University of Groningen
Bacterially derived carbon quantum dots for biofilm control Wu, Yanyan
DOI:
10.33612/diss.171939593
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Wu, Y. (2021). Bacterially derived carbon quantum dots for biofilm control. University of Groningen. https://doi.org/10.33612/diss.171939593
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General discussion
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General discussion
Antimicrobial-resistant bacterial infections remain a major threat to public health [1], as they cause a wide range of infectious diseases and the treatment is complicated due to the recalcitrance of the biofilm-mode of growth in which pathogen grow to antibiotic penetration [2]. New antimicrobial strategies focusing on biofilm dispersal and allowing antibiotic penetration, are therefore crucial for infection control. Carbon quantum dots with unique features such as good biocompatibility, excellent water suspendability, stable fluorescence, easy synthesis and modification, have attracted increasing attention for antibacterial applications. Carbon quantum dots can be synthesized through different methods and from many different carbon sources varying from organic carbon sources [3,4], inorganic carbon sources [5,6] to natural carbon sources [7]. Importantly, carbon quantum dots derived from conventional antibiotics have been found to yield a lower chance upon inducing bacterial resistance and higher bacterial killing efficacy than their source antibiotics [8]. Until now, most studies focused on killing planktonic bacteria or inhibiting bacterial growth [9–13] with only a few papers focusing on carbon dot induced biofilm dispersal, as a potential new therapy for treating infection. As the antibacterial and antibiofilm properties of carbon quantum dots are largely determined by their surface functional groups, this thesis focuses on the physico-chemical properties of carbon quantum dots, their biofilm dispersal ability, and the potential synergistic effect with antibiotics for enhanced killing efficiency in infectious biofilms.
Mechanisms of biofilm dispersal by carbon quantum dots
The mechanisms of biofilm dispersal by carbon quantum dots include electrostatic and hydrophobic interactions [14], reactive oxygen species (ROS) generation [8] and interference with the self-assembly of amyloid peptides [15]. In Chapter 2, we have designed a new pH-responsive carbon quantum dot
synthesized from polyethyleneimine and citric acid, and further modified with 2,3-dimethylmaleic-anhydride as a synthetic dispersant to staphylococcal biofilms. The pH responsive carbon quantum dots possess a surface charge-switch ability, and disperse biofilm through electrostatic and hydrophobic interactions with bacteria. We further synthesized carbon quantum dots from probiotic and pathogenic bacteria (Chapter 3), and demonstrated their ability to generate ROS
(Chapter 4). Because of the negative charge of bacterially derived carbon quantum
dots, the electrostatic double-layer attraction between these carbon dots and the negatively charged bacteria is smaller than those between pH responsive carbon quantum dots that become positively charged within an acidic biofilm and bacteria. On the other hand, the ROS generation by pH responsive carbon quantum dots is generally far less compared with bacterially derived carbon quantum dots, probably due to the different electron-donating ability of different nitrogen-carbon structures on the surface [16]. Interestingly, carbon quantum dots with positively charged groups on the surface and generating ROS ability hardly caused bacterial resistance, as the carbon quantum dots bind non-specifically through electrostatic
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107 interactions to bacteria which is different from antibiotics which have a specific interaction with bacteria [8,17].
