. Functional characterization of the nanohybrids In order to perform functional characterization of the nanohybrids, firstly a photothermal conversion study will be executed, to understand how much do the nanohybrids in solution heat after irradiation with an 808 nm laser. Afterwards, the thermal sensitivity of the nanohybrids will also be assessed, to understand how temperature influences their fluorescence lifetime. 3.2.1. Photothermal conversion study The photothermal conversion refers to the ability certain materials have to absorb photons and convert that energy into heat (124). To assess the temperature of the sample, a thermocouple was dipped on the cuvette, away from the laser’s incident point, to avoid a direct heating and incorrect measurements. This temperature assay method is based on Seebeck’s effect, which is focused on the measurement of heat by the contact between the thermocouple’s tip and the sample, converting it into electricity (75). The tip of the thermocouple is made of two metallic wires that convert the temperature into voltage, allowing the comparison of the voltage values with a temperature reference table, or automatically convert them with a digital thermometer, which is the method used and is visible on Figure 8. Experimental settings used: In frame of this project, a photothermal conversion set-up was upgraded by adding an acrylic holder designed to fit a small cuvette (provided by Filipe Camarneiro) and hold it in the 808 nm laser excitation beam (dst11-DILAS-50W-808nm-400µ-0.22NA-t19286-503 device (type 503) with M1F4S22-808.5-50C-SS5.2 laser assembled, OsTech Electro Optical Instruments), which 28 contained the nanohybrids at different concentrations (0; 50; 100; 200 and 500 µg/mL) dispersed in water. Both the sample with and without RhBITC were assessed, to understand if the RhBITC had any influence on the heating component. An 808 nm laser was used to irradiate the cuvette, in order to generate heat, and it was irradiated until the maximum temperature was achieved, at two different laser powers of 1.3 W and 1.8 W, as measured with a power meter (1917-R , Newport) at the output of the laser-coupled fiber, based on previous studies (46,125). Afterwards, the laser was turned off and the cooling down of the sample was also timed, as a mean to obtain a photothermal dependency curve on concentration, as well as the time it takes to reach the peak temperature and how long it takes to cool down the sample. The sample with the same concentration was re-heated, to verify the stability of the hybrid over multiple heatings. For the temperature recording, a thermocouple probe (type K) connected to a thermocouple data logger (421509, Extech Instruments) was used, and the results were compiled and represented using Excel. 3.2.2. Thermal sensitivity of the nanohybrids The fluorescence lifetime will be calibrated in dependency of temperature (126), with the RhB’s fluorescence lifetime being expected to decrease with the increase of temperature (68), allowing the attainment of a calibration curve. Using this calibration curve, it is possible to convert the cell’s fluorescence lifetimes after exposure to the nanohybrids into a temperature measurement, thus Figure 8 – Photothermal conversion set-up. A – Thermocouple placement and data logger with general overview of the irradiation set-up; B – Close-up of sample irradiation with 808 nm laser. 29 developing an intracellular thermometer. To achieve this goal, fluorescence lifetime characterization will be performed. 3.2.2.1. Time correlated single photon counting (TCSPC) based fluorescence lifetime microscopy The thermal sensitivity of the nanohybrids is measured using the time correlated single photon counting (TCSPC) technique, and the fluorescence lifetime of RhBITC (present in the nanohybrids in study) that differs with changes in the sample’s temperature. TCSPC is a type of fluorescence spectroscopy, based on time resolution. Since reconstructing a photon fluorescence decay from a single excitation cycle would not be achievable, TCSPC uses a periodic laser pulse as the source of excitation, occurring in several cycles in which photons may be detected and their fluorescence decay profiles reconstructed, taking into account the time resolution (127). Wahl (127) refers TCSPC as a “start-stop” measurement (Figure 9), from which the information for the photon-arrivals histogram is obtained. After a laser pulse, the time between excitation and emission of a photon is measured, and then sorted into a histogram with a range of time bins. As time passes, the counts will drop in an exponential matter, as the likelihood of finding a molecule that has yet to emit fluorescence lessens, as the population of excited molecules decreases over time. Figure 9 – Measurement of start-stop times in time-resolved fluorescence measurement with TCSPC. Adapted from Wahl (127). 30 Experimental settings used: In this experiment, a custom-built FLIM set-up developed at UBNP was used. This set-up (Figure 10) is an inverted confocal microscope (RM21TM - MCL) comprised of a microstage (with a travel distance of 25 mm in X/Y and 50 mm in Z) and a nanostage (with a 200 µm travel distance in X/Y/Z), with a top-stage UnoStage Okolab incubator. This incubator allows to place a sample on the stages and control its temperature, CO2 levels and humidity, which is useful for cells live imaging, as it maintains cells ideal environment conditions. The laser used is a 561 nm picosecond pulsed laser (BDS-561 SMY Picosecond Diode Laser, Becker & Hickl), which was chosen due to its spectral properties fitting the RhB’s excitation spectrum. This laser enters the microscope through a periscope and a beam expander, which has two lenses with different focal lengths in order to match the objective back aperture. The laser is reflected on a dichroic mirror (Di02-R561, Semrock) to the 100x oil objective (N.A. 1.45, W.D. 0.13, PlanApo, Nikon) that focuses the laser on the sample placed inside of the incubator. After hitting the sample, the emitted fluorescence is captured by the objective and sent through the dichroic mirror, which is followed by a 561 nm long pass filter (561 Edge Basic Longpass Filter, AHF). This fluorescence is focused through a 50 µm pinhole, as in accordance to the microscope’s confocal properties, and is then focused into a single photon counting Avalanche Photodiode (APD) detector, which is used to record time-dependent distribution of emitted photons after each laser pulse (128) in combination with the Single Photon Counting (SPC) electronics module (SPC130, Becker & Hickl), to which the Sync signal of the picosecond pulsed laser source is attached to allow TCSPC. 