Nitrogen functionalities of antibiofilm carbon quantum dots
Nitrogen functionalization and doping have been demonstrated to change the surface properties of carbon quantum dots. As an electron donating atom, insertion of nitrogen as heteroatom into a molecular carbon structure facilitates electron transfer and generation of ROS [16,18]. Moreover, nitrogen functional groups provide positive charge to the surface of carbon quantum dots. Therefore, nitrogen-rich sources have been widely used in the synthesis of antibacterial carbon quantum dots, to endow carbon quantum dots with a positive charge and the ability to generate ROS. Nitrogen containing carbon quantum dots can be negatively charged or positively charged. For positively charged carbon quantum dots, the cationic property plays an important role in their antibacterial activity. However, the zeta potential is not the key factor to determine their antibacterial efficiency. Carbon quantum dots with similar positive zeta potentials, but with nitrogen in different functional groups can behave differently for biofilm control, due to the different hydrophobic interactions between bacteria and carbon quantum dots [14,19]. In Chapter 2, we used branched polyethyleneimine as a nitrogen
source and citric acid as a carbon source to synthesize carbon quantum dots. The polyethyleneimine provided positively charged amine groups in the carbon quantum dot. We demonstrated that pH responsive carbon quantum dots whose surface charge changes from negative to positive possess biofilm dispersal ability in an acidic biofilm. Bacteria as a natural carbon source containing protein can be the optimal raw material for synthesizing carbon quantum dots. Additionally, probiotic bacteria have been applied for controlling gastrointestinal infection. Carbon quantum dots prepared from bacteria inherited some functional groups from the source bacteria, as well as new aromatic structures with nitrogen embedded inside the carbon structure (Chapter 3). Taking advantage of the nitrogen containing
aromatic structure, carbon quantum dots derived from Bifidobacterium breve can disperse Escherichia coli biofilms through ROS generation (Chapter 4).
Bacteria as natural carbon sources for synthesizing carbon quantum dots
Probiotics orally administered in combination with antibiotics have been demonstrated to be effective for the treatment of intestinal infections. However, commercial probiotic products have several challenges in culturing, manufacturing, life cycle management, and storage. Furthermore, pathogenic bacteria and microorganisms in a manufacturing environment can easily yield a quality problem for end-products, because small numbers of bacterial contaminants are difficult to detect yet can have major consequences for consumers and patients. Although orally administered probiotic bacteria can be used to treat intestinal infections, survival of probiotic bacteria in the gastro-intestinal tract is extremely troublesome, as probiotic bacteria are susceptible to the low pH of the gastrointestinal tract. Also, in patients simultaneously on antibiotics, probiotic bacteria can be killed by
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antibiotics along with the killing of their target pathogen [20,21]. Using carbon quantum dots with probiotic properties effectively overcomes the problems associated with the use of live probiotic bacteria, as the carbon quantum dots exhibit good water stability from pH 1 to pH 11 [22]. In order to take advantage of the infection control properties of probiotics, we have synthesized carbon quantum dots from B. breve and demonstrated their biofilm dispersal ability towards E.
coli biofilms. In contrast, carbon quantum dots synthesized from pathogenic bacteria inherited functional groups from their source bacteria that generated less ROS and possessed no biofilm dispersal ability. Nevertheless, one limitation of carbon quantum dots derived from B. breve is that they were not able to disperse Staphylococcus epidermidis biofilm, and therefore cannot qualified as a broad-spectrum biofilm dispersant and future research is necessary to optimize carbon quantum dot dispersal abilities.
Future research and perspectives
The functionalities of carbon quantum dots derived from natural materials depend on the chemical structures and heteroatoms inherited from the carbon sources [13,23,24]. We have demonstrated that carbon quantum dots derived from different source bacteria possess different physico-chemical properties. However, in our study, we only applied carbon quantum dots derived from B. breve and E. coli for antibiofilm application. It will be interesting to study the performance of carbon quantum dots derived from different probiotic bacteria in antibiofilm application and compare them with the efficacy of the probiotic source bacteria. Other probiotic bacteria, such as Bifidobacterium longum [25], Lactobacillus acidophilus [26],
Lactococcus lactis [27] and Lactobacillus salivarius [25], have demonstrated ability for infection control, but not directly on biofilms. Therefore, these probiotic bacteria can be promising natural carbon sources to synthesize antibiofilm carbon quantum dots. Furthermore, probiotics derived carbon quantum dots can be applied together with traditional antibiotics to remove and kill biofilms completely. Also the physico-chemical properties of these probiotic bacteria and the carbon quantum dots derived from them need to be determined in order to better identify the general properties that cause biofilm dispersal and better optimize their performance as a broad-spectrum biofilm dispersant, including Gram-negative, Gram-positive, and multi-drug resistant bacterial strains, whether EPS producing or not.