31 To obtain said calibration curve (that related fluorescence lifetime information to temperature), Au@MSN-RhBITC hybrids dispersed in Dulbecco’s Modified Eagle Medium (DMEM) with no phenol red at a concentration of 200 µg/mL were placed in 70 µL wells on a Petri dish, and incubated on the top-stage incubator. Several set points were programmed at the incubator (25; 30; 35; 40; 45 and 50ºC), stabilizing the temperatures for 10 minutes before measurement. FLIM images were recorded at those temperatures. Since the incubator is open on the bottom to fit the objective, a heating sleeve was placed around the objective to guarantee the least possible heat loss. Figure 10 – Diagram of the optics involved in the FLIM measurements (M represents mirrors, while L represents lenses). The 561 nm laser enters the microscope passing by two lenses with different focal lengths. The laser is reflected on a dichroic mirror, focusing it on the sample placed inside of the incubator. The emitted fluorescence from the sample is captured by the objective and sent through the dichroic mirror, which is followed by a 561 nm long pass filter. This fluorescence is focused through a 50 µm pinhole and later enters the APD detector after focusing through two lenses. 32 Data analysis Each measurement was taken three times, and using the Nanophotonics Toolbox developed at UBNP for MATLAB, with main contributions of Ricardo Adão, they were converted into fluorescence lifetimes. The algorithm used was Enderlein, using the modelled IRF function and a fit offset of 175 time bins, selecting the peak in a Gaussian fit, in which the predominant fluorescence lifetime was selected, as well as the FWHM. The Enderlein algorithm used was Figure 11 – Schematic representation of the set-up for FLIM imaging of the nanohybrids. In A is observed the 561 nm laser irradiation entering the set-up. In B the 561 nm laser is seen irradiating the sample, inside the top-stage incubator, from where the signal exits through the objective (not visible in the pictures), to the dichroic mirror and the 561 nm LP filter, as seen in C , to reach the detectors, also partially visible in A . The detector records the time-dependent distribution of emitted photons after each laser pulse, and TCSPC is performed after synchronization with the SPC module. 39 Figure 15 – Close-up of inverted FLIM set-up with 808 nm laser coupled on top for irradiation of the sample. Figure 14 – Picture of the FLIM setup at INL’s laser laboratory, with relevant components of the inverted FLIM set-up highlighted, including: the 808 nm laser added for photothermal experiments; the graphical user interface of a custom microscope control software and SPC module; the incubator controller; APD detectors alongside the inverted confocal microscope. 40 Chapter 4 - Results and discussion 4.1. Au@MSN and Au@MSN-RhBITC nanocharacterization In this sub-section will be presented the results obtained after assessing the physicochemical properties of the hybrid nanoparticles, composed of a light absorbing gold nanorod core and a mesoporous silica shell. The influence of the presence of RhBITC on the nanohybrid was also taken into account for the characterization experiments, in which relevant parameters such as morphology, size, as well as optical properties, absorption and fluorescence properties of the hybrids were studied to test their suitability for cellular uptake and in vitro studies 4.1.1. Morphological characterization using TEM For the TEM microscopy, it was of interest to observe the nanohybrids’ size, as well as to understand their morphology, verifying the presence of an integer mesoporous silica shell with a nanorod core (Figure 16 and Figure 17). Figure 16 – TEM micrographs of Au@MSN particles. Sizes of the nanohybrids are noted to be around 100 nm, with pores visible around the nanoshell. 41 In Figure 16 are represented the TEM micrographs obtained from the Au@MSN nanorods, where it is possible to verify the lack of aggregates, meaning the particles were successfully dispersed. The size of the particles is consistent with the sizes obtained through DLS analysis, with about 100 nm for each nanorod (including its shell), and the pores in said shell are notably evident, which can be valuable in future experiments that may make use of these pores to load drugs for targeted delivery (as long as their size is well-defined and very precise (137)). This confirms not only the goal in enveloping the nanorods with a shell, but also the success in forming its pores. In Figure 17 are represented the nanohybrids modified with RhBITC, showing the same size and shell properties as the unmodified hybrids, although also presenting more aggregation. Something that is common to both samples is the presence of empty silica shells, which is explained by the shell formation process. After the formation of the gold nanorods, the silica is added and it envelops the particles in solution, as CTAB is added to provide a mold for the silica structure to be formed around the gold core. This structure is derived from TEOS, which is a silica based compound, and allows the formation of a shell with pores, whose size can be tuned by changing the concentration of both TEOS and surfactant (CTAB) (138), and by changing the process of formation, as per instance, the work of Jambhrunkar et al. (139), who developed a novel way based on vapor deposition to achieve angstrom-precise pore size. The nanorods will be then Figure 17 – TEM micrographs of Au@MSN-RhBITC particles. Sizes of the nanohybrids are noted to be around 100 nm, with pores less visible around the nanoshell, due to concealing done by the RhBITC functionalization. 42 coated with CTAB and posteriorly TEOS, and the excess of these reagents will form hollow shells. However, this is an infrequent occurrence, as presented in the results obtained. Regarding the pores, both samples are considered to be mesoporous, although the pores are more easily seen in the sample without RhBITC, as the functionalization makes it harder to observe the mesoporous structure (140). Nonetheless, the structure is present in both formulations and may be used in future drug-loading applications. The results obtained by TEM show the presence of the intended mesoporous properties of the nanohybrids, as well as verifying the integrity of the silica shell and the successful synthesis, as it presents very little aggregates, few empty silica shells and a consistent and suitable size for cellular application. 4.1.2. Hydrodynamic size characterization by DLS The aim of this analysis was to confirm the nanohybrids’ size, as DLS may also be used to assess their surface charge, useful to understand interaction of the cells with the nanohybrids (Figure 18). Figure 18 – Average size distribution of the Au@MSN and Au@MSN-RhBITC samples. Particle size is expected to be about 110 nm without RhBITC, and 127 nm with RhBITC. In Figure 18, it is shown the distribution of sizes by frequency of the nanohybrids, both with and without RhBITC on their surface. To determine the average size of the nanorods, the peak sizes of 43 each repetition were averaged, and as shown in the table that accompanies the graph, the particle is expected to be about 110 nm without RhBITC, and 127 nm with RhBITC. More detailed graphs with each of the repetitions (five per sample), and graphs done with a linear scale can be found in the Annexes – 1 section (Supplementary figure 2, Supplementary figure 3 and Supplementary figure 4). The DLS measurement demonstrated that the nanorods with RhBITC are slightly larger than the ones without RhBITC, and this variance is not believed to have repercussions in terms of cellular internalization, as studies by Zhu et al. (141) suggest a maximum recommended nanoparticle diameter of 160 nm for HeLa cells specifically, while Rejman et al. (142) verified that non-phagocytic eukaryotic cells can internalize nanoparticles up to 500 nm when using energydependent processes. The sizes obtained by DLS measurement is consistent with the ones obtained in the TEM assay, thus appropriate for cellular application. 4.1.3. UV-Vis-NIR absorption spectroscopy analysis To understand further the composition of the nanohybrids, their absorbance spectroscopy was studied (Figure 19), to confirm the presence of RhBITC attached, as it was not possible to do so in DLS or TEM measurements. The complete set of results can be found in Annexes - 3 section, in Supplementary table 1. The results for both conditions show a peak in absorbance at about 780 nm, which is the region for the NIR plasmon absorption band, and one around 520 nm, which fall on the 510 – 540 nm range, characteristic for the surface plasmon of gold (140). The results from the sample containing RhBITC also show a third peak, in the 560-575 nm region, which is the same wavelength as the emission wavelength of rhodamine B. 44 Figure 19 – Absorbance spectra taken on the bare mesoporous silica particles with gold nanorod core (Au@MSN) and on the RhBITC-loaded nanohybrids (Au@MSN-RhBITC). Both conditions show a peak in absorbance at about 780 nm (NIR plasmon absorption band), and one around 520 nm which is a characteristic region for the surface plasmon of gold. In the sample with RhBITC there is a third peak of absorbance in the 560-575 nm region, which is the same wavelength as the emission wavelength of RhB. This allow the confirmation that RhBITC was successfully grafted to the nanoparticle’s surface, as the nanohybrids present an absorbance peak absent from the nanoparticles without RhBITC. 4.1.4. Fluorescence spectroscopy To complement the UV-Vis-NIR results, the presence of RhBITC on the nanohybrids was also assessed by fluorescence spectroscopy (Figure 20). The complete set of results can be found in Annexes - 4 section, in Supplementary table 2. After excitation at 560 nm, which is within RhBITC’s excitation wavelengths range, the Au@MSN-RhBITC hybrids show fluorescence, also within RhBITC’s emission range (around 590 nm), while the Au@MSN particles do not show any fluorescence in the entirety of wavelength’s range, which was expected, since they do not possess any intrinsic fluorescence by themselves. 45 This result proves the successful grafting of RhBITC to the Au@MSN particles’ surface, as nanohybrids present fluorescence, a characteristic entirely nonexistent from the nanoparticles without RhBITC. Figure 20 – Fluorescence emission spectra taken on the bare mesoporous silica particles with gold nanorod core (Au@MSN) and on the RhBITC-loaded nanohybrids (Au@MSN-RhBITC) upon excitation at 560 nm in aqueous solution at room temperature. Au@MSN-RhBITC hybrids show fluorescence within RhBITC’s emission range (around 590 nm), while Au@MSN particles do not show any fluorescence. In conclusion, the evaluated physicochemical properties of Au@MSN-RhBITC samples show an adequate size for cellular internalization, as well as optical properties that confirm the fluorescence and successful grafting of RhBITC to the nanohybrids’ surface. 46 4.2. Functional characterization of the nanohybrids After characterization, the next challenge is to confirm the nanohybrids’ thermal heating aspect, owed to their light-absorbing gold core, and their temperature sensing characteristic, via RhBITC fluorescence lifetime dependence on temperature. In the following chapter, firstly will be discussed the results regarding the nanohybrids’ ability to generate heat by irradiation with a NIR laser, and afterwards their ability to sense temperature by their fluorescence lifetime in a relevant biological temperature window (25 to 50 ºC). 4.2.1. Photothermal conversion study To understand the nanohybrids photothermal conversion, and its comparison between bare and dye-loaded particle, the sample was irradiated with a NIR laser until it reached a constant temperature, followed by blocking the laser path and allowing for the sample to cooldown. Several samples of different concentrations and irradiated at different laser powers were gathered. The time it took to reach peak temperature and cooldown points was also measured, and a representative graph of the results obtained can be found in Figure 21, while the results for every condition can be found in Annexes - 3 section (Supplementary figure 5 and Supplementary figure 6). Figure 21 – Graph representing the data obtained on the photothermal conversion assay. The sample represented refers to Au@MSN-RhBITC nanohybrids at a concentration of 500 µg/mL, irradiated by two conditions of laser power (1.3 and 1.8 W). The sample reaches its highest temperatures at 1.8 W 808 nm laser power irradiation, taking around 20 minutes to reach their maximum temperature. 