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Bacterial infections are more challenging to treat when pathogens are attached to a surface and have grown into a biofilm. Bacteria inside a biofilm may develop multidrug resistance after repeated exposure to antibiotics. Accordingly, there is an urgent need to develop alternative antimicrobial measures for prevention and treatment of infectious biofilms. In Chapter 1, we describe that nanoparticles
possess unique features due to their small size and different surface chemistries. Carbon quantum dots, in particular, possess several unique physico-chemical properties for antibacterial applications. This chapter provides an extensive overview of available methods to prepare carbon quantum dots from different carbon sources in order to provide guidelines for choosing methods and carbon sources that yield carbon quantum dots with optimal antibacterial efficacy. Antibacterial efficacy of carbon quantum dots predominantly involves cell wall damage and disruption of the matrix of infectious biofilms to cause dispersal of infecting pathogens that enhances their susceptibility to antibiotics. Carbon quantum dots derived through heating of natural carbon sources yield properties that resemble those of the carbon sources they were derived from. This makes antibiotics, natural carbon sources such as medicinal herbs and plants or probiotic bacteria ideal for the synthesis of antibacterial carbon quantum dots. Importantly antibiotic-derived carbon quantum dots have been suggested to yield a lower chance upon inducing bacterial resistance than their source antibiotics making carbon dots attractive for large scale clinical use. However, downward clinical translation of antibacterial carbon dots for infection control seems closer for synergistic use with existing antibiotics than for carbon dots as a stand-alone antimicrobial. Therefore, the aim of this thesis was to investigate biofilm dispersal ability of differently derived carbon dots, their physico-chemical and antibacterial properties. In addition, the synergistic effect of carbon dots with antibiotics was evaluated.
In Chapter 2, we report on the synthesis of novel, pH-responsive,
2,3-dimethylmaleic-anhydride modified carbon quantum dots (CDMMA-dots). CDMMA -dots, like unmodified carbon quantum dots without DMMA, were little bactericidal. However, CDMMA-dots reduced volumetric-bacterial-density within the acidic-environment of a biofilm for a non-EPS-producing Staphylococcus epidermidis strain, indicative for a more open structure. Such a structural disruption was not observed for an EPS-producing strain. Disrupted biofilms of the non-EPS-producing strain pre-exposed to CDMMA-dots at pH 5.0, were more amenable to vancomycin penetration and killing of their inhabitants than biofilms of EPS-producing-staphylococci. Herewith, we described a new role of carbon quantum dots as synthetic disruptants of biofilm structure. It is a partial success story, identifying the challenge of making carbon quantum dots that act as a universal disruptant for biofilms of strains with different microbiological characteristics, most notably the ability to produce or not-produce EPS. Such carbon quantum dots, will enable more effective clinical treatment of bacterial infections combined with current antibiotics.
Carbon quantum dots can be prepared by carbonization of highly different sources and therewith inherit different properties of the carbon sources used. In
Chapter 3, we synthesized carbon quantum dots from different bacterial strains,
with the aim to determine physico-chemical similarities between carbon quantum dots derived by pyrolytic carbonization of bacteria and their source bacteria.