47 The results obtained in the photothermal conversion experiments depending on laser power, concentration and composition of the hybrid show a heat generation dependence’ on both the concentration of nanohybrid and the laser power chosen to irradiate the sample The control sample of H2O (which was irradiated at 1.8 W laser power) heated poorly, so it was proven that the heat as generated by nanohybrids. The samples at higher concentration of nanohybrid reached higher temperatures over a similar period of time when compared to lower concentration of nanohybrid, which proves the amount of nanohybrid is directly correlated with temperature. In a similar way, a higher laser power irradiation was also proved to have an effect on reaching higher temperatures. In order to better understand how these factors impact the temperature reached by the nanohybrids, the minimum temperature was subtracted to the maximum temperature reached by the sample, so as to get the ΔT. Such is graphically represented in Figure 22 and Figure 23, with detailed results in Table 1. It is also possible to notice extreme fluctuation of temperatures in some of the first temperature recordings, which were left out when calculating the ΔT, as they were some human experimental errors concerning the adjustment of the thermocouple’s position, as it incorrectly measured higher temperatures than the solution’s actual temperature since it was directly in the laser path, with its position being adjusted in the seconds that followed. Table 1 – Results for photothermal conversion Δ T in dependence of laser power, concentration of nanohybrid and nanohybrid composition. 50 µg/mL 100 µg/mL 200 µg/mL 500 µg/mL ΔT (ºC) 1.20 2.50 9.23 9.97 STDEV 0.432 1.25 2.17 1.00 ΔT (ºC) 1.77 5.23 11.1 14.6 STDEV 0.591 2.44 3.36 1.56 ΔT (ºC) 3.00 5.07 8.57 20.5 STDEV 0.990 1.51 1.94 4.07 ΔT (ºC) 5.17 8.80 12.0 26.2 STDEV 0.974 1.14 1.40 4.21 1.3 W 1.8 W 1.3 W 1.8 W Au@MSN Au@MSN-RhBITC 48 The effect of laser power is assessed in Figure 22, in which is possible to understand that samples at the same concentration and with the same nanohybrid composition present a higher increase in temperature in every condition when irradiated at a higher laser power. Therefore, higher laser power is directly related with a temperature rise, since there is an increase in photons, thus increasing the energy available to be absorbed by the sample. Figure 22 – Comparison of Δ T depending on different sample’ concentrations and different laser power irradiation in A – Au@MSN nanohybrids and B – Au@MSN-RhBITC nanohybrids. In both samples, at higher laser power irradiation (1.8 W), higher Δ T is reached. Figure 23 – Comparison of Δ T depending on different sample’ concentrations and different nanohybrid composition in A – 1.3 W laser irradiation power and B – 1.8 W laser irradiation power. The samples containing RhBITC are shown to reach higher Δ T. 55 Contrary to what was observed on HeLa cells, A431 cells showed a noteworthy difference in growth at different time periods, considering that all wells started with a density of about 10000 cells per well, a growth of 193%, 700% and 1500% correspond to 24, 48 and 72 hours respectively (Figure 27 – A). For HeLa cells, the growth was 88%, 145% and 97% for the same conditions respectively. This may be due to differences in each cell lines’ properties, as HeLa cells have a doubling time of about 40 to 48 hours (150) , which means after that period they may have reached their maximum growth and confluency and may start to die, whilst A431 cells take 80 to 100 hours to double (151), meaning that at 72 hours, the cells are still in doubling process, showing a higher growth in the end. In terms of biocompatibility with the nanohybrids, the results obtained showed slightly better results to the ones attained from HeLa cells, with every condition showing a cellular viability of over 94% (Figure 27 - B). Figure 27 – A – Number of A431 cells per condition; B – Cell viability (percentage of alive cells). The cells retain cellular viability up to at least 72 hours, as they present percentages of living cells over 94 % for every condition of exposure time and concentration of Au@MSN-RhBITC nanohybrids tested. Figure 26 – A – Number of HeLa cells per condition; B – Cell viability (percentage of live cells). The cells retain cellular viability up to at least 72 hours, as they present percentages of living cells over 89 % for every condition of exposure time and concentration of Au@MSN-RhBITC nanohybrids tested. 56 While for both cell lines cytotoxicity seems to be very low, as per the ISO10993-5 standard that indicates that biocompatibility is achieved with a cell viability of over 70% (152), there are some issues associated with the method in which the results are obtained from. The cell viability test used, known as the Trypan Blue exclusion test of cell viability, relies on membrane integrity that prevents the dye from entering on living cells (134), and has been used for evaluating nanoparticles’ cytotoxicity in numerous articles (153,154). However, many additional factors other than nanoparticle-caused damage may destroy the membrane or disrupt its integrity, as well as other kinds of toxicity may happen and go undetected, which makes this assay fairly unreliable. Unfortunately, there were many incompatibilities with other tests available, which made this the only solution obtainable. For instance, using the MTT test meant that there could be an enhancement of exocytosis of the formazan crystals by the presence of the mesoporous silica shell (155), leading to incorrect results, while MTS test absorbs in the same wavelength as gold (at 490500 nm) (156,157), as well as Alamar Blue®, which has the same absorption wavelength as RhB (158,159) and resazurin whose absorption spectrum coincides with the emission spectra of RhB (160). In Annexes - 7 section is presented Supplementary table 4 and Supplementary table 5, that contain the full results for the biocompatibility assays. In summary, these results prove that these nanohybrids are biocompatible and well-suited for in vitro assays at concentrations of up to 200 µg/mL. 4.3.2. Live cell confocal microscopy To further understand nanohybrids’ internalization, cells were incubated with nanohybrids at 200 µg/mL, as well with a lysosomes-specific dye, to visualize the location of the nanohybrids after internalization, which was done at 12 and 24 hours for each cell line (Figure 28 and Figure 29). Figure 28 and Figure 29 show the results obtained from confocal imaging, in which both cell lines were incubated with LysoHunt Blue DND-22. The dye was excited at 405 nm laser irradiation, while laser irradiation at 561 nm was used to excite the RhBITC in the Au@MSN-RhBITC nanohybrids. The goal was to see places where the fluorescence would appear in both conditions, in order to confirm if the nanohybrids were internalized and if they were still present and maintained some integrity after 24 hours of exposure. The images shown already merge both wavelengths of irradiation, and in red are shown the results obtained by irradiation of the LysoHunt, while in blue 57 are the Au@MSN-RhBITC nanohybrids. The individual images, as well as camera images, may be found in the Annexes – 8 section (Supplementary figure 12 to Supplementary figure 15). Figure 29 – A431 cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL, exposed in A – 12 hours and B – 24 hours. In red is represented LysoHunt Blue DND-22 dye, while in blue are represented Au@MSN-RhBITC nanohybrids. Figure 28 – HeLa cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL, exposed in A – 12 hours and B – 24 hours. In red is represented LysoHunt Blue DND-22 dye, while in blue are represented Au@MSN-RhBITC nanohybrids. 58 The use of the dye allowed to stain lysosomes, which are organelles responsible for receiving and degrading macromolecules that enter the cell, due to their acid hydrolases within (161). If internalization of the nanohybrids occurred, lysosomal activity would in the same area of the nanoparticles, meaning their fluorescence would overlap. The lysosomes can be seen in red in full extension of the cell area. In Figure 28 and Figure 29 it is possible to see the fluorescence of the nanohybrids inside of the cell, as they are within the area marked in red by the lysosomes, meaning that they don’t remain exclusively in the extracellular environment. The location of the nanohybrids is also more substantial in proximity to the nucleus, but not inside of it, which suggests that while the nanohybrids penetrate the cellular membrane, they cannot penetrate the nuclear membrane. Comparing between cell lines, HeLa cells show high fluorescence surrounding the nucleus in both time conditions, while the same is not observed for A431 cells, which seem to have more concentration of nanohybrids next to the nucleus at 12 hours, and very little at 24 hours (although still present). In the HeLa cells however, the fluorescence of the Au@MSN-RhBITC seems to be higher than the lysosomes’ fluorescence, which may be likely due to a change in focal plane. In conclusion, the cells from both cell lines were confirmed to uptake the nanorods, as there are many overlapping fluorescence areas, staying inside the cell for at least 24 hours, although they seem to have their fluorescent properties heightened at 12 hours, as the particles have been less exposed to silica shell-degrading factors, when in comparison with 24 hours exposure. Further assays will be beneficial to assess uptake dynamics, Au@MSN-RhBITC intracellular stability and expulsion. This could be studied by comparing cellular membranes’ labelling, which can be labelled by free-diffusion RhBITC that has detached from the nanohybrid surface due to degradation, in comparison with localized intensity spots that correspond to intact nanohybrids inside of lysosomes. Since this degradation can take days to occur (Figure 5), it would be of interest to gather images at time points at a span of different days, to pin-point the exact time at which this degradation starts to occur intracellularly. Beyond degradation, cellular expulsion could also be tested after cellular uptake, by performing exosome labelling. 59 4.3.3. Live cell FLIM microscopy for intracellular thermometry After determining the Au@MSN-RhBITC nanohybrids heating efficacy and knowing the concentrations at which they are biocompatible with the selected cell lines, it is of interest to explore how their heating capacity and intracellular temperature reporting capability evolves upon 808 nm laser irradiation. Previously it was obtained a calibration curve (Equation 6) that will allow to convert the fluorescence lifetime into temperature, by the process illustrated in Figure 30. Figure 30 – General scheme on how the FLIM results were processed. In A is represented the intensity image, obtained directly from the APD detector. In B is shown the curve fitting, to understand if the algorithm used was adequate for the sample, obtaining C , which represents the distribution of fluorescence lifetimes. After obtaining the FLIM image, two analysis could be done: the first is visible in C, which is selecting the cell region (in black), and in D is the image obtained by converting the FLIM image into temperature by using the calibration curve. Example represented by HeLa cells without 808 nm laser irradiation at 12 hours exposure time to nanohybrids. 60 The process represented in Figure 30 was done for each condition, with at least three measurements for each one. However, due to such plentiful results, a representative sample of the cell model prior to heating, and another after heating 3 minutes at 1.3 W was chosen (while the remaining conditions may be found at the Annexes - 8 section, comprising of measurements for both cell lines at 12 and 24 hours of exposure to the Au@MSN-RhBITC, with irradiation with an 808 nm laser at either 1.3 W or 1.8 W, for 3, 6, 9 or 15 minutes). 