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113 Carbon quantum dots carbonized at a reaction temperature of 180°C had negligible quantum yields, while reaction temperatures above 220°C yielded poorly water-suspendable carbon quantum dots. FTIR absorption spectra demonstrated preservation of amide (1650 cm-1) and polysaccharide (1180-1170 cm-1) absorption bands in carbon quantum dots carbonized at 200°C or 220°C. XPS analyses of carbon quantum dots indicated that the at%N in carbon quantum dots and the prevalence of carbon in O=C and COOH functionalities increased with the amount of protein in the source bacterial surface, while the at%O increased with the amount of bacterial cell surface polysaccharide. Collectively this led to the conclusion that carbon quantum dots inherit proteins and polysaccharides from their bacterial sources. Hydrocarbon-like bacterial components were transformed upon carbonization into new heterocyclic aromatic carbon structures, indicated by the development of a broad IR absorption band (920-900 cm-1). Graphitic nitrogen and phosphorus constituted heteroatoms in the carbon structure of carbon quantum dots. As a result of their chemical inheritance, zeta potentials of bacterially derived carbon quantum dots related with the zeta potentials of their source bacteria. The physico-chemical similarity between bacterially-derived carbon quantum dots and their source bacteria paves the way for using carbon quantum dots as a substitute for hard-to-use live bacteria as done in many applications. Since carbonization conditions determine the degree upon which desirable bacterial properties are inherited by carbon quantum dots, conditions have to be fine-tuned for different bacterial strains and applications.
The physico-chemical similarities between carbon quantum dots and their source bacteria stimulated the question whether carbon quantum dots derived from probiotic Bifidobacterium breve (B-C-dots) convey more favorable effects towards infection-control than carbon quantum dots derived from Escherichia coli (E-C-dots), an intestinal pathogen. In Chapter 4, FTIR, XPS and zeta potential
measurements indicated that both strains loose proteins upon carbonization, while polysaccharides were retained. However, B-C-dots were richer in proteins and polysaccharides than E-C-dots. Unlike pathogenic E-C-dots, probiotic B-C-dots dispersed E. coli biofilms due to their higher release of reactive-oxygen-species as compared with pathogenic E-C-dots. Moreover, pre-exposure of E. coli biofilms to dots enhanced ciprofloxacin efficacy, pointing to a synergy between B-C-dots and antibiotics in the control of intestinal E. coli infections, similar as observed for viable, probiotic bacteria. Orally administered probiotic bacteria however, were killed underway to their intestinal target site by the low pH of the gastro-intestinal tract or antibiotics with which they were co-administered. This impediment for clinical use of viable probiotic bacteria for the control of intestinal infections, can be circumvented by using probiotic carbon-dots instead of viable probiotic bacteria.
In the general discussion of this thesis (Chapter 5), mechanisms of biofilm
dispersal by carbon quantum dots, the importance of nitrogen functionalities in carbon quantum dots with respect to their antibacterial properties and the possibilities of carbon quantum dots derived from probiotic bacteria are discussed. Suggestions for future research are proposed.
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Bacteriële infecties zijn moeilijker te behandelen wanneer bacteriën zich aan een oppervlak hechten en zijn uitgegroeid tot een biofilm dan wanneer ze in een vloeistof zitten (planktonisch). Bacteriën in een biofilm kunnen resistentie tegen antibiotica ontwikkelen na herhaalde blootstelling aan antibiotica. Daarom is er dringend behoefte aan de ontwikkeling van alternatieve antimicrobiële maatregelen voor de preventie en behandeling van infectieuze biofilms. In Hoofdstuk 1
beschrijven we dat nanodeeltjes unieke eigenschappen bezitten vanwege hun kleine diameter en specifieke oppervlaktechemie. Met name koolstof-nanodeeltjes bezitten verschillende unieke fysisch-chemische eigenschappen voor antibacteriële toepassingen. Dit hoofdstuk geeft een uitgebreid overzicht van beschikbare methoden om koolstof-nanodeeltjes uit verschillende koolstofbronnen te maken en om methoden en koolstofbronnen te kiezen voor optimale antibacteriële werking van koolstof-nanodeeltjes. Antibacteriële koolstof-nanodeeltjes beschadigen voornamelijk de celwand van bacteriën en maken de matrix van infectieuze biofilms kapot, waardoor er losse bacteriën vrijkomen en die zijn gevoeliger voor antibiotica dan bacteriën in een biofilm. Koolstof-nanodeeltjes die zijn verkregen door verhitting van natuurlijke koolstofbronnen, leveren eigenschappen op die lijken op die van de koolstofbronnen waarvan ze zijn afgeleid. Dit maakt antibiotica en natuurlijke koolstofbronnen zoals geneeskrachtige kruiden en planten of probiotische bacteriën ideaal voor de synthese van antibacteriële koolstof-nanodeeltjes. Tevens geeft de literatuur de suggestie dat koolstof-nanodeeltjes gemaakt van antibiotica een lagere kans op bacteriële resistentie opleveren dan hun bron-antibiotica, waardoor koolstof-nanodeeltjes aantrekkelijk kunnen worden voor grootschalig klinisch gebruik. Vooreerst echter, lijkt het gebruik van antibacteriële koolstof-nanodeeltjes in combinatie met antibiotica realistischer om te gebruiken voor de bestrijding van infectieuze biofilms in de kliniek dan koolstof-nanodeeltjes alleen. Daarom was het doel van dit proefschrift om te onderzoeken of koolstof-nanodeeltjes van verschillende koolstofbronnen, waaronder bacteriële bronnen, bacteriën los kunnen maken uit een biofilm en werden synergetische effecten van koolstof-nanodeeltjes met antibiotica geëvalueerd. Tevens werd de overerving van fysisch-chemische eigenschappen door koolstof-nanodeeltjes van bron-bacteriën verder onderzocht. In Hoofdstuk 2 rapporteren we over de synthese van nieuwe, pH-gevoelige,
2,3-dimethylmaleïnezuuranhydride gemodificeerde koolstof-nanodeeltjes (CDMMA -nanodeeltjes). CDMMA-nanodeeltjes, zoals ook koolstof-nanodeeltjes zonder DMMA, waren nauwelijks bacteriedodend. CDMMA-nanodeeltjes zorgden voor een meer open structuur, een lagere bacteriële dichtheid, in de zure omgeving van een biofilm van een niet-EPS (extracellular polymeric substances) producerende Staphylococcus epidermidis. Een dergelijke structuur verandering werd niet waargenomen in een biofilm van een EPS-producerende stam. Vancomycine kon gemakkelijker binnendringen in zo’n veranderde biofilm structuur en meer bacteriën doden in deze biofilms. Hiermee wordt een nieuwe rol van koolstof-nanodeeltjes beschreven als synthetische “dispersant” van een biofilm structuur. Het is een gedeeltelijk succesverhaal dat de uitdaging identificeert om koolstof-nanodeeltjes te maken die gebruikt kunnen worden om een meer open structuur te maken in biofilms van bacteriën met verschillende microbiologische kenmerken, met name
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117 het vermogen om al dan niet EPS te produceren. Dergelijke koolstof-nanodeeltjes zullen een effectievere klinische behandeling van bacteriële infecties mogelijk maken in combinatie met de huidige antibiotica.