4.3.3.1. Fluorescence lifetime measurements Prior to irradiation with the 808 nm laser, the cells’ fluorescence lifetime was measured only in dependence of the top-stage incubator’s temperature, as a way to initially understand how the RhBITC could report the intracellular temperature before initiating the gold nanorod mediated heating. In Figure 31 – Condition 1 are shown HeLa cells that were incubated for 12 hours with Au@MSN-RhBITC prior to 808 nm laser irradiation. The FLIM image presents fluorescence lifetime values mostly in the range of 1 to 2.5 ns, while in the selected region (which is outlined by what is seen on the intensity image), the most-occurring fluorescence lifetime value is at 2.17 ns, which using the calibration curve mentioned in Equation 6 translates into 44 ºC. Other peak values of fluorescence lifetime occur at 1.68 ns (translating into 58 ºC) and 1.28 ns (translating into 70 ºC). In Figure 31 – Condition 2 are shown HeLa cells that were incubated for 12 hours with Au@MSN-RhBITC, after undergoing 808 nm laser irradiation, at 1.3 W laser power for 3 minutes. The FLIM image presents fluorescence lifetime values mostly in the range of 1 to 2.2 ns, while in the selected region, the most-occurring fluorescence lifetime value is at 2.26 ns, which using aforementioned calibration curve translates into 42 ºC, while the other peak value of fluorescence lifetime occurs at 1.88 ns (translating into 52 ºC). After laser irradiation, the fluorescence lifetime has increased in the overall FLIM image, and consequently on the peak fluorescence lifetime values that occur within the selected areas. It is possible to see on both images a concentration of nanohybrids (presenting higher intensity values) around a structure, which correlating with confocal images previously shown, is believed to be the cell nucleus, as in these images there is a concentration of nanohybrids around it as well. This structure presents a shorter lifetime than the rest of the selected area, which can be considered as the cell’s cytoplasm. 61 In Figure 32 – Condition 1 are shown HeLa cells that were incubated for 24 hours with Au@MSN-RhBITC prior to 808 nm laser irradiation. The FLIM image presents a wide variety of fluorescence lifetime values, with higher fluorescence lifetime in the background (around 2.5 ns), while lower lifetime values are encountered in somewhat circular structures spread throughout the image. In the selected region (0 – 2 ns), the occurrences are insufficient to present a robust Gaussian fit, meaning that the structures selected do not present a prevalent fluorescence lifetime. In Figure 32 – Condition 2 are shown HeLa cells that were incubated for 24 hours with Au@MSNRhBITC, after 808 nm laser irradiation, at 1.3 W laser power for 3 minutes. The FLIM image presents fluorescence lifetime values mostly in the range of 0.5 to 1.5 ns, dominated by the selected region, with a small part of fluorescence lifetime values between 2 to 3 ns in the background. In the selected region, the most-occurring fluorescence lifetime value is at 0.90 ns, Figure 31 – Fluorescence intensity and FLIM characterization of Au@MSN-RhBITC nanohybrids: A – Intensity image, B - FLIM image that represents fluorescence lifetimes found in the measurement as well as cell region selection, C - Histogram of fluorescence lifetime values in highlighted region, which matches the extension of a cells - in accordance with the intensity distributions observed in A) . The samples are both 12 h nanohybrid incubation with HeLa cells, and in Condition 1 (top row) is represented the absence of 808 nm laser irradiation, while Condition 2 (bottom row) undergoes 808 nm laser irradiation, 1.3 W for 3 minutes. 62 which using the calibration curve translates into 80 ºC, while the other peak value of fluorescence lifetime occurs at 1.19 ns (reporting 72 ºC). For A431 cells measurement that were incubated for 12 hours with Au@MSN-RhBITC prior to 808 nm laser irradiation (Figure 33 – Condition 1), the most occurring lifetimes in the background range between 2 to 2.5 ns, which is within the same range as reported for HeLa cells in the same zone. Inside of the selected region, three fluorescence lifetimes are most prevalent, being the most occurring 0.69 ns (translating into 86 ºC), followed by 1.05 ns (translating into 76 ºC) and finally by 1.66 ns (translating into 59 ºC). In Figure 33 – Condition 2 are shown A431 cells that were incubated for 12 hours with Au@MSN-RhBITC after 808 nm laser irradiation, at 1.3 W laser power for 3 minutes. The FLIM image presents a higher fluorescence lifetime in the background (around Figure 32 – Fluorescence intensity and FLIM characterization of Au@MSN-RhBITC nanohybrids: A – Intensity image, B - FLIM image that represents fluorescence lifetimes found in the measurement as well as cell region selection, C - Histogram of fluorescence lifetime values in highlighted region, which matches the extension of a cells - in accordance with the intensity distributions observed in A) . The samples are both 24 h nanohybrid incubation with HeLa cells, and in Condition 1 (top row) is represented the absence of 808 nm laser irradiation, while Condition 2 (bottom row) undergoes 808 nm laser irradiation, 1.3 W for 3 minutes. 63 2 ns), while lower lifetime values are encountered within the selected region (0.5 – 1.5 ns). From the Gaussian fit in the fluorescence lifetimes in that region, the most occurring fluorescence lifetime is at 1.06 ns (corresponding to 76 ºC), while two more peaks are observed (1.23 ns reporting 71 ºC and 1.47 ns reporting 64 ºC). For A431 cells measurement that were incubated for 24 hours with Au@MSN-RhBITC prior to 808 nm laser irradiation (Figure 34 – Condition 1), the fluorescence lifetimes are more homogenous than on previously shown samples, with the full image having fluorescence lifetime values predominantly between 2 to 2.5 ns. The selected region presents a uniform fluorescence lifetime distribution as well, with a peak at 2.27 ns that reports 41 ºC. In Figure 34 – Condition 2 are shown A431 cells that were incubated for 24 hours with Au@MSN-RhBITC after 808 nm laser irradiation, at 1.3 W laser power for 3 minutes. The background presents itself as having Figure 33 – Fluorescence intensity and FLIM characterization of Au@MSN-RhBITC nanohybrids: A – Intensity image, B - FLIM image that represents fluorescence lifetimes found in the measurement as well as cell region selection, C - Histogram of fluorescence lifetime values in highlighted region, which matches the extension of a cells - in accordance with the intensity distributions observed in A) . The samples are both 12 h nanohybrid incubation with A431 cells, and in Condition 1 (top row) is represented the absence of 808 nm laser irradiation, while Condition 2 (bottom row) undergoes 808 nm laser irradiation, 1.3 W for 3 minutes. 