Koolstof-nanodeeltjes kunnen worden gemaakt door carbonisatie van zeer verschillende bronnen en daardoor verschillende eigenschappen van de gebruikte koolstofbronnen overerven. In Hoofdstuk 3 hebben we koolstof-nanodeeltjes
gesynthetiseerd van verschillende bacteriestammen, met als doel fysisch-chemische overeenkomsten te vergelijken van de koolstof-nanodeeltjes en hun bron-bacteriën. Koolstof-nanodeeltjes verbrand door pyrolytische carbonisatie bij een reactietemperatuur van 180°C leverden verwaarloosbare opbrengsten, terwijl reactietemperaturen boven 220°C slecht in water oplosbare koolstof-nanodeeltjes opleverden. Fourier transform infrarood (FTIR) absorptiespectra toonden aan dat de absorptiebanden van amide (1650 cm-1) en polysacharide (1180-1170 cm-1) blijven bestaan in koolstof-nanodeeltjes die werden verbrand bij 200°C of 220°C. X-ray photonelectron spectroscopy (XPS) analyses van koolstof-nanodeeltjes gaven aan dat het at% N in koolstof-koolstof-nanodeeltjes en het voorkomen van koolstof in O=C- en COOH-functionaliteiten toenamen met de hoeveelheid eiwit aan het bacteriële oppervlak die gebruikt waren om de koolstof-nanodeeltjes van te maken, terwijl het at% O toenam met de hoeveelheid polysacharide op het bacteriële celoppervlak. Dit leidde tot de conclusie dat koolstof-nanodeeltjes eiwitten en polysachariden erven van de bacteriën waar ze van gemaakt zijn. Koolwaterstofachtige bacteriële componenten werden na carbonisatie omgezet in nieuwe heterocyclische aromatische koolstofstructuren, die werden waargenomen in het infrarood-absorptiespectrum (absorptieband 920-900 cm-1). Grafiet-stikstof en fosfor vormden heteroatomen in de aromatische koolstofstructuur van koolstof-nanodeeltjes. Als resultaat van hun chemische overerving, zijn de zeta potentialen van de koolstof-nanodeeltjes gerelateerd aan de zeta potentialen van hun bron-bacteriën. De fysisch-chemische overeenkomst tussen bacterieel afgeleide koolstof-nanodeeltjes en hun bron-bacteriën maakt de weg vrij voor het gebruik van koolstof-nanodeeltjes als vervanging voor moeilijk te gebruiken levende bacteriën, zoals in veel toepassingen wordt gedaan. Aangezien carbonisatieomstandigheden bepalen in welke mate wenselijke bacteriële eigenschappen worden geërfd door koolstof-nanodeeltjes, moeten de omstandigheden worden geoptimaliseerd voor verschillende bacteriestammen en toepassingen.
De fysisch-chemische overeenkomsten tussen koolstof-nanodeeltjes en hun bron-bacteriën stimuleerden de vraag of koolstof-nanodeeltjes gemaakt van probiotische Bifidobacterium breve (B-C-nanodeeltjes) gunstiger effecten hebben op infectieuze biofilms dan koolstof-nanodeeltjes gemaakt van Escherichia coli (E-C-nanodeeltjes), een darmpathogeen. In Hoofdstuk 4 gaven FTIR-, XPS- en zeta potentiaal
metingen aan dat beide stammen zowel eiwitten als polysachariden overerven aan nanodeeltjes. B-C-nanodeeltjes waren echter rijker aan eiwitten en polysachariden dan E-C-nanodeeltjes. In tegenstelling tot pathogene E-C-nanodeeltjes, creëerden probiotische B-C-nanodeeltjes een meer open structuur in E. coli biofilms vanwege hun hogere afgifte van reactieve zuurstof componenten (ROS) in vergelijking
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met pathogene E-C-nanodeeltjes. Bovendien verhoogde blootstelling van E. coli biofilms aan B-C-nanodeeltjes de werkzaamheid van ciprofloxacine, wat wijst op een synergie tussen B-C-nanodeeltjes en antibiotica bij de bestrijding van E. coli infecties, vergelijkbaar met het effect van probiotische bacteriën. Oraal toegediende probiotische bacteriën worden gedood door de lage pH van het maagdarmkanaal die ze moeten passeren voordat ze hun doel in de darmen bereiken of ze worden gedood door antibiotica waarmee ze gelijktijdig worden toegediend. Deze belemmering voor klinisch gebruik van levende probiotische bacteriën voor de bestrijding van darminfecties kan worden omzeild door probiotische koolstof-nanodeeltjes te gebruiken.
In de algemene discussie van dit proefschrift (Hoofdstuk 5) worden
mechanismen van biofilmverstoring door koolstof-nanodeeltjes, het belang van stikstoffunctionaliteiten in koolstof-nanodeeltjes met betrekking tot hun antibacteriële eigenschappen en de mogelijkheden van koolstof-nanodeeltjes gemaakt van probiotische bacteriën besproken. Er worden suggesties gedaan voor toekomstig onderzoek.