64 homogenous fluorescence lifetime, within the range of 2 to 2.5 ns. For the selected region, two different cellular locations may be distinguished. The first, which is believed to be the nucleus due to higher concentration of nanohybrids around it (also visible in confocal images presented as Figure 29), reports a shorter fluorescence lifetime, at 0.89 ns (translating into 81 ºC). The other region, the cytoplasm, reports a higher fluorescence lifetime, at 2.29 ns (translating into 41 ºC). Lastly, from the Gaussian fit one more peak is shown, which corresponds to background that was included during cell selection, which reports 1.88 ns (translating into 52 ºC). In summary, the fluorescence lifetime reported in the different cell conditions for the background seem to be fairly consistent, which indicates that the FLIM analysis has been correctly collected. Some cellular heterogeneity has also been shown, indicating that the nanohybrids’ intracellular distribution is not uniform, concentrating mostly in the surroundings of the nucleus, as per shown in Figure 28 and Figure 29 as well. Figure 34 – Fluorescence intensity and FLIM characterization of Au@MSN-RhBITC nanohybrids: A – Intensity image, B - FLIM image that represents fluorescence lifetimes found in the measurement as well as cell region selection, C - Histogram of fluorescence lifetime values in highlighted region, which matches the extension of a cells - in accordance with the intensity distributions observed in A) . The samples are both 24 h nanohybrid incubation with A431 cells, and in Condition 1 (top row) is represented the absence of 808 nm laser irradiation, while Condition 2 (bottom row) undergoes 808 nm laser irradiation, 1.3 W for 3 minutes. 71 Chapter 5 - Conclusion and future perspectives The nanohybrids in study were successfully synthetized and characterized, and their size was shown to be appropriate for cellular application. They were verified to be biocompatible on HeLa and A431 cell lines, with viabilities over 89 % in every condition tested, which may also pave way for possible future in vivo studies. The nanohybrids were also proved to have heating properties after irradiation with an 808 nm laser, as their photothermal conversion results show heat generation dependent on laser power and nanohybrid concentration. The highest temperature increase was obtained was at the highest concentration and laser power irradiation (500 µg/mL of Au@MSN-RhBITC at 1.8 W 808 nm laser irradiation), reaching 26.23 ºC. Following the nanohybrids’ characterization and demonstration of their biocompatibility, a calibration curve of their fluorescence lifetime in dependency of temperature was obtained at relevant in vitro temperatures, in both typical and hyperthermia circumstances (from 35 to 50 ºC). Finally, after proving their internalization by fluorescence imaging, the cells were irradiated with an 808 nm laser at different laser powers and exposure times, obtaining intensity and FLIM images, as well as an intracellular temperature map, reported by resorting to the calibration curve previously obtained. The results obtained show a successful uptake of the nanohybrids by both cell lines, although the derived temperature maps report temperature values incompatible with cellular survival. This incorrect temperature reporting may have been caused by the usage of a calibration curve that has not been perfected for this application, as well as sample over-irradiation, which should be a priority for advancement when repeating this experiment. For this purpose, a more extensive research on the nanohybrids’ temperature dependence on factors other than the ones tested should be done (such as ionic strength or pH), as it is likely a key source of error in this conversion, making use of optical characterization to perfect the calibration curve as well. Additionally, the experiment should ideally be repeated in independent wells for each condition, as over-irradiation has caused cell drying and possible death that could be avoided. While fresh medium could be added throughout the experiment to replace what is evaporated, it may hinder the reliability on the 72 concentration used, which has already been proven to be an important factor for the heating obtained. Nevertheless, the results obtained in both biocompatibility and photothermal conversion experiments are encouraging, as they prove that further in vitro tests may take place using the heating properties shown by this work, and while challenges were encountered with the temperature sensing unit results, in this work is also possible to already understand some of the pitfalls and use the proposed strategies to circumvent these issues in future experiments. 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Cancer Biol. 2017;7(3):71–8. 87 3. UV-Vis-NIR absorption spectroscopy analysis Au@MSN Au@MSN-RhBITC 400 0.686 0.519 405 0.68 0.513 410 0.673 0.506 415 0.665 0.499 420 0.659 0.493 425 0.654 0.488 430 0.648 0.482 435 0.642 0.475 440 0.636 0.466 445 0.63 0.459 450 0.626 0.453 455 0.621 0.448 460 0.616 0.444 465 0.612 0.441 470 0.609 0.441 475 0.606 0.443 480 0.606 0.447 485 0.608 0.456 490 0.614 0.466 495 0.623 0.48 Wavelength Absorbance (A.U.) Au@MSN Au@MSN-RhBITC 500 0.634 0.498 505 0.648 0.525 510 0.658 0.558 515 0.661 0.59 520 0.659 0.612 525 0.652 0.629 530 0.644 0.634 535 0.633 0.632 540 0.622 0.643 545 0.614 0.667 550 0.603 0.705 555 0.591 0.743 560 0.578 0.761 565 0.566 0.749 570 0.556 0.718 575 0.544 0.663 580 0.531 0.59 585 0.519 0.523 590 0.51 0.479 595 0.501 0.442 Absorbance (A.U.) Wavelength Au@MSN Au@MSN-RhBITC 600 0.492 0.415 605 0.485 0.396 610 0.48 0.385 615 0.477 0.379 620 0.475 0.375 625 0.474 0.373 630 0.475 0.374 635 0.477 0.377 640 0.481 0.381 645 0.487 0.388 650 0.495 0.396 655 0.504 0.407 660 0.514 0.417 665 0.526 0.429 670 0.541 0.444 675 0.561 0.464 680 0.579 0.482 685 0.596 0.498 690 0.617 0.519 695 0.641 0.541 Wavelength Absorbance (A.U.) Au@MSN Au@MSN-RhBITC 700 0.664 0.563 705 0.683 0.58 710 0.704 0.598 715 0.725 0.616 720 0.749 0.634 725 0.767 0.648 730 0.785 0.66 735 0.803 0.671 740 0.82 0.68 745 0.835 0.688 750 0.845 0.691 755 0.855 0.694 760 0.865 0.695 765 0.873 0.694 770 0.879 0.691 775 0.883 0.688 780 0.886 0.683 785 0.889 0.676 790 0.889 0.67 795 0.888 0.663 Wavelength Absorbance (A.U.) Supplementary table 1 – Measured absorbance in the 400-900 nm range, for Au@MSN and Au@MSN-RhBITC (subdivided into 5 tables). 88 Au@MSN Au@MSN-RhBITC 800 0.887 0.654 805 0.884 0.645 810 0.88 0.635 815 0.876 0.626 820 0.87 0.616 825 0.865 0.607 830 0.86 0.597 835 0.853 0.588 840 0.846 0.578 845 0.838 0.568 850 0.827 0.556 855 0.818 0.547 860 0.809 0.538 865 0.8 0.529 870 0.79 0.519 875 0.781 0.511 880 0.769 0.501 885 0.76 0.492 890 0.746 0.48 895 0.734 0.47 900 0.725 0.463 Wavelength Absorbance (A.U.) 89 4. Fluorescence spectroscopy Au@MSN Au@MSN-RhBITC 590 532228 595 429372 600 425115 605 320887 610 617776 615 715618 620 913711 625 312223 630 611021 635 710279 640 12 9511 645 48442 650 14 7325 655 10 6356 660 65917 665 10 5013 670 18 4159 675 03763 680 36 3333 685 03209 690 92620 695 01466 700 18 1704 Fluorescence Intensity (A.U.) Wavelength Supplementary table 2 – Measured fluorescence intensity for Au@MSN and Au@MSN-RhBITC. 90 5. Photothermal conversion study Supplementary figure 5 – Photothermal conversion over time in dependence of nanohybrids’ concentration at 1.3 W laser power irradiation in A – Au@MSN nanohybrids and B – Au@MSNRhBITC nanohybrids. 91 Supplementary figure 6 – Photothermal conversion over time in dependence of nanohybrids’ concentration at 1.8 W laser power irradiation in A – Au@MSN nanohybrids and B – Au@MSNRhBITC nanohybrids. 92 6. Thermal sensitivity of the nanohybrids Supplementary figure 7 – FLIM and histogram image acquired in nanohybrids samples used to obtain the calibration curve. Sample at 35 ºC. 35 2.42 1.33 35 2.52 1.17 35 2.52 1.08 40 2.47 0.98 40 2.33 1.06 40 2.24 1.00 45 2.22 1.03 45 2.08 0.94 45 1.97 1.00 50 2.09 0.91 50 1.98 0.93 50 1.89 0.90 Temperature (ºC) Fluorescence Lifetime (ns) FWHM Fluorescence Lifetime (ns) - Averaged Standard Deviation 2.4867 0.4291 2.3467 0.3162 2.0900 0.3323 1.9867 0.2936 Supplementary table 3 – Fluorescence lifetime of the nanohybrids in dependence of temperature, used for obtaining the calibration curve. 93 Supplementary figure 8 – FLIM and histogram image acquired in nanohybrids samples used to obtain the calibration curve. Sample at 40 ºC. Supplementary figure 9 – FLIM and histogram image acquired in nanohybrids samples used to obtain the calibration curve. Sample at 45 ºC. 94 Supplementary figure 10 – FLIM and histogram image acquired in nanohybrids samples used to obtain the calibration curve. Sample at 50 ºC. Supplementary figure 11 – Au@MSN-RhBITC nanohybrids fluorescence lifetime’ dependence on pH. 95 7. Biocompatibility 0 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL Average 15833 18667 20000 20833 STDEV 2718.25107 3880.14891 3894.44048 1840.8935 Average 22750 28333 26167 20667 STDEV 2750 6355.22532 6599.66329 5632.24842 Average 17250 23000 23667 14667 STDEV 3250 7648.52927 4496.91252 4496.91252 Average 95.30 93.21 89.36 95.31 STDEV 4.94694 2.81317 3.52095 5.17621 Average 99.02 96.33 93.42 94.83 STDEV 0.98039 2.57759 3.31525 1.23165 Average 92.77 96.60 97.02 97.62 STDEV 0.08711 0.81682 1.30772 3.36718 HeLa cells 72 hours Cell viability (%) Nr. of Cells 24 hours 48 hours 72 hours 24 hours 48 hours Supplementary table 4 – Results regarding the number of cells and the percentage of cell viability of HeLa cells. 0 µg/mL 50 µg/mL 100 µg/mL 200 µg/mL Average 28500 30000 26333 32667 STDEV 7937.25393 4822 9292 6007 Average 86500 82167 80833 66833 STDEV 23302 10324 13475 13137 Average 157167 184500 159500 137667 STDEV 43038.7422 67506 30327 37464 Average 99.56 95.96 94.64 96.60 STDEV 0.7698 5.8221 2.1218 2.9985 Average 98.91 99.35 95.36 98.53 STDEV 1.29281 1.1175 2.9711 1.1605 Average 98.59 97.62 97.76 96.90 STDEV 0.41489 0.7504 0.7857 0.8823 A431 cells 24 hours 48 hours Nr. of Cells Cell viability (%) 24 hours 72 hours 48 hours 72 hours Supplementary table 5 – Results regarding the number of cells and the percentage of cell viability of A431 cells. 96 8. Confocal imaging Supplementary figure 12 – HeLa cells with Au@MSN-RhBITC nanohybrids at 200 µg/mL concentration – 63x Objective. A – Camera view at 12 hours exposure to Au@MSN-RhBITC nanohybrids; B – 405 nm laser irradiation at 12 hours exposure to Au@MSN-RhBITC nanohybrids; C – 561 nm laser irradiation at 12 hours exposure to Au@MSN-RhBITC nanohybrids. 103 Supplementary figure 22 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 23 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 104 Supplementary figure 24 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 25 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.3 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 105 Supplementary figure 26 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 3 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 27 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 106 Supplementary figure 29 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 28 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with HeLa cells, 808 nm laser irradiation, 1.8 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 107 Supplementary figure 30 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.3 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 31 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.3 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 108 Supplementary figure 32 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.3 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve ( * indicates the existence of an outlier pixel reporting a temperature below 0 degrees, with a fluorescence lifetime above 5 ns), D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 33 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.8 W, 3 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 109 Supplementary figure 34 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.8 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 35 – Temperature map study on FLIM characterization of Au@MSN-RhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.8 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 110 Supplementary figure 36 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 12 h with A431 cells, 808 nm laser irradiation, 1.8 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 37 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.3 W, 6 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 111 Supplementary figure 38 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.3 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 39 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.3 W, 15 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. 112 Supplementary figure 40 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.8 W, 3 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected. Supplementary figure 41 – Temperature map study based on FLIM characterization of Au@MSNRhBITC nanohybrids incubated 24 h with A431 cells, 808 nm laser irradiation, 1.8 W, 9 minutes. A – Intensity image, B - FLIM image with cell region selection, C – Temperature map study derived from calibration curve, D – Histogram of fluorescence lifetime values in cell region selected.