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Four years ago, when the first time I arrived in Groningen for the Alert-program interview, I didn’t know that I would fall in love with this place. There is no doubt that this PhD period is one of the beautiful memories in my life. I have enjoyed the pure academic research environment, obtained true friendship, and opened my mind to see the world. PhD in UMCG/RUG really shape who I am in these four years, both personally and professionally. There were tough time though, when the experiment didn’t work, or problems were very difficult to be solved. I challenged myself to start as a pure chemist with little knowledge about microbiology, and grew up as an independent researcher in the field of biomedical engineering. When I recall those experience, I do believe that what doesn’t kill me makes me stronger.
Dear Prof. Yijin, my first promotor, thank you very much for choosing me. I
remember when we met during the Alert selected days, I was super nervous and shy, and didn’t know how to open a talk with a prominent scientist. Your smile made me relax and gradually I started to share my experience. After the talk, I told myself that this is the professor that I would like to work with. In the following four years, I have learnt many things from you, the scientific way of thinking, the optimal way to present the data, and social skills. The most important thing I would like to appreciate is your belief in me. Because of your high expectations for me, I tried to improve myself to higher level to meet them. Thank you very much for believing me, and make me become a better researcher.
Dear Prof. Henk, thank you very much for not only helping me with papers and
thesis, but also for guiding me in doing science independently. I very appreciate you teaching me a way to structure research stories in a more attractive way to highlight the significance and novelty, and making me realize that science is not just about good results, “bad results” can also be valuable. Most importantly, I have learnt to see the same thing from different perspectives and gradually changed my stubborn way of thinking. I enjoyed every moments when we discussed about the results and exchanged opinions. What made me impressive and moved is that you told me “I would like to make you better when you leave Groningen”. Thank you very much for all your help, I know these skills will benefit me forever.
Dear Prof. Henny, thank you very much for your supervision during my PhD.
I’m so lucky to have you to be one of my supervisors. You not only care about my research projects, but also care about my life. When I had questions, you answered promptly. When I had problems about experiments, you were there to give me a hand. When I was depressed, you were the one who gave me support. I clearly remember the moments you helped me to check my samples in the lab, the email you sent me to ask whether I had exercised during the COVID time, and the encouragement you gave me in the last several months of my PhD. Besides, you as a model learn me how to be precise in doing work. Although I still need to improve on this part, I have realized and would always remind myself. Thank you very much for all your support. All these things mean a lot to me and I would never forget them.
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I would like to express my deepest gratitude to the reading commottee, Prof. H. Koo, Prof. H.W. Frijlink and Prof. L.A.M. Marks for agreeing to be the reading
committee for my thesis and for the valuable time to assess my thesis.
I am grateful to the entire scientific staff, Romana, Patrick, Prashant, Brandon, Theo, Jelmer, Inge, Chris for the scientific discussion in the lunch meeting and
Kolff-days.
Maria, my sweet friend, I’m very happy to know you in BME. We have so many
precious memories when I recall. I miss our vacation in Mallorca, hanging out on the weekends, and the time we had lunch together before the COVID time. Thank you very much for your company in the past three years.
Ke, thank you very much for helping me to draw pictures, helping me to buy a bike
in a good price, and ordering food with me for lunch and dinner. I like your historical story very much.
Simona, my annoying friend, thank you for taking me to explore new places in
Groningen, and laughing at me when I am lost. Thank you for “living in my phone” during the COVID time, and your words of encouragement inspired me during this difficult time. I hope you know how special you are to me.
Damla, there are so many unforgettable memories with you in the past four years. I
would like to acknowledge the many kindnesses you have shown to me, and thank you have been so supportive to me. I appreciate you more than words can say.
Mari, thank you for supporting and helping me. I’m lucky to have you by my side,
and I would like to appreciate you for understanding me and sharing your time with me whenever I need. You'll never know how much your help meant to me.
Abby, my Guimi, you simply make things brighter. You always understand me,
never judge me and accept me. You make me feel seen and heard, and I love you for that. It is so nice to have you in the office.
Yuanfeng, thank you for your warm encouragements and help. I miss the beef you
cooked, the hotpot you made, and our vacation in Greece. Your kindness means so much to me. Thais, thank you for inviting me to your wonderful wedding, and
that is one of the excellent memories in my PhD. Although now there are thousand miles apart, you are still my good old friend. Hao and Guang, thank you for helping
me with experiments, especially in the COVID time with a lot of inconvenient.
Jacopo, thank you for helping me on the fluorescence spectrophotometer. I very
appreciate you came to the lab at seven o' clock on Saturday morning for helping my experiment.
Lu Yuan, thank you for your help with cell culture experiments, and also sharing
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happiness in the office. Huaiying, thank you very much for cooking and sharing
your place with me, and your food was super delicious. Aryan, thank you for
learning me how to measure ROS and always answer my question patiently.
Jeroen, thank you for showing me your garden and giving me beautiful chicken
feathers. Yong, thank you very much for sharing your ideas and opinions with me. Olga C, Valentina, Guangyue, Hongping, Can, Linzhu, Lu Ge, Liangliang, many
thanks for your encouragements and support. I miss the time when we have coffer break in the kitchen. Xiaoxiang, thank you for checking and choosing the place
for vacation, and I have enjoyed the trip very much. Ruifang, many thanks for
your company in Finland; I have a beautiful memory with you there. Chengxiong, Masha and Kechen, thank you for traveling with me to Keukenhof, what a beautiful
tulips garden. Nathali, thank you for joining us in Crete, it was nice to have you in
the trip. Kaiqi, thank you for your company on the weekends in the lab.
I would like to show my grateful to the people who I met in the department: Olga S, Yuchen, Torben, Tarreneh, JP, Qihui, Lais, Zhengya, Zhiwei, Linyan, Lei Li, Chuang Li, Yi Yang, Yue Zhang, Runrun, Yiruo, Derly, Yafei, Eliza, Vera, Rebecca, Yori, Kiran, Alina, Alejandro, Neda, Simon, Colin, Gwenda, Devlina, Thea, Clio, Klaudia, Fenghua. Many thanks for your help in the department. I am
very happy to work with Manali and be involved in her Mater project.
Special thanks for Alert people, Patricia, Jan, Laszlo, Roberto, Piermichele, Miancheng, Harita, Nicola, Francis, Elisa, Xintong, it is so nice to meet you in
Groningen.
Debby, Vanessa, and Wya, thank you for helping me with different forms and
reimbursement. Ina, thank you for inviting me to your house, the cake was very
delicious, and thank you for your help in the department. Willy, thank you for
sharing with me your beautiful garden.
I would like to acknowledge our technical staff: Reinier, thank you for helping
me with the cell culture experiments. Melissa, thank you for helping me with
experiments in my first year, I have learn a lot from you. Gesinda, thank you for
helping me with OCT and freeze dryer. The OCT images are amazing. Joop, thank
you for helping me on XPS measurement. Willem, thank you for helping me in the
lab. Ed, thank you for the help on OCT software. Corien, thank you for helping
me on TEM. Jelly and Marja, thank you for helping me in the lab. Betsy, thank
you for helping me with ordering. Hans, thank you for helping me on Zeta and
DLS measurement, and sharing your knowledge of fish with me. Marc, thank you
for helping me on taking super nice TEM images, and giving me a training about preparing different kinds of TEM samples. I have learn a lot about TEM from you. 谢谢爸爸妈妈还有姐姐，非常感谢你们鼓励我去追求自己的梦想。我知道这四年里 你们的担心和牵挂，因为有你们的支持，我才能毫无后顾之忧地去尝试人生中的各 种可能。人生短暂，希望我不负韶华，更不负你们的期望。
